Dissertationen: „Immune tumor microenvironment“

1

Jiménez, Bernal Isabel. „Tumor immune microenvironment in B-cell lymphoid malignancies“. Doctoral thesis, Universitat Autònoma de Barcelona, 2020. http://hdl.handle.net/10803/671173.

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El microambient immune tumoral juga un paper fonamental en les etapes inicials de la formació dels tumors i en la progressió d’aquests. Teràpies dirigides a aquest microambient ofereixen noves opcions terapèutiques i també serveixen per a millorar les teràpies actuals enfront de molts càncers, incloent els que afecten les cèl·lules B. No obstant això, són necessàries més recerques per a entendre en major profunditat els mecanismes d’evasió del sistema immune que afavoreixen la progressió dels tumors i dissenyar immunoteràpies més precises. Els nostres principals objectius són aportar noves evidències sobre mecanismes immunes associats a la progressió tumoral i les bases pre-clíniques per al desenvolupament de noves estratègies terapèutiques amb potencial immunomodulador. Per a això, ens centrem en la leucèmia limfàtica crònica (LLC) i en el limfoma cerebral primari (LCP). Els mecanismes de progressió en LLC des d’estadis inicials no són coneguts íntegrament. Encara que l’adquisició d’alteracions moleculars és escassa suggerint que la LLC no progressa exclusivament per mecanismes d’evolució clonal, encara no s’ha dut a terme una anàlisi exhaustiva del microambient immune que demostri que la progressió sí que pugui deure’s a canvis immunes. Per això, hem realitzat un estudi longitudinal abastant tant els escenaris genètics com immunològics en pacients de LLC sense tractar que han progressat clínicament i en pacients asimptomàtics durant un llarg període de temps. Els nostres resultats mostren que els pacients que progressen experimenten un increment de cèl·lules T CD8+ efectores de memòria i terminalment exhaustes T-betmid/-*EomeshiPDhi a la progressió. Aquest increment no s’observa en els pacients de LLC que no han progressat. A més, les cèl·lules T a la progressió adquireixen un perfil transcripcional diferent. Això va acompanyat d’un augment en les propietats immunosupressores de les cèl·lules leucèmiques a la progressió. Vam demostrar que les cèl·lules de LLC en el moment de la progressió tenen major capacitat d’induir exhaustió tant en cèl·lules T CD8+ de LLC com aquelles procedents d’individus sans, i que ho fan mitjançant un mecanisme dependent de factors solubles que inclou IL-10. Els escassos canvis genètics que trobem després de seqüenciar el exoma dels nostres pacients ens permeten concloure que les variacions immunes que hem identificat són fonamentals per a la progressió de la LLC. El desenllaç dels pacients diagnosticats amb LCP és normalment desfavorable a causa de l’escassetat d’opcions terapèutiques efectives. Les cèl·lules malignes de LCP presenten amb freqüència una desregulació de la via del receptor de la cèl·lula B (de l’anglès, BCR), però la seva inhibició mitjançant ibrutinib mostra respostes molt breus en pacients. No obstant això, la via del BCR també pot bloquejar-se mitjançant la inhibició de la exportina nuclear XPO1 amb selinexor. Selinexor travessa la barrera hemato-encefàlica i ha mostrat activitat en un pacient diagnosticat amb limfoma difús de cèl·lules grans B amb recaiguda en el sistema nerviós central. Per consegüent, decidim avaluar els efectes de selinexor en monoteràpia i combinat amb ibrutinib en models preclínics murinos de LCP. La nostra anàlisi mostra que selinexor bloqueja el creixement tumoral i prolonga la supervivència en un model de ratolí bioluminiscent i la combinació amb ibrutinib prolonga encara més la supervivència. Vam demostrar que els limfomes cerebrals en ratolí estan infiltrats amb macròfags pro-tumorals M2 que expressen PD-1 i SIRPα. A més, el tractament amb selinexor i ibrutinib afavoreix la resposta immune anti-tumoral induint un canvi en la polarització dels macròfags cap a un perfil pro-inflamatori i reduint l’expressió de PD-1 i SIRPα en els macròfags M2 associats al tumor.
El microambiente inmune tumoral juega un papel fundamental en las etapas tempranas de la formación de los tumores y en la progresión de éstos. Terapias dirigidas a este microambiente ofrecen nuevas opciones terapéuticas y también sirven para mejorar las terapias actuales frente a muchos cánceres, incluyendo los que afectan a las células B. Sin embargo, son necesarias más investigaciones para entender en mayor profundidad los mecanismos de evasión del sistema inmune que favorecen la progresión de los tumores y diseñar inmunoterapias más precisas. Nuestros principales objetivos son aportar nuevas evidencias sobre mecanismos inmunes asociados a la progresión tumoral y las bases pre-clínicas para el desarrollo de nuevas estrategias terapéuticas con potencial inmuno-modulador. Para ello, nos centramos en la leucemia linfática crónica (LLC) y en el linfoma cerebral primario (LCP). Los mecanismos de progresión en LLC desde estadios tempranos no son conocidos en su totalidad. Aunque la adquisición de alteraciones moleculares es escasa sugiriendo que la LLC no progresa exclusivamente por mecanismos de evolución clonal, todavía no se ha llevado a cabo un análisis exhaustivo del microambiente inmune que demuestre que la progresión sí pueda deberse a cambios inmunes. Por ello, hemos realizado un estudio longitudinal abarcando tanto los escenarios genéticos como inmunológicos en pacientes de LLC sin tratar que han progresado clínicamente y en pacientes asintomáticos durante un largo periodo de tiempo. Nuestros resultados muestran que los pacientes que progresan experimentan un incremento de células T CD8+ efectoras de memoria y terminalmente exhaustas T-betmid/-EomeshiPDhi a la progresión. Este incremento no se observa en los pacientes de LLC que no han progresado. Además, las células T a la progresión adquieren un perfil transcripcional diferente. Esto va acompañado de un aumento en las propiedades inmunosupresoras de las células leucémicas a la progresión. Demostramos que las células de LLC en el momento de la progresión tienen mayor capacidad de inducir exhaustión tanto en células T CD8+ de LLC como aquellas procedentes de individuos sanos, y que lo hacen mediante un mecanismo dependiente de factores solubles que incluye IL-10. Los escasos cambios genéticos que encontramos tras secuenciar el exoma de nuestros pacientes nos permiten concluir que las variaciones inmunes que hemos identificado son fundamentales para la progresión de la LLC. El desenlace de los pacientes diagnosticados con LCP es normalmente desfavorable debido a la escasez de opciones terapéuticas efectivas. Las células malignas de LCP presentan con frecuencia una desregulación de la vía del receptor de la célula B (del inglés, BCR), pero su inhibición mediante ibrutinib muestra respuestas muy breves en pacientes. Sin embargo, la vía del BCR también puede bloquearse mediante la inhibición de la exportina nuclear XPO1 con selinexor. Selinexor atraviesa la barrera hemato-encefálica y ha mostrado actividad en un paciente diagnosticado con linfoma difuso de células grandes B con recaída en el sistema nervioso central. Por consiguiente, decidimos evaluar los efectos de selinexor en monoterapia y combinado con ibrutinib en modelos pre-clínicos murinos de LCP. Nuestro análisis muestra que selinexor bloquea el crecimiento tumoral y prolonga la supervivencia en un modelo de ratón bioluminiscente y la combinación con ibrutinib prolonga aún más la supervivencia. Demostramos que los linfomas cerebrales en ratón están infiltrados con macrófagos pro-tumorales M2 que expresan PD-1 y SIRPα. Además, el tratamiento con selinexor e ibrutinib favorece la respuesta inmune anti-tumoral induciendo un cambio en la polarización de los macrófagos hacia un perfil pro-inflamatorio y reduciendo la expresión de PD-1 y SIRPα en los macrófagos M2 asociados al tumor.
The tumor immune microenvironment (TIME) plays a critical role in the early formation of tumors and their progression. Targeting the TIME has offered new therapeutic approaches and improved current ones in several cancers, including B-cell malignancies. Nonetheless, further investigation is needed in order to more deeply understand immune evasion mechanisms that lead to tumor progression and to design therapies that modulate the immune system more precisely. Here, our main objectives are to provide new insights into immune mechanisms that favor tumor progression and a pre-clinical rationale for the design of new therapeutic strategies with immunomodulatory potential. To accomplish these goals our study will focus on chronic lymphocytic leukemia (CLL) and primary central nervous system lymphoma (PCNSL). Mechanisms driving the progression of CLL from its early stages are not fully understood. This hampers detecting progression in advance and developing therapies that could intervene in the early stages. Although the limited acquisition of molecular changes suggests that CLL progression is not mainly driven by clonal evolution, a deeper analysis of the immune microenvironment that demonstrates immune variations over time that contribute to progression has not been performed. Hence, we longitudinally studied the immune and genetic landscapes of untreated progressing and non-progressing patients. Our results show that progressed CLL patients experience an increase in effector memory and terminally exhausted T-betmid/-EomeshiPDhi CD8+ T cells over time, not observed in non-progressing patients. In addition, T cells at progression acquire a distinct transcriptional profile. This is accompanied by enhanced immunosuppressive properties in leukemic cells at progression. We prove that progressed CLL cells are intrinsically more capable of inducing CD8+ T-cell exhaustion in T cells affected by CLL and healthy T cells by a mechanism dependent on soluble factors including IL-10. In addition, the reduced genetic changes we found by whole-exome sequencing in our cohort indicate these immune variations are fundamental for progression in CLL. Patients diagnosed with PCNSL often face dismal outcomes due to the limited availability of therapeutic options. PCNSL cells frequently have deregulated B-cell receptor (BCR) signaling, but its inhibition using ibrutinib only offers a brief effective response in PCNSL patients. Nonetheless, the BCR pathway can also be blocked by inhibiting the nuclear exportin XPO1 using selinexor. Selinexor is able to cross the blood–brain barrier and has shown positive clinical activity in a patient with refractory diffuse large B-cell lymphoma in the CNS. Accordingly, we evaluated the effects of selinexor alone and also combined it with ibrutinib in pre-clinical mouse models of PCNSL. Our analysis shows that selinexor blocks tumor growth and prolongs survival in a bioluminescent mouse model and its combination with ibrutinib further increases survival. We demonstrate that CNS lymphomas in mice are infiltrated by tumor-promoting M2-like macrophages expressing PD-1 and SIRPα. Moreover, the treatment with selinexor and ibrutinib favors an anti-tumoral immune response by shifting macrophage polarization toward an inflammatory phenotype and diminishing the expression of PD-1 and SIRPα in M2 tumor-associated macrophages.

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TANASKOVIC, OLGA. „LACK OF P21 EXPRESSION IN TUMOR-ASSOCIATED APCS TRIGGERS THE ACTIVATION OF A POTENT ANTI-TUMOR IMMUNE RESPONSE“. Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/608993.

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Over the last decade, the cell-cycle inhibitor p21 has been shown to sustain leukaemia propagation with two distinct mechanisms. On one hand, p21 was shown to be critical for maintaining increased self-renewal capacities of leukaemia stem cells. Indeed, the absence of p21 in leukaemia stem cells leads to their functional exhaustion, which results in loss of leukaemia transplantability in syngeneic mice. On the other hand, p21 expression is crucial for evading the surveillance mechanisms of the immune system, thus ensuring tumor growth. Specifically, lack of p21 in the leukemic microenvironment activates a potent CD4+ T-cell mediated immunological response against tumor in syngeneic context (un-published data from the host laboratory). To translate the observed p21-dependent anti-tumoral immunity into novel immune-therapies against cancer, underlying mechanisms needed to be unrevealed. In my thesis work, I dissected the cellular bases of the p21-dependent anti-tumor immuni-ty. I disclosed a crucial role of the p21-/- tumor microenvironment in triggering activation of an anti-tumor immunological response. In particular, for the first time I identified rare iron-loaded CD68+ tumor-associated macrophages (iTAMs) in the p21-null context as key me-diators of a potent immunological mechanisms of cancer clearance. By unravelling crucial players of the p21-dependent anti-tumor immunity, my work set the basis for the future de-sign of novel anti-cancer vaccines. Such vaccines will grant more efficient and less toxic treatment for cancer patients. To further transpose such immunological mechanism of cancer clearance in humans, the us-age of a proper humanized mouse model is needed. Actually, humanized mice allow to study the interaction between human immune system and cancers of human origin. I gener-ated hCB-CD34+ NSG mice containing all the cellular components of human immunity. However, I showed that these mice are fully accessible to human tumor growth, demon-strating the inadequacy of hCB-CD34+ NSG model in immuno-oncology. Thus, the devel-opment of a proper humanized mouse model to study p21-dependent anti-tumoral immune response in human context remains necessary.

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Giallongo, Cesarina. „Immune escape mechanisms in hematological diseades: role of the myeloid derived suppressor cells and tumor microenvironment“. Doctoral thesis, Università di Catania, 2017. http://hdl.handle.net/10761/3889.

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The interactions between the immune system and the tumor cells occur through complex events that lead to tumor eradication or immune evasion by cancer. Recently, the prognostic role of Myeloid derived suppressor cells (MDSC) accumulation has been documented for some hematological malignancies where they correlates with disease progression and persistence of minimal residual disease. We first evaluated the change of MDSC frequency in hematological patients during therapy founding a significant correlation between the number of persistent monocytic-MDSC and major molecular response (MMR) value in chronic myeloid leukemia patients treated with dasatinib. Moreover, our data demonstrated that tumor cells, through the release of soluble factors and exosomes, are able to expand monocytic-MDSC, creating an immunotolerant environment that results in T cell anergy and facilitates tumor growth. In addition, cancer cells are also able to promote immune dysfunction in MSC with their consequent commitment, via TLR4 signaling, toward an activated status promoting immune escape through the polarization of neutrophils in immunosuppressive cells.

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Ma, Yuting. „The crosstalk between dying tumor cells and immune effectors within tumor microenvironment elicited by anti-cancer therapies dictates the therapeutic outcome“. Phd thesis, Université Paris Sud - Paris XI, 2011. http://tel.archives-ouvertes.fr/tel-00636891.

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Besides exerting cytostatic or cytotoxic effects on tumor cells, some anti-cancer therapies (anthracyclines, oxaliplatin, X-Rays) could trigger an immunogenic cell death modality, releasing danger signals to alert immune system. We have shown that tumor-specific IFN- producing CD8+ T cells (Tc1) are mandatory for the success of chemotherapy to prevent tumor outgrowth. Priming of Tc1 response depends on IL-1β secretion by DC confronted with anthracycline-treated tumor cells releasing ATP. To identify the inflammatory components which link innate and cognate immune responses, we analyzed the influence of immunogenic chemotherapy on tumor microenvironment. We found an upregulated Th1- and Th17-related gene expression pattern in growth-retarded tumor after anthracycline treatment. By interfering with IFN- or IL-17A pathways, therapeutic effect of doxorubicin and oxaliplatin was abolished and dying tumor cell-based vaccine lost its efficacy to protect mice from live tumor cell rechallenge. Interestingly, we discovered that distinct subsets of  T lymphocytes (V4+ and V6+) colonized tumors shortly after chemotherapy, where they proliferated and became the dominant IL-17 producers within tumor beds. In three tumor models treated with chemotherapy or radiotherapy, a strong correlation between the presence of IL-17-producing  T ( T17) and IFN--producing CD8+ TIL (Tc1) was discovered. IL-17A signaling acts as upstream of IFN- since defect in IL-17RA led to complete loss of antigen specific Tc1 priming. The contribution of  T17 cells (V4+ and V6+) to chemotherapy is critical as V4/6-/- mice showed reduced sensitivity to chemotherapy and vaccination. Also, tumor infiltrating  T17 and Tc1 cells were reduced to basal level in this strain. IL-1β/IL-1R, but not IL-23/IL-23R, is pivotal for IL-17 production by  T cells and the success of chemotherapy. Importantly, adoptive transfer of  T cells could restore the efficacy of chemotherapy in IL-17A-/- mice and ameliorate the effect of chemotherapy in wild type host, provided that they retain the expression of IL-1R and IL-17A. Our research suggest a DC (IL-1β) →  T cells (IL-17) → Tc1 (IFN-) immune axis triggered by chemotherapy-induced dying tumor cells, which is critical for the favorable therapeutic response. To boost the immune system, we try to combine immunogenic chemotherapy with tumor vaccine in the presence of TLR3 agonist Poly (A:U). This sequential combined therapy, which we named VCT, could significantly retard tumor growth or even completely eradicate tumor and establish long-term protection against rechallenge in highly tumorigenic models. To dissect the effect of Poly (A:U) on immune system and that on TLR3 expressing-tumor cells, we performed VCT treatment in nude mice, TRIF-/- mice and with TRIF-silencing tumors. Interestingly, our results suggested that anti-tumor effect of VCT required T cells and intact TRIF signaling pathway at the level of the host and that of tumor cells. Poly (A:U) treatment could induce high level of CCL5 and CXCL10 production from tumor cells both in vitro and in vivo, which could negatively and positively influence the therapeutic outcome. By uncoupling the effect of CCL5 from that of CXCL10, the VCT treatment can be ameliorated. Our study emphasizes that both tumor and host derived inflammatory factors participate in regulating anti-tumor response. We also highlight that therapeutic application of TLR agonists can be optimized through regulating the profile of chemokines and their downstream signaling events.

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Yuting, Ma. „The crosstalk between dying tumor cells and immune effectors within tumor microenvironment elicited by anti-cancer therapies dictates the therapeutic outcome“. Thesis, Paris 11, 2011. http://www.theses.fr/2011PA11T033/document.

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En dehors des effets cytostatiques ou cytotoxiques sur les cellules tumorales, certaines thérapies anti-cancéreuses peuvent déclencher la mort cellulaire immunogénique, libérant les signaux de danger pour alerter le système immunitaire. Les cellules T CD8+ T (Tc1) productrices d’IFN- et spécifiques de la tumeur sont nécessaires pour le succès de la chimiothérapie et la diminution de la croissance tumorale. L’amorçage d’une réponse bénéfique Tc1 dépend de la sécrétion d'IL-1β par les cellules dendritiques confrontées à des cellules tumorales traitées avec de l’anthracycline libérant de l’ATP. Pour identifier les composants inflammatoires qui lient les réponses immunitaires innées et adaptatives, nous avons analysé l'influence de la chimiothérapie immunogène sur le microenvironnement de la tumeur. Nous avons identifié une up-régulation de gènes associés à la réponse Th1 et Th17 dans un modèle de tumeur répondant au traitement par les anthracyclines par un retard de croissance. En interférant avec les voies IFN- ou l'IL-17A, l'effet thérapeutique de la doxorubicine et l'oxaliplatine a été aboli et le vaccin à base de cellules tumorales mortes ne protège plus les souris de la réintroduction de cellules tumorales vivantes. Nous avons également découvert que des sous-populations distinctes de lymphocytes T  (V4+ et V6+) colonisent des tumeurs peu de temps après la chimiothérapie, où ils ont proliféré et sont devenus producteurs majeurs de l’IL-17 au sein de la tumeur. Nous avons constaté une forte corrélation entre la présence de lymphocytes T  producteurs d’IL-17 ( T17) et de TIL CD8+ (Tc1) dans trois modèles différents de tumeurs traitées par la chimiothérapie ou la radiothérapie. IL-17A agit sur la signalisation en amont de l'IFN- puisqu’un défaut d’expression d’IL-17RA conduit à la perte complète de la production des Tc1 spécifiques de l’antigène. La contribution des cellules  T17 (V4+ et V6+) dans l’effet bénéfique de la chimiothérapie est essentielle puisque les souris V4/6-/-. L’axe IL-1β/IL-1R, mais pas IL-23/IL-23R, est essentielle pour la production d'IL-17 par les cellules T et l’effet bénéfique de la chimiothérapie. Le transfert adoptif de lymphocytes  T peut rétablir l'efficacité de la chimiothérapie dans le modèle de souris IL-17A-/- et améliorer l'effet de la chimiothérapie chez la souris wt, s'ils conservent l'expression de l'IL-1R et de l'IL-17A. Nos résultats suggèrent l’existence d’un axe fonctionnel: DC (IL-1β) → cellules T (IL-17) → Tc1 (IFN-), déclenché par la chimiothérapie induisant la mort des cellules tumorales, phénomène essentiel pour une réponse thérapeutique favorable. Pour renforcer la réponse immunitaire, nous essayons de combiner la chimiothérapie « immunogène » avec le vaccin anti-tumoral en présence d’adjuvants (poly (A:U), l'agoniste de TLR3).Ce type de thérapie séquentielle combinée, appelé VCT, pourrait retarder considérablement la croissance des tumeurs, voire l’éradiquer complètement et établir une protection spécifique à long terme. Pour décortiquer l'effet de la poly (A:U) sur le système immunitaire et sur les cellules tumorales exprimant le TLR3, nous avons effectué un traitement VCT chez la souris nude, TRIF-/- et les souris présentant une diminution de l’expression de TRIF au niveau des cellules tumorales. Ainsi l'effet anti-tumoral de VCT requiert les lymphocytes T et la voie de signalisation TRIF intacte au niveau de l'hôte et des cellules tumorales. Le traitement poly (A:U) peut induire un niveau élevé de production de certaines chimiokines associées à la réponse de type Th1 (CCL5 et CXCL10 ) par les cellules tumorales in vitro et in vivo, ce qui peut influencer négativement et positivement les résultats thérapeutiques. Le découplage de l’action de CCL5 et de XCL10, pourrait améliorer le traitement par la VCT. Notre étude souligne ainsi le rôle des facteurs inflammatoires dérivés de la tumeur et de l’hôte dans la régulation de la réponse immunitaire anti-tumorale
Besides exerting cytostatic or cytotoxic effects on tumor cells, some anti-cancer therapies (anthracyclines, oxaliplatin, X-Rays) could trigger an immunogenic cell death modality, releasing danger signals to alert immune system. We have shown that tumor-specific IFN- producing CD8+ T cells (Tc1) are mandatory for the success of chemotherapy to prevent tumor outgrowth. Priming of Tc1 response depends on IL-1β secretion by DC confronted with anthracycline-treated tumor cells releasing ATP. To identify the inflammatory components which link innate and cognate immune responses, we analyzed the influence of immunogenic chemotherapy on tumor microenvironment. We found an upregulated Th1- and Th17-related gene expression pattern in growth-retarded tumor after anthracycline treatment. By interfering with IFN- or IL-17A pathways, therapeutic effect of doxorubicin and oxaliplatin was abolished and dying tumor cell-based vaccine lost its efficacy to protect mice from live tumor cell rechallenge. Interestingly, we discovered that distinct subsets of  T lymphocytes (V4+ and V6+) colonized tumors shortly after chemotherapy, where they proliferated and became the dominant IL-17 producers within tumor beds. In three tumor models treated with chemotherapy or radiotherapy, a strong correlation between the presence of IL-17-producing  T ( T17) and IFN--producing CD8+ TIL (Tc1) was discovered. IL-17A signaling acts as upstream of IFN- since defect in IL-17RA led to complete loss of antigen specific Tc1 priming. The contribution of  T17 cells (V4+ and V6+) to chemotherapy is critical as V4/6-/- mice showed reduced sensitivity to chemotherapy and vaccination. Also, tumor infiltrating  T17 and Tc1 cells were reduced to basal level in this strain. IL-1β/IL-1R, but not IL-23/IL-23R, is pivotal for IL-17 production by  T cells and the success of chemotherapy. Importantly, adoptive transfer of  T cells could restore the efficacy of chemotherapy in IL-17A-/- mice and ameliorate the effect of chemotherapy in wild type host, provided that they retain the expression of IL-1R and IL-17A. Our research suggest a DC (IL-1β) →  T cells (IL-17) → Tc1 (IFN-) immune axis triggered by chemotherapy-induced dying tumor cells, which is critical for the favorable therapeutic response. To boost the immune system, we try to combine immunogenic chemotherapy with tumor vaccine in the presence of TLR3 agonist Poly (A:U). This sequential combined therapy, which we named VCT, could significantly retard tumor growth or even completely eradicate tumor and establish long-term protection against rechallenge in highly tumorigenic models. To dissect the effect of Poly (A:U) on immune system and that on TLR3 expressing-tumor cells, we performed VCT treatment in nude mice, TRIF-/- mice and with TRIF-silencing tumors. Interestingly, our results suggested that anti-tumor effect of VCT required T cells and intact TRIF signaling pathway at the level of the host and that of tumor cells. Poly (A:U) treatment could induce high level of CCL5 and CXCL10 production from tumor cells both in vitro and in vivo, which could negatively and positively influence the therapeutic outcome. By uncoupling the effect of CCL5 from that of CXCL10, the VCT treatment can be ameliorated. Our study emphasizes that both tumor and host derived inflammatory factors participate in regulating anti-tumor response. We also highlight that therapeutic application of TLR agonists can be optimized through regulating the profile of chemokines and their downstream signaling events

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Khan, Sarwat Tahsin. „An Interleukin-12-Expressing Oncolytic-Virus Infected Autologous Tumor Cell Vaccine Generates Potent Anti-Tumor Immune Responses“. Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/37940.

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Zhang, Yahan. „THE EFFECT OF PEGYLATION ON THE CELLULAR UPTAKE OF AN IMMUNOSTIMULATORY NANOPARTICLE IN THE TUMOR IMMUNE MICROENVIRONMENT“. Case Western Reserve University School of Graduate Studies / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case1618916816447844.

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Sullivan, Camille. „Epithelial and Macrophage RON Receptor Signaling Regulates the Antitumor Immune Response in Prostate Cancer“. University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin159524743258716.

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Lima, Joanna Darck Carola Correia. „O papel do infiltrado inflamatório no tumor e sua contribuição para inflamação sistêmica e desenvolimento da caquexia“. Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/42/42134/tde-12082016-100836/.

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A caquexia associada ao câncer é caracterizada pela perda de peso severa e um desequilíbrio metabólico.Acredita-se que resulta da interação entre o hospedeiro e tumor que induz a inflamação sistêmica,portanto compreender essa relação é fundamental para a descoberta de marcadores efetivos para diagnóstico.O objetivo do trabalho foi caracterizar diferenças nos infiltrados imunitários do tumor e analisar aspectos moleculares ligados à inflamação,a fim de avaliar se a presença da caquexia é determinada pelo perfil inflamatório do tumor.O estudo envolveu pacientes com câncer colorretal e posteriormente distribuídos em dois grupos:Câncer sem caquexia(WSC) e Câncer com caquexia (CC).A análise histopatológica mostrou que o estadiamento independende da caquexia e caracterização dos macrófagos infiltrantes resultou M2 menor em CC,já a expressão proteica de citocinas indicou IL-13 menor em CC e citocinas pró-iflamatórias estavam aumentadas em CC. A correlação de macrófagos com citocinas foi positiva com M1 e negativa com M2.Esses resultados fornecem evidências de que o tumor possui um perfil de secreção diferente entre os grupos no que diz respeito a fatores inflamatórios e diferentes percentuais de fenótipo de macrófagos.
Cancer cachexia is characterized by severe weight loss and large metabolic imbalance.It is a result of the interaction between the host\'s cells and the tumour, which induces systemic inflammation.Understand the relationship is required for the discovery of diagnostic markers.The aim of the present study was to characterize differences in inflammatory tumour infiltrate and molecular aspects in order to assess whether the presence of cachexia is determined by the inflammatory tumour profile. The study involved patients diagnosed with colorectal cancer and then distributed into two groups: cancer without cachexia(WSC) and cancer cachexia(CC).Histopathological analysis showed that the presence of cachexia in patients with colo-rectal cancer was independent from tumour staging.Characterization of infiltrating macrophages revealed a lower percentage of M2 profile in CC.Protein expression of cytokines demonstrated lower of IL-13 in CC and pro-inflammatory cytokines is higher in CC. Correlation between macrophages and cytokines was shown positive with macrophages type M1.These results provide evidence that tumor has a different secretion profile between the groups with regard to inflammatory factors and different percentages of macrophage phenotype.

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VENETIS, KONSTANTINOS. „BREAST CANCER DURING PREGNANCY AS A SPECIAL TYPE OF EARLY-ONSET BREAST CANCER: INSIGHTS INTO THE TUMOR MICROENVIRONMENT AND IMMUNE TRANSCRIPTOME“. Doctoral thesis, Università degli Studi di Milano, 2023. https://hdl.handle.net/2434/951469.

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Breast cancer during pregnancy (PrBC) is a rare tumor with limited information on its immune landscape. Here, we sought to characterize the cellular composition of the tumor microenvironment (TME) and the immune transcriptome of PrBC to identify their differences from early-onset breast cancer (EOBC) in non-pregnant women. Eighty-three PrBC and 89 EOBC were selected and subjected to tumor-infiltrating lymphocytes (TILs) profiling and immunohistochemistry for CD4, CD8, forkhead box P3 (FOXP3), and programmed death-ligand 1 (PD-L1) (clone 22C3). A subset of 28 PrBC and 23 EOBC was selected for transcriptome profiling. RNA extracted from tumor (T) and corresponding normal (N) tissues was subjected to gene expression analysis using a next-generation sequencing assay (Oncomine™ Immune Response Research Assay) targeting 395 immune-related genes. A significantly lower frequency of hormone receptor (HR)-positive tumors was found in PrBC. The prevalence of low/null PD-L1 and CD8+TILs was higher in PrBC than in the controls, specifically in HR+/HER2– breast cancers. PrBC had a significantly higher risk of relapse and disease-related death, compared to EOBC. The presence of TILs and each TIL subpopulation were significantly associated with disease relapse. The death rate was higher in PrBC with CD8+ TILs. Twenty-three differentially expressed genes (DEGs) were found in the comparison between T (PrBC and EOBC), 3 (IFNA17, IFNB1, and PECAM1) upregulated and 20 downregulated. Compared to corresponding N, PrBCT had 46 upregulated and 18 downregulated genes. Four DEGs were PrBCT-specific, one (PECAM1) upregulated and 3 (CXCL1, CCL21, and HGF) downregulated. The TME and immune transcriptome of PrBC are characterized by specific patterns of TIL subpopulations and distinct gene expression patterns. Assessment of TILs and TILs subtyping in these patients might help to identify clinically relevant subsets of women with PrBC. Our findings suggest that immune regulation takes different genomic pathways in PrBC, and PrBCT has a unique immune transcriptome.

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Kostine, Marie. „Defining the immune microenvironment in sarcoma : could immunotherapy be part of the treatment strategy in sarcoma patients ?“ Thesis, Bordeaux, 2018. http://www.theses.fr/2018BORD0387/document.

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La chirurgie est la pierre angulaire du traitement curatif des sarcomes, lorsqu’elle est possible. En revanche, en cas de maladie avancée ou métastatique, les traitements systémiques ont une efficacité assez limitée avec un réel besoin de nouvelles options thérapeutiques. Le récent succès de l’immunothérapie dans les tumeurs épithéliales soulève donc la question de la possibilité d’une telle approche dans les sarcomes, et surtout pour quels sous-types histologiques. L’objectif de ce travail de thèse était d’obtenir des données précliniques en caractérisant le microenvironnement immunitaire au sein de trois types de sarcomes potentiellement candidats à l’immunothérapie, prérequis indispensable avant d’envisager une application clinique : 1) Dans le chondrosarcome, l’expression de PD-L1 a été retrouvée exclusivement dans près de 50% des chondrosarcomes dédifférenciés, et s’associait à une infiltration lymphocytaire T et l’expression des molécules HLA de classe I. Ces données incitent donc à inclure les patients avec ce sous type de chondrosarcome dans des essais cliniques évaluant un traitement anti PD-1/PD-L1. 2) Dans l’ostéosarcome, un infiltrat lymphocytaire T était observé de façon bien plus importante dans les lésions métastatiques que dans lésions primitives ou rechutes locales. De plus, l’expression de PD-L1 était retrouvée dans presque 50% des métastases mais pas ou peu dans la tumeur primitive correspondante, traduisant ici une dynamique d’échappement au système immunitaire lors de la progression de la maladie. Une stratégie ciblée sur les lymphocytes T visant à amplifier et potentialiser cette réponse immune préexistante dans les lésions métastatiques pourrait donc offrir un bénéfice clinique. 3) Dans le léiomyosarcome, les molécules HLA de classe I étaient fortement exprimées et l’expression de PD-L1 retrouvée dans 30% des tumeurs de haut grade, également très infiltrées par des macrophages immunosuppresseurs CD163+. Une importante infiltration de macrophages CD163+ était un marqueur indépendant de mauvais pronostic pour la survie, indiquant l’intérêt de d’une approche ciblée visant les macrophages dans ce type de sarcome, éventuellement en association avec un traitement anti PD-1/PD-L1
Local control with adequate surgery is the cornerstone of sarcoma treatment. However, most sarcoma lack effective systemic therapies in case of advanced disease, emphasizing an unmet medical need for new therapeutic targets. The recent success of immunotherapy in epithelial malignancies raises the question whether such therapies, and which ones, would be applicable in sarcomas. As a prerequisite for therapeutic applications, we characterized the immune microenvironment in three sarcoma subtypes potentially candidate to immunotherapy: 1) In chondrosarcoma, PD-L1 expression was exclusively found in nearly 50% of the dedifferentiated subtype, in association with immune-infiltrating cells and HLA class I expression. These data provide rationale for including such patients in clinical trials with PD-1/PD-L1-targeted therapies. 2) In osteosarcoma, we observed a high density of tumor-infiltrating T cells in metastatic lesions compared to primary tumors and local relapses. Furthermore, PD-L1 positivity in almost half of metastases while mainly negative in the associated primary tumors, emphasises the dynamics of an adaptive mechanism of immune escape. Enhancing the preexisting immune response in metastatic lesions using T-cell-based immunotherapy may offer clinical benefit. 3) In leiomyosarcoma, HLA class I molecules were strongly upregulated and PD-L1 expression found in 30% of high-grade tumors, which were also highly infiltrated with CD163+ immunosuppressive macrophages. CD163+ was found to be an independent poor prognostic factor for overall survival, indicating the need for assessing a macrophage-targeted approach in this tumor type, as single agent or in combination with anti PD-1/PD-L1agents

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Leruste, Amaury. „Immune context of malignant rhabdoid tumors : description and identification of new therapeutic targets“. Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS050.

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Les tumeurs rhabdoïdes (TR) constituent un rare cancer indifférencié du jeune enfant et du nourrisson, avec un âge médian au diagnostic de 20 mois. Ces tumeurs sont caractérisées par une inactivation biallélique du gène suppresseur de tumeur SMARCB1, un des membres du complexe SWI/SNF, acteur majeur du remodelage de la chromatine, sans autre altération génomique récurrente. Le pronostic des TR est péjoratif, le taux de survie globale atteignant 30% dans la plupart des séries, malgré des approches thérapeutiques conventionnelles particulièrement agressives. Les approches d’immunothérapies ont obtenu un succès certain dans certains cancers de l’adulte, et récentes analyses de l’infiltrat immun des cancers pédiatriques ne montrent pas un fort taux de tumeurs infiltrées à l’exception de rare types de cancers dont les TR intracrâniennes. Nous avons donc procédé à une analyse multimodale de l’infiltrat immun de cohortes de patients ainsi que d’un modèle de TR murines établi dans notre laboratoire. Nous avons identifié une forte proportion de tumeurs infiltrées dans certains sous-groupes de TR. Cet infiltrat était composé à la fois de cellules myéloïdes incluant des populations au phénotype immunosuppresseur, et lymphocytaires T notamment de phénotype résident mémoire caractérisées par une forte expansion clonale probablement spécifique d’un antigène tumoral. Nous avons identifié des cibles thérapeutiques communes aux tumeurs humaines et au modèle murin syngénique, et trouvé que cibler l’infiltrat lymphocytaire T ou myéloïde était susceptible d’induire une réponse tumorale complète avec induction d’une mémoire immunitaire, confirmant le caractère immunogénique des TR, et apportant de nouvelles stratégies thérapeutiques utiles en clinique. Enfin, nous avons identifié que les TR étaient le site d’une réexpression de rétrovirus endogènes, dépendante de celle de SMARCB1, avec activation des voies de l’interféron, apportant une base à une immunogénicité des TR issue du génome non codant
Rhabdoid tumors (RT) are highly undifferentiated cancers occurring in infancy and early childhood, with a median age at diagnosis about 20 months. These tumors are characterized by the biallelic inactivation of SMARCB1 tumor suppressor gene, core member of the SWI/SNF complex, one major chromatin remodeling actor, in an otherwise highly stable genome. The prognosis of RT is dismal with overall survival hardly reaching 30% in most series, despite particularly aggressive conventional treatment. Immunotherapy approaches has gained a striking success within some adult cancer types and recent analyses of immune cell content of pediatric cancers don’t reveal a high rate of infiltrated tumors, except in few tumor types such as intracranial rhabdoid tumors. Then, we conducted a comprehensive analysis of the immune context of both human RT cohorts and a mouse RT model, including at single cell level. We identified a high recurrence of infiltrated tumors, in a RT-subgroup related manner, composed of both myeloid cells including cells with immune suppressive phenotypes, and T cells with notably a tissue resident memory phenotype demonstrating a high clonal expansion highly suggestive of immunogenicity. We identified common targetable immune populations between human and mouse RTs, and found that targeting both T and myeloid infiltrating cells was able to induce complete anti-tumor response with induced memory, confirming the immunogenic properties of RTs, and identifying new therapeutic strategies of clinical relevance. We finally identified that RTs were the site of SMARCB1-dependent endogenous retroviruses reexpression, with subsequent activation of interferon signaling, likely triggering the immune response in the context of RT, and providing a basis of non-coding genome-driven immunogenicity for these tumors

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Seitz, Christoph [Verfasser], Udo [Akademischer Betreuer] Gaipl, Benjamin [Akademischer Betreuer] Frey, Udo [Gutachter] Gaipl und Benjamin [Gutachter] Frey. „Tumor Cell-Based Vaccine Generated With High Hydrostatic Pressure Synergizes With Radiotherapy by Generating a Favorable Anti-tumor Immune Microenvironment / Christoph Seitz ; Gutachter: Udo Gaipl, Benjamin Frey ; Udo Gaipl, Benjamin Frey“. Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2021. http://d-nb.info/1233484281/34.

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Wierz, Marina. „Characterization of the Tumor Microenvironment in Chronic Lymphocytic Leukemia by Mass Cytometry : Implications for Immunotherapy Dual PD1/LAG3 Immune Checkpoint Blockade Limits Tumor Development in a Murine Model of Chronic Lymphocytic Leukemia High-dimensional Mass Cytometry Analysis Revealed Microenvironment Complexity in Chronic Lymphocytic Leukemia“. Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL020.

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La leucémie lymphoïde chronique (LLC), la leucémie la plus fréquente chez l'adulte, est caractérisée par l'accumulation de lymphocytes B matures dans le sang périphérique et les tissus lymphoïdes. La progression de la LLC est fortement dépendante des interactions complexes au sein du microenvironnement tumoral (TME) et malgré les récentes avancées dans le traitement de la LLC ciblant la TME, la LLC reste une maladie incurable. Par conséquent, nous voulions caractériser en profondeur le paysage immunitaire dans le TME dans la LLC murine et humaine afin d'identifier de nouvelles cibles potentielles pour une approche immunothérapeutique. À cette fin, nous avons effectué une caractérisation complète et approfondie par cytométrie de masse à haute dimension pour établir une cartographie approfondie des sous-populations de cellules immunitaires. Nous démontrons que les changements pertinents dans la composition des cellules immunitaires, en particulier l'expansion de sous-populations spécifiques de cellules immunitaires lymphoïdes et myéloïdes, sont associés à une forte suppression immunitaire contribuant ainsi à un phénotype d'échappement dans la LLC. Ces changements associés à la LLC peuvent être restaurés dans les modèles précliniques par un double blocage du point de contrôle immunitaire PD1/LAG3. De plus, nous démontrons une forte hétérogénéité des cellules T entre les patients qui peut être stratifiée en fonction de leur profil de cellules T, et la corrélation de sous-ensembles de cellules T spécifiques avec le temps jusqu'au traitement initial, mettant en évidence leur valeur pronostique potentielle. En conclusion, avec cette première étude CyTOF dans la LLC, nous avons élargi les connaissances actuelles sur la complexité phénotypique du TME. Nous avons démontré que le double ciblage des points de contrôle immunitaires contrôlait efficacement le développement de la LLC dans les modèles précliniques et pouvait donc avoir des avantages potentiels dans la LLC pour restaurer une immunité anti-tumorale fonctionnelle
Chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults, is characterized by the accumulation of mature B lymphocytes in peripheral blood and lymphoid tissues. The progression of CLL is highly dependent on complex interactions within the tumor microenvironment (TME) and despite recent advances in CLL treatment targeting the TME, CLL remains an incurable disease. Therefore, we wanted to deeply characterize the immune landscape in the TME in murine and human CLL to identify novel potential targets for an immunotherapeutic approach. For this purpose, we performed a comprehensive and extensive characterization by high-dimensional mass cytometry to establish an extensive cartography of immune cell subsets. We demonstrated that relevant changes in the immune cell composition, especially the expansion of specific lymphoid and myeloid immune cell subsets, are associated with strong immune suppression thereby contributing to an escape phenotype in CLL. These CLL-associated changes can be restored in preclinical models by a dual PD1/LAG3 immune checkpoint blockade. Moreover, we demonstrated a high T cell heterogeneity between patients that can be stratified according to their T cell profile, and the correlation of specific T cell subsets with time to initial treatment, highlighting their potential prognostic value. In conclusion, with this first CyTOF study in CLL, we expanded the current knowledge of the phenotypic complexity of the TME. We demonstrated that dual targeting of immune checkpoints efficiently controlled CLL development in preclinical models and therefore could have potential benefits in CLL to restore a functional anti-tumor immunity

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Labani, Motlagh Alireza. „Immune modulation in serous epithelial ovarian cancer : focus on the role of tumor-derived exosomes“. Doctoral thesis, Umeå universitet, Institutionen för klinisk mikrobiologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-138264.

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Serous epithelial ovarian cancer (EOC) is a potent suppressor of the immune defense. Here, we studied interactions between EOC and the immune system that lead to escape from tumor immune surveillance. We explored: 1) tumor escape from cytotoxicity by exosome-mediated modulation of the NK-cell receptors NKG2D and DNAM-1; 2) cytokine mRNA profiles in the EOC microenvironment and peripheral blood and their role in the suppression of the anti-tumor immune responses; 3) expression of long non-coding (lnc) RNAs in EOC tumors and exosomes. We found that EOC-secreted exosomes carried MICA/B and ULBP1-3, ligands of NKG2D, and could downregulate the NKG2D receptor and impair NKG2D-mediated cytotoxicity. In contrast, the DNAM-1 receptor ligands PVR and nectin-2 were seldom found in exosomes and were not associated with the exosomal membrane leaving the DNAM-1 receptor-mediated cytotoxicity intact. We compared cytokine mRNA expression in the tumor microenvironment and in immune cells of peripheral blood in EOC patients and patients with benign ovarian conditions. EOC patients were unable to mount an IFN-gamma mRNA response needed for tumor cell elimination. Instead, there was a significant up-regulation of inflammation and immune suppression i.e. responses promoting tumorigenesis and T-regulatory cell priming that suppress anti-tumor immunity. In addition, we studied lncRNAs in tissues and sera exosomes from EOC and benign ovarian conditions aiming to assess the lncRNA(s) expression profile and look for lncRNA(s) as possible marker(s) for early diagnosis. We found a deregulated lncRNAs expression in EOC tissues that correlated well with the lncRNAs expression in exosomes. Candidate lncRNAs with the highest expression and abundance were suggested for evaluation as EOC diagnostic markers in a future large cohort study. Our studies of EOC tissue and EOC exosomes highlight the immunosuppressive tumor microenvironment and the complex tumor exosome-mediated network of immunosuppressive mechanisms, and provide a mechanistic explanation of the observation that NKG2D-mediated cytotoxicity does not function in EOC patients and is partially replaced by the accessory DNAM-1 dependent cytotoxic pathway. The deregulated lncRNAs expression in EOC tissues and exosomes might serve for diagnostic purposes but could also be a potential risk of spreading tumor-derived lncRNAs in EOC exosomes to recipient cells throughout the body.

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PARISI, ERICA. „Immune response against Wilms Tumor: characterization of cellular and molecular interactions and identification of novel therapeutic targets“. Doctoral thesis, Università degli studi di Genova, 2022. http://hdl.handle.net/11567/1078738.

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Wilms tumor (WT) is a pediatric kidney tumor that accounts for 6-7% of children’s malignant tumors. WT cells arise from embryonic mesenchymal renal progenitors, not differentiated, remaining in the organ after birth. The classic triphasic WT contains a mixture of undifferentiated blastemal, differentiated epithelial cells, and stromal elements, whereas individual cell types may predominate in some tumors. Moreover, as human mesenchymal stem cells (MSCs), these cells have the ability to differentiate in many tissue lineages. In particular, stromal-like WT (str-WT) display morphological, phenotypic, and biological features comparable to MSCs, suggesting they could play a significant role in the interactions with immune cells in the tumor microenvironment. The role of immune cells in the response against WT is still unclear, because of the very few data available; it is of note, however, that the presence of leukocyte infiltrate and a pro-inflammatory milieu have been described. In view of the similarity between MSCs and stromal-like WT cells, the aim of this study is to investigate the immunoregulatory properties of WT cells towards Natural Killer (NK) cells and the immunoregulatory mechanisms taking place in the tumor microenvironment. Our data showed the molecular interactions between str-WT cells and NK lymphocytes, resulting in different outcomes presumably occurring in the WT microenvironment.

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Berchem, Guy. „Rôle du stress hypoxique dans la régulation de la réponse immunitaire anti-tumorale des lymphocytes "Natural Killer"“. Thesis, Paris 11, 2014. http://www.theses.fr/2014PA11T105/document.

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Le microenvironnement tumoral, et notamment le stress hypoxique, joue un rôle immunosuppressif permettant l’échappement des cellules tumorales à la surveillance du système immunitaire. Des études récentes ont montré que l’échange de microvésicules (MVs) entre les cellules tumorales et les cellules du système immunitaire peut être responsable de l’établissement d’un microenvironnement immunosuppressif. Dans ce contexte, nous avons étudié l’effet des MVs issues des cellules tumorales hypoxiques sur la cytotoxicité des cellules «Natural Killer» (NKs). Nos résultats démontrent clairement que les cellules NKs sont capables d’internaliser les MVs issues des cellules tumorales normoxiques et hypoxiques. Cependant, seules les MVs hypoxiques sont capables de diminuer significativement la cytotoxicité des cellules NKs. Ainsi, nous avons déterminé que les MVs dérivées des cellules tumorales hypoxiques séquestrent deux immunomodulateurs, le TGF- et le miR-23a. Nous avons montré que le transfert de TGF- et miR-23a aux cellules NKs était responsable de la diminution respective de l’expression du récepteur activateur NKG2D à leur surface et de la protéine membranaire associée aux lysosomes (LAMP-1/CD107a) impliquée dans la dégranulation des granules cytotoxiques. Dans la deuxième partie de cette étude nous avons montré que les cellules tumorales soumises à un stress hypoxique étaient capables de déjouer un système immunitaire fonctionnel et d’échapper ainsi à la surveillance immunitaire des cellules NKs. En effet, nos résultats ont clairement démontré que la résistance des cellules tumorales hypoxiques à la lyse par les cellules NKs n’était pas liée à un défaut de reconnaissance, mais plutôt à l’activation d’un mécanisme de résistance intrinsèque dans les cellules tumorales. Ce mécanisme de résistance implique l’activation de l’autophagie qui opère dans les cellules tumorales pour dégrader le granzyme B, une protéase à sérine secrétée par les cellules NKs dont l’internalisation par les cellules tumorales cibles est nécessaire pour induire leur mort. Les expériences d’imagerie cellulaire combinées à des approches biochimiques ont confirmé que le niveau de granzyme B dans les cellules tumorales hypoxiques était significativement mois élevé par rapport à celui des cellules tumorales normoxiques. Ces résultats suggèrent fortement que le granzyme B est destiné à être dégradé par autophagie dans les cellules tumorales hypoxiques. En effet, l’inhibition génétique et pharmacologique de l’autophagie dans les cellules tumorales hypoxiques était suffisante pour contrecarrer la dégradation de granzyme B et ainsi restaurer la sensibilité des cellules tumorales hypoxiques à la lyse par les cellules NKs. Nos résultats ont clairement établi que l’inhibition de l’autophagie pouvait améliorer la réponse immunitaire antitumorale dépendante des cellules NK. Nous avons validé ce concept in vivo chez la souris en utilisant deux modèles syngéniques de cancer du sein et de mélanome. L’ensemble de nos travaux indiquent clairement que le stress hypoxique, qui est une caractéristique majeure du microenvironnement tumoral, peut favoriser l’établissement d’un microenvironnement immunosuppressif par plusieurs mécanismes qui ne s’excluent pas mutuellement. En effet, le stress hypoxique modifie les caractéristiques des cellules tumorales et active des mécanismes de résistance à la surveillance immunitaire. De plus, les cellules tumorales modifiées peuvent éduquer et exporter leur phénotype hypoxique aux cellules immunitaires présentes dans le microenvironnement afin d’affaiblir leur pouvoir cytotoxique. Nos résultats ouvrent ainsi la voie à la mise en place de nouvelles applications cliniques en immunothérapie anticancéreuse basées sur la réactivation des lymphocytes cytotoxiques et l’inhibition simultanée de l’autophagie
The tumor microenvironment, including hypoxic stress plays an immunosuppressive role in tumor cell escape from immune surveillance. Recent studies have shown that the exchange of microvesicles (MVs) between tumor cells and cells of the immune system could be responsible for the establishment of an immunosuppressive microenvironment. In this context, we investigated the effect of MVs derived from hypoxic tumor cells on the cytotoxicity of Natural Killer (NK) cells. Our results clearly demonstrated that NK cells are able to internalize MVs derived from both normoxic and hypoxic tumor cells. However, only hypoxic MVs are able to significantly reduce the cytotoxicity of NK cells. Thus, we revealed that MVs derived from hypoxic tumor cells sequester two immunomodulators, TGF- and miR-23a. We have shown that the transfer of TGF- and miR-23a to NK cells was responsible for the respective reduction of the expression of NKG2D activating receptor on their surface and lysosomal-associated membrane protein (LAMP-1 / CD107a) involved in degranulation of cytotoxic granules.In the second part of this thesis we have shown that tumor cells subjected to hypoxic stress were able to outmaneuver a functional immune system and thus escape NK-mediated immune surveillance. Indeed, our results clearly demonstrated that the resistance of hypoxic tumor cells to NK-mediated lysis was not related to the impairment of recognition by NK cells, but rather to the activation of an intrinsic resistance mechanism in tumor cells. We showed that the resistance mechanism involves the activation of the autophagy which operates in the tumor cells to degrade the granzyme B, a serine protease secreted by NK cells and internalized by target tumor cells to induce cell death. Cell imaging experiments combined to biochemical approaches have confirmed that the level of granzyme B in hypoxic tumor cells was significantly higher compared to normoxic tumor cells. The analysis of the subcellular distribution of granzyme B reveals that it is predominantly present in the endosomes and autophagosomes of hypoxic tumor cells. These results strongly suggest that granzyme B is subjected to be degraded by autophagy in hypoxic tumor cells. Genetic and pharmacological inhibition of autophagy in hypoxic tumor cells was sufficient to block the degradation of granzyme B and thus restore the sensitivity of hypoxic tumor cells to NK-mediated lysis. Our results clearly demonstrated that inhibition of autophagy could improve NK-mediated antitumor immune response. We validated this concept in vivo using two syngeneic mice model of breast cancer and melanoma.Taken together, our work clearly shows that hypoxic stress, which is a major feature of the tumor microenvironment, can promote the establishment of an immunosuppressive microenvironment by several mechanisms which are not mutually exclusive. Thus, hypoxic stress changes the characteristics of tumor cells and activates the mechanisms of resistance to immune surveillance. In addition, tumor cells can educate and export their hypoxic phenotype to the immune cells in the microenvironment in order to impair their cytotoxicity. Our findings pave the way for the development of new clinical applications in cancer immunotherapy based on the reactivation of cytotoxic lymphocytes and simultaneous inhibition of autophagy

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Lequeux, Audrey. „Impact du ciblage du domaine de liaison de HIF-1α avec HIF-1β sur le paysage immunitaire du mélanome Targeting HIF-1 Alpha Transcriptional Activity Drives Cytotoxic Immune Effector Cells into Melanoma and Improves Combination Immunotherapy Hijacker of the Antitumor Immune Response: Autophagy is Showing its Worst Facet Impact of Hypoxic Tumor Microenvironment and Tumor Cell Plasticity on The Expression of Immune Checkpoints Improving Cancer Immunotherapy by Targeting the Hypoxic Tumor Microenvironment: New Opportunities and Challenges“. Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL026.

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L’hypoxie est une caractéristique majeure des tumeurs solides et elle est capable d’induire un microenvironnement tumoral immunosuppressif. Nous avons étudié l’impact de l’inhibition du domaine de liaison de HIF-1α avec HIF-1β sur le paysage immunitaire du mélanome murin B16-F10. Le ciblage de ce domaine de liaison inhibe l’activité transcriptionnelle de HIF-1α dans les cellules B16-F10 in vitro. In vivo, l’inhibition de l’activité transcriptionnelle de HIF-1α dans le mélanome B16-F10 montre une diminution significative de la croissance tumorale et une amélioration consistante de la survie des souris. La croissance tumorale est restaurée dans les souris immunodéficientes, soulignant l’importance du système immunitaire dans le contrôle de la croissance du mélanome. L’étude du phénotype des cellules immunitaires intra-tumorales révèle une augmentation de l’infiltration des cellules Natural Killer (NK), des lymphocytes T CD4+, des T régulateurs, des macrophages de type M1 et M2 et des cellules dendritiques lorsque l’activité transcriptionnelle de HIF-1α est inhibée. La déplétion de cellules NK dans notre modèle expérimental restaure la croissance tumorale, soulignant le rôle des NK dans la surveillance du mélanome B16-F10. Le changement du paysage immunitaire observé dans notre modèle corrèle également avec une modification du réseau de cytokines caractérisé par une nette augmentation de la sécrétion de CCL5 et de CCL2. En conclusion, cette étude met en évidence le rôle de HIF-1α dans le contrôle de la croissance et le remodelage du paysage immunitaire du mélanome B16-F10. Elle indique la possibilité de combiner un inhibiteur de HIF-1α avec une immunothérapie basée sur le blocage des points de contrôle immunitaire pour étendre leur efficacité et leur bénéfice thérapeutiques à un plus grand nombre de patients cancéreux
Hypoxia is a major feature of solid tumors and is able to induce a tumor immunosuppressive microenvironment. Here, we investigated the impact of inhibiting of the binding domain of HIF-1α to HIF-1β on the immune landscape of B16-F10 melanoma. Targeting this binding domain inhibits the transcriptional activity of HIF-1α in B16-F10 cells in vitro. In vivo, inhibiting the transcriptional activity of HIF-1α in B16-F10 melanoma shows a significant decrease in tumor growth and a consistent improvement in mice survival. Tumor growth is restored in immunodeficient mice, highlighting the critical role of the immune system in controlling melanoma growth. The phenotyping of intra-melanoma immune cells reveals an increase in Natural Killer (NK), CD4+ T cells, regulatory T cells, M1 and M2 macrophages and dendritic cells. NK depletion restores tumor growth in our experimental model, highlighting the role of NK cells in melanoma surveillance. The alteration of the immune landscape that we observed also correlates with a clear increase of secreted CCL5 and CCL2. In conclusion, this study highlights the role of HIF-1α in controlling the growth and the immune landscape of B16-F10 melanoma. It indicates the opportunity of combining HIF-1α inhibitors with immune checkpoint blockade to extend immune checkpoint blockade efficiency and therapeutic benefit to a larger number of cancer patients

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Cole, Lauren. „Primary Melanoma tumor immune contexture analysis: T regulatory cell to T effector cell ratio as related to MHC class II and GILT expression“. Thesis, The University of Arizona, 2017. http://hdl.handle.net/10150/623292.

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A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine.
Histopathologic examination of the tumor microenvironment demonstrates the presence of a vast repertoire of infiltrating lymphocytes and antigen presenting cells (APC’s). Recent studies establish a strong correlation between the tumor microenvironment cell composition and prognostic value in terms of cell type, location and ratio, referred to as a tumor’s immunoscore. More specifically, the relationship between T regulatory (Treg) cell to T effector (Teff) cell percentage predominates as a mechanism of tumor immune evasion. Further investigation of the factors influencing the development of Treg and Teff cells is therefore warranted. Gammainterferon‐inducible lysosomal thiol reductase (GILT) acts to influence antigenic processing and presentation by MHC class II cells, ultimately impacting lymphocyte development. Evaluation of the role of GILT expression in MHC class II+ APC’s with respect to Treg and Teff cell development in primary melanoma lesions, to our knowledge, has not been reported. Therefore our investigation focuses on elucidating a plausible relationship between GILT presence and Treg to Teff cell ratio. The aim of our study is to examine a possible association between GILT expression in APC’s and Treg:Teff cell ratio. We hypothesized GILT expression in melanoma cells would result in a decreased Treg to Teff ratio or an enhanced T cell‐mediated response. Our study included 17 de‐identified primary melanoma specimens previously stained and scored for Treg, Teff, CD8, MHC class II and GILT. Scoring was performed through identification of four areas per specimen with highest Treg and Teff cell density. These four areas were then averaged with ± standard deviation (SD). With use of landmark association, these four areas were identified and scored for MHC class II and GILT in APC’s and tumor cells with consideration to presence/absence, intensity and frequency of staining. Statistical significance was not reached relative to our hypothesized relationship of a decreased Treg to Teff cell ratio in the presence of GILT+ MHC class II. Similarly, we did not reach statistical significance when comparing individual cell types to GILT, MHC class II and GILT + MHC class. In our study, we were unable reach statistical significance relative to our proposed correlation between MHC class II and GILT presence leading to a decreased Treg to Teff cell ratio or enhanced T‐cell mediated immune response. A major limitation of our study included the small sample size leading to a probable type II error, prompting the need for further investigation of the factors influencing the Treg to Teff cell ratio within the melanoma tumor microenvironment on a larger scale.

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Petitprez, Florent. „Integrated analysis and clinical impact of immune and stromal microenvironments in solid tumors Quantitative analyses of the tumor microenvironment composition and orientation in the era of precision medicine Transcriptomic analysis of the tumor microenvironment to guide prognosis and immunotherapies Tumor microenvironment quantification tool draws a comprehensive map of the tumor microenvironment of non-hematologic human cancers The mMCP-counter method to estimate abundance of tissue-infiltrating immune and stromal cell populations using gene expression in murine samples Immune sub-classes in sarcoma predict survival and immunotherapy response Intra-tumoral tertiary lymphoid structures are associated with a low risk of hepatocellular carcinoma early recurrence Association of IL-36γ with tertiary lymphoid structures and inflammatory immune infiltrates in human colorectal cancer Immune-based identification of cancer patients at high risk of progression Tumor-infiltrating and peripheral blood T-cell immunophenotypes predict early relapse in localized clear cell renal cell carcinoma PD-L1 expression and CD8+ T-cell infiltrate are associated with clinical progression in patients with node-positive prostate cancer Intratumoral classical complement pathway activation promotes cancer progression“. Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCB104.

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Les tumeurs sont composées de cellules malignes et d'une grande variété de cellules non-tumorales, en particulier des cellules immunitaires qui forment le micro-environnement tumoral (MET). Il a été démontré que la composition du MET était associée au devenir clinique des patients, en termes de survie et de réponses thérapeutiques. Avec le développement récent des immunothérapies qui ciblent des éléments spécifiques du MET, l'immunité anti-tumorale a soulevé un intérêt majeur. Plusieurs méthodologies ont été mises au point afin d'étudier la composition du MET, avec une précision toujours plus grande. En particulier, des méthodes comme MCP-counter permettent d'exploiter les données transcriptomiques de la tumeur entière afin de quantifier les différentes populations qui composent le MET. Le volet méthodologique de ce travail de thèse a ainsi consisté à proposer une amélioration de MCP-counter, en particulier pour l'analyse de données RNA-Seq. Une adaptation de la méthode pour des données issues de modèles murins (mMCP-counter) est également proposée. MCP-counter permet d'analyser rapidement le MET de larges séries de tumeurs. Un second volet de cette thèse consiste en l'application de cette méthode pour établir une classification immunitaire des sarcomes des tissus mous, un type de cancer rare, hétérogène et agressif. Cette classification immunitaire a permis de mettre en évidence des groupes de tumeurs faiblement ou fortement infiltrés, ainsi qu'un groupe marqué par une forte vascularisation. De manière intéressante, la classification immunitaire permet de prédire la réponse des patients aux immunothérapies. Ce travail a aussi démontré un rôle important des structures lymphoïdes tertiaires (SLT). Les SLT sont des structures de type noeud lymphatique composées de lymphocytes B et T qui se forment dans la tumeur ou à proximité de celle-ci. Au sein des SLT, une réponse immunitaire anti-tumorale peut se former et maturer. L'intérêt porté aux SLT est de plus en plus important pour de nombreux types de cancers. Dans la plupart des types de cancer, une forte infiltration de la tumeur par des lymphocytes T, en particulier CD8+, est associée à une meilleure survie des patients. Cependant, le carcinome rénal à cellules claires et le cancer de la prostate sont des exceptions à cette règle. En effet, dans ces deux cancers urologiques, la présence dans la tumeur de lymphocytes T est associée à une survie plus courte des patients, ainsi qu'à une rechute et une progression plus précoce. Ces exceptions sont détaillées dans une troisième partie de cette thèse, par une description minutieuse du MET, ainsi que par l'analyse de l'implication du système du complément. Dans leur ensemble, les résultats présentés dans cette thèse démontrent qu'en combinant différentes méthodes d'analyse, in silico, in situ et in vivo, il est possible d'obtenir une vision extrêmement complète du MET. La connaissance des types cellulaires présents dans la tumeur ainsi que leur orientation fonctionnelle permet de guider le soin apporté aux patients et d'améliorer leur devenir clinique. La description complète du MET ouvre la voie à une médecine personnalisée pour les patients atteints de cancer
Tumors are composed not only of malignant cells but also contain a vast variety of non-malignant cells, notably immune cells forming the tumor microenvironment (TME). The composition of the TME was shown to be associated with clinical outcome for cancer patients, in terms of survival and therapeutic responses. With the relatively recent development of immunotherapies targeting specific elements of the TME, tumor immunology has risen a strong interest and holds a strong therapeutic potential. Several methodologies have been developed to study the composition of the TME with an increased precision. Notably, some methods such as MCP-counter enable the use of the tumor bulk transcriptome to quantify cell populations composing the TME. The methodological aspect of this PhD project consisted in setting up an enhanced version of MCP-counter that can be readily applied to RNA-Seq data, as well as propose an adaptation of the method for mouse models. Using MCP-counter, the TME of large series of tumors can be easily analyzed. The application part of this PhD work consisted of applying MCP-counter to establish an immune-based classification of soft-tissue sarcoma, a rare, aggressive and heterogeneous cancer type. The immune classification notably allowed to identify immune low and high groups, and a group characterized by a strong vasculature. Interestingly, the classification was notably found to be predictive of the patients' response to immunotherapies. It also highlighted an important role of tertiary lymphoid structures (TLS). TLS are lymph-node-like structures composed of T and B cells that form within the tumor or in close proximity. They are a site of formation and maturation of antitumoral immune responses. TLS are raising a growing interest in many malignancies. In most cancer types, a strong infiltration by T cells, in particular CD8+ T cells, is associated with a favorable clinical outcome. However, clear-cell renal cell carcinoma and prostate cancer are exceptions to this general rule. Indeed, in these urological cancers, an increased infiltration by T cells is associated with a decreased patient survival and with earlier relapse and disease progression. In a third part of this thesis, these exceptions are investigated with more details by scrutinizing the TME, and questioning the implication of the complement system. Overall, this thesis presents how the combination of several analysis methods, in silico, in situ and in vivo, can help achieve an extremely precise description of the TME. Knowing accurately what cell populations and what their functional orientation can help guide patients care and improve clinical outcome. Complete description of the TME opens the way towards personalized medicine for cancer patients

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Augustin, Jérémy. „Caractérisation du microenvironnement tumoral immunitaire des carcinomes hépatocellulaires réséqués“. Electronic Thesis or Diss., Sorbonne université, 2021. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2021SORUS409.pdf.

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Les carcinomes hépatocellulaires (CHC) répondent mal à l’immunothérapie et leur environnement immun n’est pas bien caractérisé. Notre objectif était de faire le pont entre les aspects morphologiques, viraux et immuns et analyser leur impact sur la sensibilité à l’immunothérapie. Nous avons réalisé une analyse transcriptomique de l’environnement immun de 170 CHC: 23% d’étiologie virale B (VHB), 29% d’étiologie virale C (VHC), 16% d’étiologie métabolique, 17% d’étiologie alcoolique, 14% d’étiologie indéterminée. Nous avons corrélé les profils transcriptomiques aux données cliniques, morphologiques et virales (ARN prégénomique du VHB). Nous n’avons pas observé de différence globale dans l’environnement immun des CHC en fonction des étiologies, mais avons identifié 3 clusters du VHB donc aucun n’exprimait d’interféron-γ (contre 25% des CHC toutes étiologies confondues). Le Cluster 1, associé à une récurrence élevée, avait un profil immun ambivalent et chaud, surexprimait des marqueurs d’épuisement mais montrait morphologiquement une faible densité de lymphocytes T et était associé à la présence d’une transcription du VHB. Le Cluster 2, aussi associé à une récurrence élevée, était enrichi en sous-type macrotrabéculaire et avait profil immun froid. Le Cluster 3, de meilleur pronostic, était plus développé sur foie cirrhotique et montrait un niveau intermédiaire d’infiltration de cellules immunes, sans expression de marqueurs d’épuisement. En conclusion, nous montrons des spécificités immunes au sein des CHC liés à l’HBV, en relation avec des facteurs viraux transcriptionnels, et avec une portée pronostique
Hepatocellular carcinoma (HCC) shows globally low response to immunotherapy and HCC immune microenvironment is not well characterized. Our objective was to connect immune, viral and morphologic aspects of HCC and understand how they intervene in sensitivity to immune checkpoint blockade. In this study, we performed a transcriptomic analysis of onco-immune genes to characterize the tumor microenvironment of 170 HCC: 23% hepatitis B (HBV), 29% hepatitis C (HCV), 16% metabolic syndrome, 17% alcohol consumption related, and 14% of undetermined etiology. We correlated gene expression profiles with clinical, morphological and viral features. We did not observe difference of immune microenvironment at a global scale, between etiologies. But within HBV group, we identified 3 Clusters. None of of these clusters expressed ϒ-interferon (compared to 25% of HCC of all etiologies combined). Cluster 1 showed an ambivalent « hot » and exhausted profile with higher expression of exhaustion markers but lower densities of T lymphocytes by immunostaining. This Cluster was associated with HBV transcription and patients from this Cluster showed higher recurrence. Cluster 2 was enriched with macrotrabecular massive subtype and was immunologically “cold” and was also associated with higher recurrence. At last, Cluster 3 was developed much more on cirrhotic liver and showed an intermediate level of immune cells infiltration, with no marker of exhaustion. It was associated with lower recurrence. In conclusion we highlight viral related specificities within HBV HCC, associated with prognostic significance

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Hall, Charles. „Ex vivo reprogramming of tumor-reactive immune cells from FVBN202 mice bearing lung metastatic mammary carcinoma: an immunotherapeutic opportunity revealed against recurrence“. VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/3176.

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Metastatic breast cancer treatment has seen few advances in recent years, yet treatment resistance continues to rise, causing disease recurrence. A pilot study was performed to determine the efficacy of ex vivo expansion and reprogramming of tumor-reactive immune cells from experimental metastatic tumor-sensitized mice. Also, phenotypic changes in tumors due to metastasis or tumor microenvironment influences were characterized. Metastatic neu+ mouse mammary carcinoma (mMMC) and its distant relapsing neu-antigen-negative variant (mANV) were investigated in FVBN202 mice. Tumor-reactive central memory CD8+ T cells and activated NK/NKT cells were successfully reprogrammed and expanded during 6-day expansion from mMMC- and/or mANV-sensitized mice, resulting in tumor-specific cytotoxicity. mMMC exhibited a flexible neu-expression pattern and acquired stem-like, tumorigenic phenotype following metastasis while mANV remained stable except decreased tumorigenicity. Myeloid-derived suppressor cell (MDSC) levels were not increased. Adoptive cellular therapy (ACT) with reprogrammed tumor-reactive immune cells may prove effective prophylaxis against metastatic or recurrent breast cancer.

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Angell, Helen K. „Immune modulation of the tumour microenvironment“. Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12641/.

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Regulatory T cells (Tregs) are a distinct lymphocyte lineage, functionally defined as T cells that inhibit an immune response, which is crucial for maintaining peripheral tolerance and the prevention of autoimmunity. Tregs have been implicated in tumour immune evasion, suppressing the anti-tumour immune response, resulting in tumour progression. The aim of this PhD was to recognise how Tregs function and understand how they are able to modulate tumour immunology. In order to research their proposed role in cancer, it was necessary to be able to phenotype, sort and expand functional Tregs. In preliminary research, strategies were designed to phenotype and test the functionality of ex vivo Tregs and commercial isolation kits were tested and compared in terms of cost efficiency and effectiveness. The mechanisms of Treg-mediated suppression were investigated, including the necessity for cell-to-cell contact and the involvement of key cytokines. The importance of particular cytokines in Treg-mediated suppression was not clear; but cell contact appears to be required for optimal suppression. The project then aimed to address whether or not the importance of Tregs highlighted in the literature was reflected in a clinical setting. The significance of immune cell orientation within the tumour microenvironment was researched, investigating the co- localisation between key immune cell subtypes and their correlation with areas of tumour proliferation, apoptosis, hypoxia and vasculature coverage. In order to achieve this successfully, immunohistochemical techniques were optimised and image analysis algorithms were constructed to facilitate rigorous and systematic quantification of immune cell infiltrates in primary colorectal and metastatic liver cancer patients. Significant increases in the prevalence of Tregs, CD8 cells, macrophages and natural killer (NK) cells were observed within the stroma, compared with the tumour. A metastatic phenotype was alluded to; encompassing elevated Tregs and reduced numbers of CD8 cells. Further to this, the level of Tregs in the peripheral blood and tumour tissue of liver- metastatic patients were assessed to investigate whether any correlation existed between circulating and tumour-infiltrating Tregs. It was shown that within peripheral blood, patients exhibited a TreghighCD8lowNKlow phenotype, when compared with healthy volunteers. Finally, the project aimed to build an in vitro human Treg tumour-killing suppression model to examine the ability of Tregs to inhibit NK cell-mediated cytotoxicity. The mechanism for this was investigated, evaluating the importance of the NK group 2 member D (NKG2D) receptor ligand interaction, to mediate cell killing in a transforming growth factor-β dependent manner. This study adds to the ongoing discussion on the role of immune cells in metastatic development. Accumulating evidence points to a critical role of immune infiltrates in allowing metastatic development, where Tregs support tumour growth by suppressing the host anti-tumour immune response.

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Czerwińska, Urszula. „Unsupervised deconvolution of bulk omics profiles : methodology and application to characterize the immune landscape in tumors Determining the optimal number of independent components for reproducible transcriptomic data analysis Application of independent component analysis to tumor transcriptomes reveals specific and reproducible immune-related signals A multiscale signalling network map of innate immune response in cancer reveals signatures of cell heterogeneity and functional polarization“. Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCB075.

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Les tumeurs sont entourées d'un microenvironnement complexe comprenant des cellules tumorales, des fibroblastes et une diversité de cellules immunitaires. Avec le développement actuel des immunothérapies, la compréhension de la composition du microenvironnement tumoral est d'une importance critique pour effectuer un pronostic sur la progression tumorale et sa réponse au traitement. Cependant, nous manquons d'approches quantitatives fiables et validées pour caractériser le microenvironnement tumoral, facilitant ainsi le choix de la meilleure thérapie. Une partie de ce défi consiste à quantifier la composition cellulaire d'un échantillon tumoral (appelé problème de déconvolution dans ce contexte), en utilisant son profil omique de masse (le profil quantitatif global de certains types de molécules, tels que l'ARNm ou les marqueurs épigénétiques). La plupart des méthodes existantes utilisent des signatures prédéfinies de types cellulaires et ensuite extrapolent cette information à des nouveaux contextes. Cela peut introduire un biais dans la quantification de microenvironnement tumoral dans les situations où le contexte étudié est significativement différent de la référence. Sous certaines conditions, il est possible de séparer des mélanges de signaux complexes, en utilisant des méthodes de séparation de sources et de réduction des dimensions, sans définitions de sources préexistantes. Si une telle approche (déconvolution non supervisée) peut être appliquée à des profils omiques de masse de tumeurs, cela permettrait d'éviter les biais contextuels mentionnés précédemment et fournirait un aperçu des signatures cellulaires spécifiques au contexte. Dans ce travail, j'ai développé une nouvelle méthode appelée DeconICA (Déconvolution de données omiques de masse par l'analyse en composantes immunitaires), basée sur la méthodologie de séparation aveugle de source. DeconICA a pour but l'interprétation et la quantification des signaux biologiques, façonnant les profils omiques d'échantillons tumoraux ou de tissus normaux, en mettant l'accent sur les signaux liés au système immunitaire et la découverte de nouvelles signatures. Afin de rendre mon travail plus accessible, j'ai implémenté la méthode DeconICA en tant que librairie R. En appliquant ce logiciel aux jeux de données de référence, j'ai démontré qu'il est possible de quantifier les cellules immunitaires avec une précision comparable aux méthodes de pointe publiées, sans définir a priori des gènes spécifiques au type cellulaire. DeconICA peut fonctionner avec des techniques de factorisation matricielle telles que l'analyse indépendante des composants (ICA) ou la factorisation matricielle non négative (NMF). Enfin, j'ai appliqué DeconICA à un grand volume de données : plus de 100 jeux de données, contenant au total plus de 28 000 échantillons de 40 types de tumeurs, générés par différentes technologies et traités indépendamment. Cette analyse a démontré que les signaux immunitaires basés sur l'ICA sont reproductibles entre les différents jeux de données. D'autre part, nous avons montré que les trois principaux types de cellules immunitaires, à savoir les lymphocytes T, les lymphocytes B et les cellules myéloïdes, peuvent y être identifiés et quantifiés. Enfin, les métagènes dérivés de l'ICA, c'est-à-dire les valeurs de projection associées à une source, ont été utilisés comme des signatures spécifiques permettant d'étudier les caractéristiques des cellules immunitaires dans différents types de tumeurs. L'analyse a révélé une grande diversité de phénotypes cellulaires identifiés ainsi que la plasticité des cellules immunitaires, qu'elle soit dépendante ou indépendante du type de tumeur. Ces résultats pourraient être utilisés pour identifier des cibles médicamenteuses ou des biomarqueurs pour l'immunothérapie du cancer
Tumors are engulfed in a complex microenvironment (TME) including tumor cells, fibroblasts, and a diversity of immune cells. Currently, a new generation of cancer therapies based on modulation of the immune system response is in active clinical development with first promising results. Therefore, understanding the composition of TME in each tumor case is critically important to make a prognosis on the tumor progression and its response to treatment. However, we lack reliable and validated quantitative approaches to characterize the TME in order to facilitate the choice of the best existing therapy. One part of this challenge is to be able to quantify the cellular composition of a tumor sample (called deconvolution problem in this context), using its bulk omics profile (global quantitative profiling of certain types of molecules, such as mRNA or epigenetic markers). In recent years, there was a remarkable explosion in the number of methods approaching this problem in several different ways. Most of them use pre-defined molecular signatures of specific cell types and extrapolate this information to previously unseen contexts. This can bias the TME quantification in those situations where the context under study is significantly different from the reference. In theory, under certain assumptions, it is possible to separate complex signal mixtures, using classical and advanced methods of source separation and dimension reduction, without pre-existing source definitions. If such an approach (unsupervised deconvolution) is feasible to apply for bulk omic profiles of tumor samples, then this would make it possible to avoid the above mentioned contextual biases and provide insights into the context-specific signatures of cell types. In this work, I developed a new method called DeconICA (Deconvolution of bulk omics datasets through Immune Component Analysis), based on the blind source separation methodology. DeconICA has an aim to decipher and quantify the biological signals shaping omics profiles of tumor samples or normal tissues. A particular focus of my study was on the immune system-related signals and discovering new signatures of immune cell types. In order to make my work more accessible, I implemented the DeconICA method as an R package named "DeconICA". By applying this software to the standard benchmark datasets, I demonstrated that DeconICA is able to quantify immune cells with accuracy comparable to published state-of-the-art methods but without a priori defining a cell type-specific signature genes. The implementation can work with existing deconvolution methods based on matrix factorization techniques such as Independent Component Analysis (ICA) or Non-Negative Matrix Factorization (NMF). Finally, I applied DeconICA to a big corpus of data containing more than 100 transcriptomic datasets composed of, in total, over 28000 samples of 40 tumor types generated by different technologies and processed independently. This analysis demonstrated that ICA-based immune signals are reproducible between datasets and three major immune cell types: T-cells, B-cells and Myeloid cells can be reliably identified and quantified. Additionally, I used the ICA-derived metagenes as context-specific signatures in order to study the characteristics of immune cells in different tumor types. The analysis revealed a large diversity and plasticity of immune cells dependent and independent on tumor type. Some conclusions of the study can be helpful in identification of new drug targets or biomarkers for immunotherapy of cancer

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Sidot, Emmanuelle. „Rôle des cellules tuft dans l'homéostasie et les cancers intestinaux“. Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT057.

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Au cours de ma thèse, je me suis intéressée à une population cellulaire rare et peu étudiée de l’épithélium intestinal ; les cellules tuft. La fonction de ces cellules fut longtemps débattue dans la littérature, jusqu’à ce que nous découvrions leur fonction dans l’initiation de la réponse immune de type II en réponse à une infection parasitaire. De manière intéressante, ces cellules sont présentes de manière massive et transitoire au sein des lésions adénomateuses précoces et certains groupes ont suggéré l’implication de ces cellules en tant que cellules souches tumorales. Les principaux objectifs de ma thèse ont été de déterminer le rôle des cellules tuft au cours de la tumorigenèse intestinale et colorectale.Nous avons montré que l’absence de cellules tuft impacte le processus de tumorigenèse à la fois dans l’intestin grêle, dans des souris de la lignée Apc14/+, et au niveau du colon, après traitement avec un agent carcinogène. Nos données indiquent que si les cellules tuft n’agissent pas en tant que cellules souches tumorales, leur absence impacte certaines populations de cellules immunitaires. Afin de déterminer les mécanismes permettant aux cellules tuft de moduler le microenvironnement immunitaire, nous avons identifié par analyse transcriptomique de cellules tuft isolées par cytométrie en flux, des gènes codant pour des médiateurs connus pour être impliqués dans la communication avec le système immunitaire. Des analyses in-vivo, permettront de valider d’un point de vue fonctionnel l’implication de ces médiateurs immunitaires dans la fonction immuno-régulatrice des cellules tuft ainsi que dans le développement tumoral.L’ensemble de ces travaux a permis d’identifier une fonction immuno-régulatrice des cellules tuft au cours d’une infection parasitaire, mais aussi très probablement lors du développement tumoral. La compréhension des mécanismes permettant aux cellules tuft de moduler certaines populations de cellules immunitaires permettra d’identifier des cibles d’intérêt thérapeutique potentiel pour le traitement de patients atteints d’un cancer colorectal
I focused my PhD project on a scare epithelial cell population referred as tuft cells. Their function has been debated for decades in the literature, until we discovered their crucial role in the initiation of the so-called type-2 immune response following parasitic infection. Interestingly, tuft cells are present in early adenomatous intestinal lesions and literature suggested that these cells could act as cancer stem cells. The main objective of my PhD was to determine the tuft cell function during intestinal and colorectal cancer.We showed that tuft cells deficiency impacts both intestinal and colorectal tumorigenesis process, using Apc14/+ mouse strain and chemically induced carcinogenesis model, respectively. Our data indicate that tuft cells are not cancer stem cells, but that these cells are able to regulate immune cell populations. To get more insights into mechanisms allowing tuft cells to modulate the immune microenvironment, we identified, by transcriptomic analysis of FACS-isolated tuft cells, specific genes encoding mediators involved in the crosstalk with the immune system. Functional in-vivo validation of the most relevant candidates will identify tuft cells derived factors crucial for the immune-regulatory tuft cell function and for tumor development.This work allowed to highlight the immune-regulatory function of tuft cells during parasitic infection and likely during tumor development. A better knowledge of the mechanisms allowing tuft cells to shape either a pro- or an anti-tumoral microenvironment, will potentially paves the way for new therapeutic strategies regarding intestinal and colorectal tumorigenesis

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Hodkinson, Philip Simon. „Tumour microenvironment interactions of small cell lung cancer“. Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4254.

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Small cell lung cancer (SCLC) is characterised by rapid growth, early metastatic spread and poor long-term survival. The tumour is initially sensitive to chemotherapy and thus objective response rates are high. Unfortunately, this response is often short-lived and SCLC recurs with acquired drug resistance, resulting in early patient death. Despite intensive chemo- and radiotherapy regimes survival has not improved significantly in 20 years. Prior research suggests a critical role for the tumour microenvironment in the pathogenesis of other cancers. Therefore, investigating interactions between SCLC cells and components of the tumour stroma may identify novel therapeutic targets. This thesis demonstrates that extracellular matrix (ECM) proteins present in the tumour microenvironment protect SCLC cells in vitro from chemo- and radiotherapy induced cell cycle arrest and apoptosis via cell surface β1 integrins. Pharmacological and genetic inhibition of phosphoinositol-3 kinase signalling abrogates this effect, defining a central role for this pathway in SCLC de novo drug resistance. Furthermore, the protective effect of ECM occurs without alteration in chemotherapy-induced DNA damage allowing SCLC cells to survive with new genetic defects. Integrin-mediated drug resistance has been shown to be important in other tumours and thus development of strategies to inhibit this pathway may yield new anti-cancer treatments. The design of targeted agents to down-regulate integrin-ECM interaction requires an in depth understanding of the intracellular signals that modulate integrin affinity. Two such pathways are investigated in this thesis. 1) H-Ras, a dominant suppressor of integrin affinity, acts in part through phosphorylation of Erk. Data presented here demonstrate that H-Ras also suppresses integrins through a phospholipase-C epsilon (PLCε)-dependent pathway, thus explaining discrepancies in prior data and confirming a physiological role for this recently identified phospholipase. 2) The Notch signalling pathway has been shown to have important roles in both development and cancer. It is shown here that activation of Notch signalling increases β1 integrin affinity and can protect SCLC cells from chemotherapyinduced apoptosis. However the mechanisms appear to be different; Notch-1 modulates integrin activation through the small GTPase R-Ras and Notch-2 promotes SCLC cell survival. These results define a new Notch pathway, a novel integrin modulator and a potential therapeutic target in SCLC cells. In addition to ECM proteins, the tumour microenvironment contains immune cells that may contribute to cancer growth. The cellular composition of the SCLC stroma is poorly understood. The data presented here indicate that the microenvironment of SCLC is infiltrated by lymphocytes and macrophages, the degree of which independently predicts patient survival. This suggests that the host immune system may be able to suppress SCLC growth. It is well recognised that patients with SCLC have defects in cellular immunity which correlate with survival. An in vitro coculture model was used to investigate the underpinning mechanisms, showing SCLC cells can suppress CD4+ T-cell proliferation and macrophage CD86 expression. Furthermore, preliminary data suggest a role for a soluble factor released by SCLC cells that up-regulates CD4+ T-cell production of IL-10. The work in this thesis implies a complex interaction between SCLC cells, ECM and immune cells in the tumour microenvironment. Manipulation of these pathways may have important therapeutic implications. Further investigation is required to understand the mechanisms of this interplay, which may in part be aided by prospective analysis of patient tumour samples and an in vivo model of SCLC.

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Kubasch, Anne Sophie, Rebekka Wehner, Serena Bazzurri, Antje Tunger, Sebastian Stasik, Marlene Garzarolli, Jörn Meinel et al. „Clinical, molecular, and immunological responses to pembrolizumab treatment of synchronous melanoma and acute myeloid leukemia“. American Society of Hematology, 2018. https://tud.qucosa.de/id/qucosa%3A34908.

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Cancer cells use the interaction between immune-checkpoint receptor programmed cell death protein 1 (PD-1) on activated T cells and programmed cell death ligand 1 (PD-L1) on tumor cells and various cell types of the tumor microenvironment to evade immune surveillance. Blocking the interaction between PD-1 and PD-L1 with checkpoint inhibitors can improve T-cell reactions and mediate efficient antitumor activity in a variety of solid tumors including melanoma. However, clinical data from patients with myeloid diseases that are treated with these agents are limited to clinical trials in advanced disease. Pembrolizumab (PEM) is a humanized monoclonal antibody of the immunoglobulin G4/κ isotype designed to block PD-1/PD-L1 interactions and is approved for various solid tumors including advanced melanoma.

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Harris, Jennifer Nicole. „The role of lymph node-derived lymphatic endothelial cells in immune modulation in the tumour microenvironment“. Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/287480.

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The lymphatic vasculature is a key player in progression of many cancers, with lymphangiogenesis at the primary tumour and tumour-draining lymph nodes (TDLNs) associated with poor patient prognosis. As well as providing a highway for metastatic tumour cells, recent reports propose lymphatics as modulators of immunity, highlighting a need for greater understanding of immune regulation by lymphatics. The specific role of lymphatic endothelial cells (LECs) in this context, particularly in TDLNs, is unknown. As TDLNs are immune hubs, yet anti-tumour immune responses are often ineffective, this thesis aimed to investigate functional changes to lymphatics in TDLNs and the role of TDLN-derived LECs in anti-tumour immunity. I hypothesised that factors from the tumour microenvironment alter functionality of TDLN-LECs from early stages of tumour development. I further hypothesised that these changes would promote immune tolerance, with this thesis exploring specific impact on dendritic cell (DC) mediated immunity. Using the B16-F10 melanoma model, this work confirmed expansion of TDLN-LECs prior to metastasis and demonstrated transcriptional reprogramming of immune-associated pathways in LECs isolated from early TDLNs. This was accompanied by differentially localized migratory DCs, clustered at lymphatic subcapsular sinuses. In vitro using co-culture assays revealed mature DCs undergo prolonged interactions with LECs conditioned with B16-F10 tumour-conditioned media, suggesting a change in the physical interactions occurring in vivo in early TDLNs. Additionally, we investigated possible mechanistic contributors, demonstrating using in vitro and in vivo blockade and knockout models, a role for lymphatic expressed Podoplanin in DC interactions and migration. Prolonged physical interactions were further found to facilitate antigen transfer from ovalbumin-loaded LECs to DCs yet inhibit DC priming of T-cells, with DCs found capable of acquiring TDLN-LEC archived antigen in vivo. These results show that in lymph nodes conditioned by factors derived from the tumour microenvironment, prolonged physical interactions between LECs and DCs impact DC migration and T-cell priming. As immune tolerance is a key feature of the tumour microenvironment, this work has highlighted lymphatics as key modulators of the anti-tumour immune response. Furthermore, this work provides new insight into lymphatic involvement during tumour development, identifying lymphatics as a potential target for early intervention therapies.

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Becht, Etienne. „Transcriptomic analysis of the immune microenvironment of non-hematopoietic human tumors“. Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015PA05T029/document.

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Le microenvironnement des tumeurs est composé de cellules immunitaires, de fibroblastes et de cellules endothéliales, ainsi que d’autres cellules non-malignes. Son étude a permis d’établir des classifications qui ont une valeur pronostique et théranostique, ainsi que de développer des traitements modulant la composition et l’orientation fonctionnelle du microenvironnement. En parallèle, des classifications moléculaires des tumeurs ont proposé des taxonomies stratifiant les cancers humains en sous-groupes associés à des différences de survie des patients et leur réponse aux traitements. Des études récentes suggèrent que le phénotype de la cellule cancéreuse est un facteur critique dans le façonnement du microenvironnement tumoral, suggérant un possible consensus entre les classifications immunitaires et moléculaires. Le but de cette thèse était donc de caractériser le microenvironnement des sous-groupes moléculaires de tumeurs humaines. Le cancer colorectal a été le premier cancer humain dans lequel il a été mis en évidence qu’une réponse immunitaire adaptative était associée à un contrôle de la croissance tumorale, et représente ainsi un exemple type pour l’immunologie des tumeurs. A l’inverse, le carcinome du rein à cellules claires est une exception vis-à-vis de l’immunologie des tumeurs, puisqu’une forte réponse immunitaire adaptative y est associée à des tumeurs plus agressives. Des classifications transcriptomiques ont été récemment établies pour ces deux cancers, qu’à première vue tout oppose sur le plan immunitaire. Dans ce travail, je propose une méthode permettant l’étude du microenvironnement tumoral à partir de données transcriptomiques, et décris son application à l’étude du contexte immunitaire des cancers colorectaux et du rein à cellules claires. Ces analyses suggèrent qu’une unification des classifications moléculaires et immunitaires des tumeurs humaines est possible, remettent en cause notre conceptualisation des liens entre la composition du microenvironnement tumoral et le pronostic du patient, et évoque des pistes immunothérapeutiques potentiellement adaptées à certains sous-groupes de patients dans ces cancers
Tumors grow within a complex microenvironment composed of immune cells, fibroblasts, endothelial cells and other non-malignant cells. The study of the composition of tumor microenvironments has led to classifications with prognostic and theranostic values, as well as the discovery of treatments modulating the composition and the functional orientation of the microenvironment. Concurrently, molecular classifications of tumors have proposed taxonomies within cancers that define groups of patients with different prognoses and are associated with response to treatments. Recent evidence suggest that the phenotype of the malignant cell is a critical determinant in the shaping of its microenvironment, suggesting potential correlations between immune and molecular classifications. The goal of this PhD project was therefore to analyze the microenvironment of molecularly-classified human tumors. Colorectal cancer represents a paradigm for tumor immunology, as it is the humancancer in which it was exemplified that an adaptive immune response can control tumor Growth and metastasis. Conversely, clear-cell renal cell carcinoma represents an exception in tumor immunology, as an extensive adaptive immune response is associated with more aggressive diseases. Molecular transcriptomic classifications were recently proposed for both of these apparently immunologically contrasted cancers. In this work, I propose a methodology that enables the characterization of the tumor microenvironment using transcriptomic data, and apply it to describe the immune contexture of molecular subgroups of colorectal and clear-cell renal cell carcinomas. These analyses argue in favor of the unification of molecular and immune classifications of human cancers, challenge our current views of the relationship between the composition of the tumor microenvironment and patient’s prognosis, and suggest immunotherapeutic approaches that could benefit subgroups of patients in these two cancers

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Ahmady, Farah. „The role of immune cells in ovarian cancer“. Thesis, Federation University Australia, 2022. http://researchonline.federation.edu.au/vital/access/HandleResolver/1959.17/184111.

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Ovarian cancer remains the leading cause of gynaecological disease related death in women worldwide. Patients given standard treatment have low survival rates and immunotherapy remains unsuccessful. This is largely due to the suppressive tumour microenvironment (TME) and the interaction of the different cells being poorly understood. This has sparked interest in understanding the functions of immune cells and their contribution to the TME. In chapter 3, B and T cells were characterised in samples collected from chemo-naïve highgrade serous ovarian cancer (HGSOC) patients and compared to benign and healthy controls. B cells were able to express the T cell exhaustion marker T cell immunoglobulin and mucin domain-3 (TIM-3) on their surface. Blood derived B cells of benign patients expressed less TIM-3 compared to high-grade and healthy donors. Alterations in TIM-3 expression on B cells suggests that the TME may not be causing B cells to harbour an exhaustion phenotype. The frequency of circulating mucosal-associated invariant T cells (MAIT cells) was reduced in benign and high-grade patients compared to healthy donors. Blood derived MAIT cells of healthy donors stimulated with 5-OP-RU antigen and cultured with ovarian cancer cell line supernatants had a reduced capacity of producing tumour necrosis factor (TNF)-a compared to control. The reduced frequency of MAIT cells at the premalignant state of the disease indicates that malignancy of the disease is not necessarily the contributing factor in the reduction observed. As TNF-a is a key anti-tumour cytokine expressed by MAIT cells, the impaired production may indicate defects in their function. Low dose-dense (LDD) chemotherapy has recently shown promise as depicted in a clinical study by Kannourakis et al. where LDD treated HGSOC patients had longer survival than those treated with maximum tolerated dose (MTD). It is postulated that this may be due to aspects of the immune system, however the effects of LDD chemotherapy on immune cells remains unknown. In chpter 4, circulating B and T cells of LDD treated patients were characterised and compared to untreated controls. The key finding of this study was the immunostimulatory attributes of LDD regimen in a range of B and T cell populations via their increased expression of TNF-a compared to untreated controls and healthy donors. The frequency of MAIT cells, however, was reduced in LDD treated patients, suggesting that these cells may not contribute to the immunostimulatory aspects, and that this regimen may impact their frequency by creating an environment which may not be compatible for MAIT cell survival. In chapter 5, expression of immuno-oncology protein markers in benign and high-grade primary and metastasised tumours were determined for alterations in hot and cold tumour regions using Digital Spatial Profiling (DSP). An increased expression of immune cell, costimulatory and inhibitory markers were apparent in high-grade tumours compared to benign. The stimulator of interferon genes (STING) marker was studied further, and its expression was also higher in high-grade tumours compared to benign, with cisplatin further enhancing its expression in ovarian cancer cell lines. There were alterations in the expression of some downstream molecules, however interferon (IFN)-b, the key cytokine produced in the activated STING pathway, was virtually non-existent. This suggests a defect in the pathway which if restored, may contribute to some of the known anti-tumour functions of the pathway. The results from these studies provide an understanding of differences in the immune profile of non-malignant and malignant ovarian cancer. These findings work together to improve our understanding of the unique TME with the aim to identify potential immune therapeutic targets for future study.
Doctor of Philosophy

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Baloche, Valentin. „Contributions négatives et positives de la galectine-9 au développement tumoral : étude dans des modèles tumoraux murins syngéniques In the MB49 Murine Model, Genetic Ablation of Galectin-9 Enhances Anti-Tumor Immune Response: Possible Role of a Greater CXCL9/Il-6 Production Tumor Exosomal Micrornas Thwarting Anti-Tumor Immune Responses in Nasopharyngeal Carcinomas Interferon β and Anti-PD1/PD-L1 Checkpoint Blockade Cooperate in NK Cell-Mediated Killing of Nasopharyngeal Carcinoma Cells Interferon Beta Increases NK Cell Cytotoxicity against Tumor Cells in Patients with Nasopharyngeal Carcinoma via Tumor Necrosis Factor Apoptosis-Inducing Ligand Emerging Therapeutic Targets for Nasopharyngeal Carcinoma: Opportunities and Challenges Galectin-9 Promotes a Suppressive Microenvironment in Human Cancer by Enhancing STING Degradation“. Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASS117.

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Comme les autres galectines, la galectine-9(gal-9) est une lectine animale qui interagit avec un sous-groupe défini de polysaccharides portés par des glycoprotéines ou des glycolipides. La gal-9 associée aux cellules exerce de multiples fonctions dans le cytoplasme, dans le noyau et à la surface de la membrane plasmique. Quelques publications suggèrent que la gal-9 intra-cellulaire inhibe la mobilité des cellules malignes et exerce un effet antimétastatique. En outre la gal-9 peut être sécrétée dans le milieu extra-cellulaire où elle se comporte comme une cytokine avec des effets principalement immunosuppresseurs. Ces effets ont été mis en évidence dans un contexte tumoral chez l’homme et dans des modèles murins. Cependant, on ne disposait pas jusqu’à présent d’un modèle tumoral murin permettant d’évaluer les effets pro-tumoraux ou antitumoraux de la gal-9 indépendamment de la gal-9 des cellules infiltrantes. Pour résoudre ce problème, nous avons dérivé, en employant la technologie CRISPR/Cas9, des clones isogéniques invalidés ou non pour la gal-9 à partir de 2 lignées tumorales murines : CT26 (fond génétique BALB/c) et MB49 (fond génétique C57BL/6). Dans le cas de la lignée MB49, nous avons pu mettre en évidence un phénotype remarquable in vivo. Lors de transplantations itératives, on assiste pour les tumeurs dérivées des clones invalidées à une réduction drastique de la croissance tumorale au bout de 3 ou 4 passages sur les souris syngéniques mais pas sur les souris immunodéficientes. L’émergence de la réponse immunitaire responsable de cet arrêt de la croissance tumorale a été étudiée par immunohistochimie, dosage de cytokines en multiplex dans les extraits tumoraux et analyse du transtriptome par RNAseq. L’augmentation de la production intra-tumorale d’interféron-γ, de CXCL9 et d’Il-6 semble jouer un rôle important dans le renforcement de la réponse immunitaire contre les tumeurs KO-gal-9
Like other galectins, galectin-9 (gal-9) is an animal lectin which interacts with a defined subgroup of glycans carried by glycoproteins or glycolipids. Gal-9 associated with cells performs multiple functions in the cytoplasm, in the nucleus and at the surface of the plasma membrane. Some publications suggest that intracellular gal-9 inhibits the mobility of malignant cells and exerts an anti-metastatic effect. In addition, gal-9 can be secreted into the extracellular medium where it behaves like a cytokine with mainly immunosuppressive effects. These effects have been demonstrated in the context of human tumors and in mouse tumor models. However, so far there was no murine tumor model available to assess the pro-tumor or anti-tumor effet of gal-9 independently of gal-9 produced by infiltrating cells. To address this issue, we derived isogenic clones invalidated or not for gal-9 from 2 murine tumoral lines : CT26 (BABL/c genetic background) and MB49 (C57BL/6 genetic background), using CRISPR/Cas9 technology. In the case of the MB49 line, we were able to demonstrate a remarkable phenotype in vivo. During serial transplantations, we saw, for tumors derived from invalidated clones, a dramatic reduction in tumor growth after 3 or 4 passages in syngenic mice but not in immunodeficient mice. The emergence of the immune response responsible for this arrest of tumor growth was investigated by immunohistochemistry, multiplex cytokine assay in tumor extracts and transcriptome analysis by RNAseq. Increased intra-tumor production of interferon-γ, CXCL9 and Il-6 appears to play an important role in enhancing the immune response against KO-gal-9 tumors

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Thomas, Audrey. „Effet sur le microenvironnement tumoral d’une modulation pharmacologique du stress oxydant“. Thesis, Paris 5, 2012. http://www.theses.fr/2012PA05T086/document.

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Les Formes réactives de l’oxygène (FRO) ont un rôle bien établi dans l’oncogénèse en augmentant les capacités de prolifération et d’invasion des cellules tumorales. Mais les FRO exercent aussi un effet important sur le microenvironnement tumoral, en particulier en participant à l’échappement des tumeurs au système immunitaire. Une modulation pharmacologique de l’équilibre oxydo-réductif tumoral est donc susceptible d’influencer la progression tumorale. Il a été montré que l’induction pharmacologique d’un stress oxydant dans les cellules tumorales peut induire un effet cytotoxique mais ses effets sur le microenvironnement sont moins bien connus. L’objectif de nos travaux était d’étudier l’effet d’une modulation du stress oxydant sur les cellules immunitaires du microenvironnement tumoral et in fine de préciser les conséquences potentielles sur la progression tumorale. L’arsenic trioxyde (As2O3), inducteur de FRO, présentait à faible dose, dénuée d’effet cytotoxique direct sur les cellules malignes, un effet antitumoral dans un modèle de cancer colique murin. Cet effet était lié à une déplétion sélective en lymphocytes T régulateurs (Tregs) et était médié par la génération d’anions superoxyde (O2°-) et de monoxyde d’azote (NO), eux même responsables de la formation de peroxynitrite. Les Tregs présentaient un niveau basal de FRO plus élevé que les autres populations lymphocytaires, qui pourrait expliquer leur plus grande sensibilité à un surcroît de stress oxydant induit par l’As2O3. Un cytotoxique antitumoral, la vinorelbine, s’est également montré capable d’exercer un effet sur le microenvironnement tumoral. Par co-cultures, nous avons montré que la vinorelbine induisait un effet bystander toxique sur les cellules immunitaires effectrices voisines des cellules tumorales. In vivo, le prétraitement par vinorelbine de cellules malignes implantées à la souris était responsable d’une perte de la réactivité anti-tumorale des cellules mono-nuclées. Cet effet était dépendant de la production d’O2°- et de NO par les cellules malignes. Un modulateur du stress oxydant, le mangafodipir, inhibait cet effet, permettant ainsi de restaurer la réponse immunitaire antitumorale locale. Notre travail a donc permis de mettre en évidence que des modulateurs du stress oxydant peuvent agir sur le microenvironnement, et spécialement sur les cellules immunitaires. Ils pourraient être utilisés en clinique pour restaurer la réponse immunitaire antitumorale. Une meilleure compréhension du rôle du stress oxydant dans la défaillance de l’immunité antitumorale est nécessaire
Several reports have demonstrated the involvement of reactive oxygen species (ROS) in carcinogenesis, through promotion of cancer cell proliferation and invasion. But ROS could also have consequences on non cancerous cells which are part of the tumor microenvironment, such as immune cells. Therefore, a pharmacological modulation of oxidative stress can induce a cytotoxic effect on tumor cells but its consequences on microenvironment are unknown. The aim of our studies was to evaluate the effects of a pharmacological modulation of oxidative stress on immune cells from the tumor microenvironment. At low dose, Arsenic trioxide (As203), an oxidative stress modulator, was shown to exert antitumor effects in colon tumor-bearing mice. We observed that this effect was related to As203-induced regulatory T cells (Tregs) -selective depletion in vitro and in vivo and was mediated by oxidative and nitrosative bursts. The differential effect of As203 on Tregs versus other CD4 cells was related to difference in the cells’redox status. We also observed that vinorelbine, an anticancer agent, could interfere with the antitumor immune response. We showed that vinorelbine could alter the function of immune cells surrounding tumor cells by a bystander toxic effect against tumor effector cells. In vivo experiment in A549 tumor bearing nude mice showed that adoptive transfer of A549 immune splenocytes was not able to delay tumor growth when vinorelbine-pretreated A549 cells were used for immunization. This effect was mediated by ROS and was inhibited by an oxidative stress modulator, mangafodipir, which restored antitumor immune function. Therefore, our work showed that oxidative stress modulators can influence tumor microenvironment and more specifically, immune cells. They could be used to restore antitumor immune response

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Girard, Pauline. „Pathophysiologie des pDCs et des Lymphocytes Tγδ en contexte de mélanome, et potentiel de leur interaction pour le développement de nouvelles thérapies The features of circulating and tumor-infiltrating gdT cells in melanoma patients display critical perturbations with prognostic impact on clinical outcome Potent Bidirectional Cross-Talk Between Plasmacytoid Dendritic Cells and γδT Cells Through BTN3A, Type I/II IFNs and Immune Checkpoints“. Thesis, Université Grenoble Alpes, 2020. http://www.theses.fr/2020GRALV042.

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.Les pDCs et Tγδ ont des rôles cruciaux dans l’initiation et l’orientation des réponses immunitaires. Leurs fonctions uniques, leur grande plasticité et leur capacité d’interagir avec de nombreux acteurs immunitaires leur permettent de créer un lien entre l’immunité innée et l’immunité adaptative. Elles contribuent donc grandement aux réponses immunitaires protectrices et pathogéniques, et sont de ce fait très prometteuses pour le développement d’immunothérapies anti-tumorales, autant en tant que vecteurs que cibles. Cependant, les lymphocytes Tγδ n’ont pas été étudiés de manière approfondie en contexte de mélanome, et les interactions entre pDCs et Tγδ n’ont été explicitées ni en contexte sain, ni en contexte mélanome, ou le contrôle immunitaire de la tumeur ‡ long terme est encore un défi. Nous avons réalisé une étude détaillée du phénotype et de la fonction des lymphocytes Tγδ circulant et infiltrant le mélanome, et analysé leur impact sur l’évolution clinique. Nous avons aussi caractérisé les interactions bidirectionnelles entre les pDCs et les Tγδ issus de sang de donneurs sains, et de sang ou de tumeur de patients. Nous avons mis en évidence que le mélanome détourne les fonctions effectrices des Tγδ dans le but d’échapper au contrôle immunitaire, et que les caractéristiques des Tγδ issus de sang ou de tumeurs de patients peuvent étre des bio-marqueurs prometteurs d’évolution clinique. Nous avons également montré que les interactions entre pDCs et Tγδ sont médiées par les IFNs de type I et II et par le récepteur BTN3A, essentiel pour l’activation des Td2+, et sont dérégulées en contexte de mélanome. L’administration de cytokines et d’anticorps ciblant les points de contrôle immunitaire peut rétablir des interactions fonctionnelles entre les deux populations cellulaires. De façon intéressante, nous avons observé une augmentation de l’expression de BTN3A sur les pDCs et Tγδ issus de sang de patients ou de tumeurs, tout en soulignant une potentielle dysfonction de cette molécule. Notre étude révèle que le mélanome détourne les interactions entre pDCs et Tgd notamment via la dérégulation de BTN3A. De tels résultats motivent l’exploitation de ces effecteurs immunitaires ainsi que de leur synergie, pour développer de nouvelles approches thérapeutiques exploitant leur potentiel anti-tumoral tout en évitant leur détournement par la tumeur pour améliorer l’évolution clinique des patients. Nos découvertes soutiennent l’exploitation de ces partenaires puissants et prometteurs pour élaborer de nouvelles stratégies thérapeutiques et restaurer des réponses immunes appropriées en contexte de cancer, infections et auto-immunité
Both pDCs and γδT cells harbor critical roles in immune responses induction and orientation. Their unique features, high functional plasticity and ability to interact with many immune cell types allow them to bridge innate and adaptive immunity. They actively contribute to protective and pathogenic immune responses, which render them very attractive both as targets and vectors for cancer immunotherapy. Yet, γδT cells have not been extensively explored in melanoma, and despite strategic and closed missions, cross-talks between pDCs and γδT cells have not been deciphered yet, neither in healthy context nor in cancers, especially in melanoma where the long-term control of the tumor still remains a challenge. We provided here a detailed investigation of the phenotypic and functional properties of circulating and tumor-infiltrating γδT cells in melanoma patients, as well as their impact on clinical evolution. We also characterized the bidirectional cross-talks between pDCs and γδT cells both from healthy donor’s blood, patient’s blood and tumor micro-environment. Our study highlighted that melanoma hijacked γδT cells to escape from immune control, and revealed that circulating and tumor-infiltrating γδT cell features are promising potential biomarkers of clinical evolution. We also demonstrated crucial bidirectional interactions between these key potent immune players though type I and II IFN and BTN3A that are dysfunctional in the context of melanoma. Reversion of the dysfunctional bidirectional cross-talks in melanoma context could be achieved by specific cytokine administration and immune checkpoint targeting. We also revealed an increased expression of BTN3A on circulating and tumor-infiltrating pDCs and γδT cells from melanoma patients but stressed out its potential functional impairment.Thus, our study uncovered that melanoma hijacked pDCs/ γδT cells bidirectional interplay to escape from immune control, and pointed out BTN3A dysfunction. Such understanding will help harnessing and synergizing the power of these potent immune cells to design new therapeutic approaches exploiting their antitumor potential while counteracting their skewing by tumors to improve patient outcomes. Our findings pave the way to manipulate these potent and promising cell partners to design novel immunotherapeutic strategies and restore appropriate immune responses in cancers, infections and autoimmune diseases

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Makdissi, Fabiana Baroni Alves. „Influência do microambiente no prognóstico do câncer da mama“. Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-01042014-112230/.

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Introdução: Os cânceres de mama subtipos Luminal A e B (HER2 negativo) podem apresentar prognóstico variável, a depender do índice de proliferação, avaliado pelo Ki67. As células malignas e as células estromais adjacentes (fibroblastos e células de resposta imune ) podem interagir tanto pelo contato célula a célula como por fatores secretados por elas, ambas influenciando no comportamento tumoral. Já foi demonstrado que as células estromais podem aumentar a proliferação das células do câncer da mama. Objetivo: Nosso objetivo foi avaliar o perfil de expressão gênica de células do estroma em câncer de mama luminal A e luminal B e analisar se este se correlaciona com o prognóstico da doença. Pacientes e Métodos/ Resultados: Amostras de tumores de 11 pacientes na pós menopausa foram analisadas, todas elas HER2 negativas. A expressão de Ki67 foi <= 10 % em 5 pacientes (luminal A) e >= 30 % em outras 6 amostras(Luminal B ). Células estromais foram microdissecadas para a extração de RNA, que posteriormente foi hibridizado na plataforma de microarray Agilent G485 -1A GE 8x60K. Após a normalização, 50 % dos genes com a maior variância foram selecionados para análise por SAM duas classes desemparelhado (software TMEV ) e aceitando FDR 14.1%, 35 sequências foram identificadas como diferencialmente expressas, incluindo 16 genes conhecidos, entre as células estromais das amostras de Luminal A versos Luminal B, todos mais expressos nas amostras B. Dentre as funções biológicas enriquecidas em genes diferencialmente expressos encontram-se regulação positiva do sistema imune, incluindo genes como ZAP70 (proteína quinase 70kDa associada a cadeia zeta (TCR)), CD38 (molécula CD38); UBASH3A (ubiquitina associada e SH3 domínio que contém A); PLA2G7 (fosfolipase A2, grupo VII (fator acetil ativador de plaquetas no plasma)); NCR3 (citotoxicidade natural, provocando receptor 3). Nosso próximo passo foi avaliar se a expressão de alguns genes selecionados estava associada com prognóstico de tumores luminais. Para tal selecionamos amostras de outro grupo de 89 pacientes com seguimento de pelo menos 5 anos, cujos tumores eram ER(+), HER2(-), para análise de expressão proteica em Tissue microarray. Caracterizamos os fibroblastos destas amostras com 3 marcadores de fibroblastos: actina de músculo liso (AML), S100A4 e caveolina-1 (CAV1) e analisamos a marcação da proteína ZAP70. Correlacionamos a expressão proteica de todos os marcadores com as características anatomopatológicas da amostra. Observamos que fibroblastos de todas as amostras de tumor de mama expressam AML, S100A4 e CAV1, em diferentes proporções, entretanto não detectamos diferença entre os tumores luminais A e B. Também não obsevamos diferença de expressão de AML, S100A4 e CAV1 em relação a grau histológico, comprometimento linfonodal e estadiamento clínico. Nestas amostras não detectamos expressão proteica de ZAP70 em fibroblastos tumorais. Conclusão: Houve expressão diferencial de 16 genes relacionados a processos imunes, todos eles mais expressos em células estromais de tumores Luminal B em relação a luminal A
Introduction: Luminal breast cancer subtypes A and B (HER2 negative) may present a variable prognosis, depending on tumor proliferation index, evaluated by Ki67 expression. Malignant cells and adjacent stromal cells (fibroblasts and immune response cells) may interact by both cell contact and secreted factors and influence tumor behavior. It was shown that stromal cells may enhance breast cancer cells proliferation. Objective: Our aim was to evaluate stromal cells gene expression profile in luminal A and luminal B tumors and to evaluate whether selected transcripts expressed in stromal cells may be associated with prognosis in breast cancer. Material/ Methods and Results: Hormone receptor positive tumor samples from 11 post menopausal patients were analyzed, all of them Her2 negative. Ki67 expression <= 10% (luminal A) was observed in five and Ki67 >= 30% (luminal B) in six samples. Stromal cells were microdissected for RNA extraction, which was hybridized in Agilent G485-1A GE 8x60K microarray platform. After normalization, 50% of the genes with the highest variance were selected for further analysis by two class unpaired SAM (TMEV software) and accepting FDR 14,1%, 35 sequences, including 16 known genes, were found differentially expressed between stromal cells from luminal A vs luminal B breast cancer samples, all of them more expressed in luminal B. Among biological functions enriched in genes found differentially expressed were positive regulation of immune system process, including genes as: ZAP70 (zeta-chain (TCR) associated protein kinase 70kDa); CD38 (CD38 molecule); UBASH3A (ubiquitin associated and SH3 domain containing A); PLA2G7 (phospholipase A2, group VII (platelet-activating factor acetylhydrolase, plasma); NCR3 (natural cytotoxicity triggering receptor 3). Our next step was evaluate whether expression of selected genes was associated with prognosis in another group of patients. Tumor samples from 89 patients with at least 5 years of follow up, all of them estrogen receptor positive and HER2 negative, were selected. Tissue microarray was prepared with stromal tumor compartment from paraffin embedded tumor samples. Fibroblasts were characterized for the expression of 3 fibroblasts markers (alfa-SMA, alpha smooth muscel actin; S100A4 and CAV1, caveolin 1), and ZAP70. Correlation of expression of these markers with prognostic variables was determined. Expression of alfa-SMA, S100A4 and CAV1 was detected in fibroblasts from all tumor samples in different proportions, however no differential expression was observed between luminal A and B tumors. Neither difference was detected on the expression of these proteins in relation with histological grade, lymph node involvement and clinical stage. Conclusion: A differential expression of 16 genes involved in immune process was found, all of them more expressed in fibroblasts from luminal B as compared with luminal A tumors

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Elaldi, Roxane. „Carcinomes épidermoïdes cutanés : caractérisation du micro-environnement immunitaire tumoral et identification de biomarqueurs moléculaires pronostiques“. Electronic Thesis or Diss., Université Côte d'Azur, 2023. https://intranet-theses.unice.fr/2023COAZ6054.

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Les carcinomes épidermoïdes cutanés (CEC) sont les deuxièmes tumeurs cutanées les plus fréquentes après les carcinomes basocellulaires. Elles sont majoritairement traitées par chirurgie et de bon pronostic. Mais certaines tumeurs à « haut risque », et particulièrement celles situées au niveau de la face et du cou, récidivent fréquemment dans les 2 ans après la chirurgie. Ces récidives, parfois inopérables, sont de mauvais pronostic et l'identification des patients atteints d'une tumeur à « haut risque » n'est pas consensuelle, ce qui constitue un problème majeur pour la prise en charge de ces cancers et l'inclusion de ces patients dans des essais cliniques. De plus, les CEC présentent une réponse globale de 50% aux immunothérapies anti-PD1 et plusieurs mécanismes d'immunosuppression caractérisant ces tumeurs inflammatoires ont été mis en évidence par mon laboratoire, soutenant l'importance du micro-environnement immunitaire dans leur carcinogenèse et leur progression.Mon hypothèse de travail de thèse a été que la caractérisation spatiale et intégrative des cellules immunitaires du microenvironnement tumoral ainsi qu'une comparaison des profils immunologiques de tumeurs ayant un pronostic différent permettrait de mieux appréhender la complexité de ces tumeurs.Pour répondre à cette question, je me suis appuyée sur des coupes de tissus conservés en paraffine (FFPE) provenant de tumeurs primitives et récidivantes de patients appartenant à une cohorte que j'ai constituée en collaboration avec le Centre de Ressources Biologiques du Centre Antoine Lacassagne (Nice), et sur une approche d'imagerie par cytométrie de masse (IMC).J'ai tout d'abord créé une cohorte de 160 patients opérés de 217 CEC de la face et du cou au Centre Antoine Lacassagne (Nice) et j'ai comparé la performance des 4 classifications histo-pronostiques existantes actuellement les plus utilisées pour identifier les patients présentant un CEC à « haut risque ». Je n'ai pas mis en évidence de classification significativement supérieure en terme de discrimination pour l'identification des patients présentant une récidive, et la valeur prédictive positive retrouvée la plus élevée était de 30% (Elaldi et al, J Clin Med, 2023). Ces résultats ont donc confirmé la nécessité d'affiner la sélection des patients à « haut risque » via l'identification d'un nouveau marqueur pronostic prenant en compte l'organisation spatiale du microenvironnement tumoral de ces tumeurs.J'ai donc constitué et validé un panel de 39 marqueurs permettant la caractérisation du microenvironnement des CEC humains de la face et du cou sur une seule coupe de tissu FFPE par imagerie par cytométrie de masse (Elaldi et al, Front Immunol, 2021). Puis, à partir de la base de données clinico-histologiques précédemment décrite, j'ai constitué une cohorte exploratoire de tumeurs primitives et récidivantes chez des patients ayant ou non récidivé dans les 2 ans après chirurgie. J'ai ensuite caractérisé les peaux péri-lésionnelles saines et les tumeurs de cette cohorte avec le panel IMC 39 marqueurs précédemment développé. J'ai ainsi identifié des motifs immunologiques permettant de distinguer ces deux types de tumeurs avec notamment une sous-population de macrophages caractéristique de la peau saine et enrichie dans les tumeurs de bon pronostic (Elaldi et al, en préparation).Mes travaux de thèse permettent d'améliorer la connaissance des mécanismes immunitaires mis en place pour lutter contre la progression tumorale des CEC et des mécanismes d'immunosuppression possiblement impliqués dans le phénomène d'échappement de ces tumeurs. A court terme, l'analyse d'une cohorte de validation permettra de valider la valeur pronostique de la population de macrophages décrite, afin d'améliorer la sélection des patients à « haut risque » et d'améliorer leur prise en charge
Cutaneous squamous cell carcinomas (cSCC) are the second most common skin tumors after basal cell carcinomas. Most are treated surgically, and their prognosis is good. However, some "high-risk" tumors, particularly those on the face and neck, frequently relapse within 2 years of surgery. These relapses, sometimes inoperable, have a poor prognosis, and the identification of patients with "high-risk" tumors is universally is a major challenge in managing these cancers and including these patients in clinical trials. Additionally, cSCC show an overall response rate of 50% to anti-PD1 immunotherapies, and several immunosuppression mechanisms characterizing these inflammatory tumors have been identified by our laboratory, emphasizing the impact of the immune microenvironment in their carcinogenesis and progressionMy work hypothesis was that the spatial and integrative characterization of immune cells of the tumor microenvironment, together with a comparison of immunological profiles of tumors with different prognoses, would provide a better understanding of the complexity of these tumors.To answer this question, I worked with formalin fixed paraffin embedded tissue sections (FFPE) from primary and recurrent tumors of patients belonging to a cohort I set up in collaboration with the Centre de Ressources Biologiques of the Centre Antoine Lacassagne (Nice), and an imaging mass cytometry (IMC) approach.First, I created a cohort of 160 patients who underwent surgery for 217 face and neck cSCC at the Centre Antoine Lacassagne. I compared the performance of the four currently most widely used classifications system to identify patients with "high-risk" cSCC. I did not find a significantly superior classification in terms of discrimination for identifying patients with recurrence, and the highest positive predictive value found was 30% (Elaldi et al, J Clin Med, 2023). These results confirmed the need to refine the selection of "high-risk" patients by identifying a new prognostic marker that takes into account the spatial organization of the tumor microenvironment of these tumors.I therefore set up and validated a panel of 39 markers enabling the characterization of the microenvironment of human cSCC on a single FFPE tissue section by IMC (Elaldi et al, Front Immunol, 2021). Then, using the previously described clinico-histological database, I set up an exploratory cohort of primary and recurrent tumors in patients who had or had not relapsed within 2 years following the surgery. I then characterized the healthy peri-lesional skin and tumors of this cohort with the previously developed IMC 39-marker panel. I identified immunological patterns that distinguish these two types of tumors, notably a macrophage subpopulation characteristic of healthy skin and enriched in tumors with a good prognosis (Elaldi et al, in preparation).My thesis work is improving our understanding of the immune mechanisms at work in the fight against cSCC relapse. In the short term, the analysis of a validation cohort will validate the prognostic value of the macrophage population we described in order to improve the selection of "high-risk" patients and their management

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Li, Ran Ph D. Massachusetts Institute of Technology. „Tumor microenvironmental control of metastasis : effects of the immune cells and physical forces on cell migration“. Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/109664.

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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February 2017.
"January 2017." Cataloged from PDF version of thesis.
Includes bibliographical references.
Metastasis, which accounts for 90% of cancer deaths, critically depends on the ability of cancer cells to migrate through the dense extracellular matrix (ECM) surrounding the solid tumor. Recent advances in cancer biology have revealed that non-cancerous cells and biophysical forces in tumor microenvironment can influence metastasis. Specifically, macrophage, one of the most abundant tumor-associated stromal cell types, has been shown to assist cancer cell invasion. However, exactly how macrophages affect the different aspects (e.g. speed and persistence) of cancer cell migration, especially in 3D ECM that mimics the in vivo tumor microenvironment, remains largely unknown. In addition to macrophages, elevated interstitial flow (the flow of tissue fluid) within the tumor tissue has been shown to influence cancer cell and fibroblast migration. Nevertheless, the effects of this tumor-associated biophysical force on macrophages are still unknown. In this thesis, we first explored how macrophages control the subtle details (speed vs. persistence) of cancer cell migration. Using a microfluidic migration assay, we found that macrophage-released TNFa and TGF1 enhance cancer cell migration speed and persistence in 3D ECM in an MMP-dependent fashion via two distinct pathways. Specifically, macrophagereleased TGF1 enhances cancer cell migration speed via the induction of MTl-MMP expression in cancer cells. In contrast, macrophage-released TNFa and TGFp1 synergistically enhance cancer cell migration persistence via the induction of NF-KB-mediated MMP1 expression. Therefore, these results suggest that macrophages control different aspects of cancer cell migration in 3D differently, and both TNFa and TGFp1 released by macrophages need to be simultaneously inhibited to effectively limit macrophage-assisted cancer cell metastasis. In a separate study, we investigated how tumor-associated interstitial flow (IF) affects macrophage migration and protein expression. We discovered that IF promotes macrophage migration in 3D ECM via the flow-induced activation of FAK and Akt. Interestingly, IF also directs the preferential migration of macrophages against the direction of flow (upstream). Moreover, we show that IF polarizes macrophages toward a pro-metastatic M2 phenotype via integrin/Src-dependent STAT3/6 activation. Since IF emanates from tumor core to stromal tissue surrounding the tumor, these results suggest that IF can promote metastasis by not only recruiting macrophages from stroma into tumor, but also enhancing the M2 polarization of macrophages in the tumor microenvironment. Keywords: Tumor Microenvironment, Macrophages, Interstitial Flow, Migration, and Polarization.
by Ran Li.
Ph. D.

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Trimaglio, Giulia. „An orthotopic syngeneic mouse model to study the role of DCIR in colorectal cancer“. Thesis, Toulouse 3, 2020. http://www.theses.fr/2020TOU30053.

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Le cancer colorectal (CCR) est le troisième cancer le plus fréquent et le deuxième cancer le plus mortel dans le monde. En conséquence, de nouveaux biomarqueurs diagnostiques ainsi qu’une amélioration des traitements actuels sont nécessaires. Les tumeurs se développent dans des microenvironnements complexes où les cellules cancéreuses interagissent et modulent la réponse immunitaire locale pour persister et se multiplier. Les lectines de type C, exprimées notamment par les cellules de l’immunité, régulent la réponse anti-cancéreuse, et donc le développement tumoral. Parmi elles, l'immunorécepteur des cellules dendritiques (DCIR), a été montré comme jouant un rôle immunitaire majeur au cours des maladies infectieuses et auto-immunes. À l’inverse, son rôle dans l'immunité tumorale reste méconnu. L'analyse des données transcriptomiques de deux cohortes de patients atteints de CCR a révélé un lien entre une expression élevée de DCIR et une meilleure survie des patients. Dans ce contexte, l'objectif principal de ma thèse était de déterminer l'impact de DCIR sur le développement du CCR et la réponse immunitaire associée. Dans ce but, j’ai établi un modèle murin préclinique, orthotopique et syngénique du CCR consistant en l'injection intracaecale de cellules tumorales MC38 exprimant la luciférase (MC38-fLuc+) dans des souris C57BL/6. Le suivi de la croissance tumorale par bioluminescence a montré que, malgré l’acquisition initiale de tumeurs solides par toutes les souris, seulement 30% des souris ont développé un CCR progressif et mortel, tandis que les autres animaux ont spontanément rejeté leurs tumeurs. Aucun rejet des tumeurs CCR MC38 n'a été observé en l'absence d'immunité adaptative, ni lors de l'injection de cellules MC38 dans d'autres sites anatomiques. L'immunophénotypage par transcriptomique et cytométrie de flux a révélé que les souris développant des tumeurs progressives présentaient une réponse immunitaire pro-tumorale, définie par une signature caractéristique des lymphocytes T régulateurs, détectable peu après l'implantation tumorale, et par une infiltration de cellules myéloïdes suppressives connues pour favoriser la croissance tumorale. En revanche, les souris rejetant les tumeurs présentaient une signature pro-inflammatoire précoce et un microenvironnement anti-tumoral enrichi en lymphocytes T CD8+. Ainsi, nos résultats démontrent un rôle du microenvironnement spécifique du côlon dans la régulation de l'équilibre entre les réponses immunitaires anti- ou pro-tumorales et souligne l'importance d'utiliser des modèles murins orthotopiques pour les études in vivo. Dans la seconde partie de ma thèse, j’ai utilisé ce modèle murin de CCR pour comparer le développement tumoral dans des souris C57BL/6 de type sauvage ou des souris déficientes pour l’expression de mDcir1 (mDcir1-KO), un homologue murin du DCIR humain. Bien que l'absence de mDCIR1 n'ait aucune incidence sur le pourcentage de souris développant ou rejetant les tumeurs du CCR, nous avons observé que les animaux mDcir1-KO développaient des tumeurs plus importantes que les sauvages En accord avec ce résultat, nous avons constaté une infiltration plus faible de lymphocytes CD8+ cytotoxiques et une activation moindre des lymphocytes T CD4+ et CD8+ (c'est-à-dire T-BET+, CD44haut, CTLA-4+) dans les tumeurs des souris mDcir1-KO par rapport aux souris sauvages. Ainsi, nos données indiquent un rôle protecteur et anti-tumoral de DCIR pendant le développement du CCR, probablement dû à une dérégulation de l'équilibre existant entre la tumeur et la réponse immunitaire. Dans l'ensemble, cette étude ouvre la voie à la mise au point éventuelle de biomolécules pharmacologiques ciblant DCIR pour déclencher une réponse immunitaire anti-tumorale efficace dans le contexte du CCR et au-delà
Colorectal cancer (CRC) is the third most common and second deadliest cancer worldwide accounting for 900.000 deaths in 2018. Consequently, there is a strong need for new biomarkers as well as an improvement of the current treatments. Tumors develop in complex microenvironments where cancer cells constantly crosstalk with, and modulate, the local immune response to persist and replicate. C-type lectins receptors, expressed in particular by immune cells, actively regulate the immune response to cancer cells and, therefore, tumor development. Dendritic cell immunoreceptor (DCIR), a C-type lectin expressed by myeloid cells, has been shown to play a major role in immunity to infectious and autoimmune diseases. Yet, the role played by DCIR in tumor immunity remains unknown. Analysis of publicly available transcriptomic data from two cohorts of CRC patients revealed an association between high DCIR gene expression and improved survival of patients. In this context, the principal objective of my PhD thesis was to determine the role played by DCIR in the immune response during CRC development. First, I developed an orthotopic syngeneic pre-clinical CRC mouse model consisting in the intra-caecal injection of engineered MC38 tumor cells expressing firefly luciferase (MC38-fLuc+) in C57BL/6 mice. Monitoring of the tumor growth by bioluminescence revealed that, despite an initial growth of solid tumors in all the mice, only 30% of mice developed a progressive lethal CRC, while the remaining animals spontaneously rejected their solid tumor and survived more than 100 days. No rejection of tumors was observed in the absence of adaptive immunity, nor when MC38-fLuc+ cells were injected in other anatomical locations (i.e., liver and skin). Immunophenotyping by transcriptomic and flow cytometry showed that mice with progressive CRC tumors exhibited a pro-tumor immune response, characterized by a regulatory T cell pattern, discernible shortly post-tumor implantation, as well as myeloid suppressor cells that are well-known to favor tumor growth. By contrast, tumor-rejecting mice presented an early pro-inflammatory response and an anti-tumor microenvironment enriched with CD8+ T cells. Taken together, our results demonstrate a preponderant role of the colon-specific microenvironment in regulating the balance between anti- or pro-tumor immune responses and underline the importance of using orthotopic mouse models for in vivo studies. In a second part of my thesis, we used this CRC mouse model to compare the tumor development in wild-type (WT) C57BL/6 mice or mice deficient for mDcir1 (mDcir1-KO), a murine homologue of human DCIR. While the lack of mDCIR1 has no impact on the percentage of mice developing or rejecting CRC tumors, we observed that mDcir1-KO animals developed bigger tumors than their WT counterparts. In line with this result, we found a lower infiltration of cytotoxic CD8+ and decreased activation of both CD4+ and CD8+ T cells (i.e., T-BET+, CD44high, CTLA-4+) in CRC tumors from mDcir1-KO mice compared to WT mice. Altogether, our data point to a protective and anti-tumor role of DCIR during CRC development, probably due to a dysregulation of the balance existing between the tumor and the immune response. Overall, this study paves the way for the potential future development of pharmacological biomolecules targeting DCIR to trigger an efficient anti-tumor immune response in the context of CRC and beyond

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Tuccitto, Alessandra. „pH regulatory molecules in the tumour microenvironment : modulators of aggressiveness and immune profile of human hepatocellular carcinoma“. Thesis, Open University, 2018. http://oro.open.ac.uk/55985/.

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Background: Hepatocellular carcinoma (HCC) arises in an inflammatory, hypoxic/acidic microenvironment that favours tumour progression and fosters immunosuppression. Tumour cells survive this hostile environment by over-expressing pH regulatory molecules such as carbonic anhydrase (CA) IX, XII and V-ATPase complex, but the relevance of these molecules in HCC is poorly defined. Aim: The aim of this study was to dissect the relationships between pH regulatory molecules and the aggressive behaviour of malignant hepatocytes, and to evaluate how pH regulatory molecules influence the immune microenvironment of HCC. Methods: HCC, non-tumour and normal liver tissue samples were analysed by qRT-PCR for the expression of genes encoding the pH regulatory molecules (CAIX, CAXII and V-ATPase), of genes associated to epithelial-to-mesenchymal-transition (EMT) (TWIST, CDH1, VIM) and those encoding for HCC stem cell-associated markers (CD13, CD24, CD44, CD90, EpCAM, CD133, KRT19, OCT4, NANOG and SOX2). Selected HCC, non-tumour and normal liver tissue samples were evaluated by immunohistochemistry (IHC) to detect the presence and localization of CAIX, CAXII and VATPase and to assess the distribution of macrophages and T cells. Confocal microscopy and flow cytometry were implemented to assess the co-expression of selected markers. HCC cell lines, characterised for the expression of pH regulators, were tested for the sensitivity to the CAIX, CAXII, and V-ATPase specific inhibitors. The effects of V-ATPase specific drug were also studied ex vivo in primary human HCC tumour explants by qRT-PCR and by flow cytometry in HCC single cell suspensions obtained by the enzymatic digestion of HCC specimens. Results: Our mRNA analysis showed that the expression of CA9 was significantly correlated with the expression of the hypoxia-inducible factor 1α-related gene (HIF1A) and of the stem cell-associated markers CD24, CD133, EpCAM and KRT19. Moreover, mRNA for CA9 and for the different CA12 isoforms were associated with tumour grading, thus indicating their possible role in tumour malignancy. Applying a machine learning tool known as the ‘Adaptive Index Model’ the combined expression of different CA12 isoforms, CD209 and CDH1 defined a ‘signature’ classifying HCC patients in groups at different risk of recurrence, thus indicating a link between pH regulators, myeloid and EMT markers likely influencing HCC prognosis. IHC analysis indicated that HCC displays a complex expression pattern for the pH regulatory proteins. Both CAIX and CAXII were detected in transformed, but not in normal hepatocytes. CAIX protein had a focal distribution in the tumour, thus supporting its possible association with hypoxic and the most aggressive tumour area. Conversely, CAXII was homogeneously expressed by all tumour hepatocytes, but mainly retained in the endoplasmic reticulum (ER). The majority of HCC expressed V-ATPase which, importantly, was also present in immune infiltrating cells. This expression pattern qualified the CAIX, CAXII and V-ATPase as possible targetable molecules. Our in vitro data indicated that blockage of their enzymatic activities by specific drugs affected the viability of HCC cell lines in a dose dependent fashion, although with the CAXII specific inhibitor showing low efficacy, likely related to the preferential ER localization of CAXII molecules inside the HCC cells. Ex vivo experiments with HCC tissue explants and HCC cellular suspensions showed that inhibition of VATPase modulated the epithelial/mesenchymal features of HCC cells and the levels of pro- and anti-tumour cytokines expressed by M2 macrophages and T cells infiltrating HCC. Conclusions: Herein, our data demonstrated that the pH regulatory molecules, CAIX, CAXII and V-ATPase are over expressed in the HCC microenvironment and interfering with their pathways exerted anti-tumour activities, although these data also lead to the conclusion that more effective CAXII specific drugs should be designed. The results of this thesis also suggest that pH regulatory molecules might have a role in HCC aggressiveness and prognosis. Importantly, one of these pH regulators, namely V-ATPase complex, influences the mesenchymal features of tumour cells and the immunosuppressive tumour microenvironment (TME). Interfering with tumour metabolism is an emerging strategy for treating cancers that are resistant to standard therapies. Thus, targeting the unique crosstalk between tumour cells and the microenvironment, played by the pH regulatory molecules, can be considered as a new option for HCC treatment and the blockage of the V-ATPase complex might represent a multi-task strategy for the treatment of HCC patients.

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VILIA, MARIA GIOVANNA. „Development of a Liver-specific immune 3D microenvironment for the study of primary and metastatic liver tumour“. Doctoral thesis, Università degli Studi di Cagliari, 2020. http://hdl.handle.net/11584/285114.

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Two dimensional (2D) in vitro models have been insufficient in replicating the complex interplay that occurs in the in vivo microenvironment between cancer and immune cells. The main reason for this discrepancy is due to the lack of cell-cell interactions and the cell-tumour microenvironment in conventional 2D plastic models. Indeed, cancer is a product of the complex interaction between cancer cells and tumour microenvironment, which comprises a three-dimensional (3D) extracellular matrix (ECM) together with tissue-resident cells as well as immune cells. The aim of this project was to evaluate immune and cancer cells interaction, cell infiltration and the ability of immune cells to target cancer cells, within tissue-specific and disease-specific human ECM scaffolds. Additionally, we aimed to test a 3D liver model of primary and metastatic liver cancer for therapy and immunotherapy. The results revealed that 3D cultures induced a mesenchymal-like phenotypes depending on disease-specific biochemical and biomechanical composition of the scaffold (Healthy or Cirrhotic). In fact, 3D, unlike 2D cultures of hepatocellular carcinoma (HCC) cell line, induced E-cadherin downregulation in healthy and cirrhotic scaffolds, suggesting the activation of the epithelial to mesenchymal transition (EMT). Current models for testing new immunotherapeutic drugs in solid tumours are not satisfactory. One of the potential limitations affecting the translation of therapies towards effective treatments for solid tumours is due to the lack of reliable in vitro and in vivo models. Therefore, another aim of my thesis is to investigate the impact of tissue-specific and disease-specific ECM scaffolds in modulating tumour and immune cells behaviours as well as response to immunotherapy. Our results demonstrated that the disease-specific 3D environment promoted a protective microenvironment for cancer cells as well as an immune cells exclusion phenotype, accounting for the low therapeutic efficacy found in clinical practice. Based on the finding that 3D healthy and cirrhotic scaffolds recreate molecular features of immunological “hot” and “cold” tumour, respectively, we propose 3D ECM cultures as a novel and powerful platform for drug screening. Finally, the observation that the efficacy of sorafenib and immunotherapy (anti-PD1 and anti-PDL1) 13 is strongly reduced in 3D (especially cirrhotic ECM) compared to 2D models suggests that 3D scaffolds can represent also a useful tool for the study of the mechanisms underlying cancer cell resistance.

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George, Courtney M. „Medulloblastoma: New animal models, preclinical drug testing, and characterising immune infiltrates“. Thesis, Edith Cowan University, Research Online, Perth, Western Australia, 2022. https://ro.ecu.edu.au/theses/2575.

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Medulloblastoma is the most common malignant brain tumour in children. The current treatment for medulloblastoma consists of surgery, radiation, and chemotherapy. Although these therapies have their merits, the outcome for some patients, particularly those with MYC amplified tumours, is poor, and the damaging nature of these therapies results in morbidities that significantly impact on a patient’s quality of life. To improve outcome and reduce adverse side effects, several strategies have been employed, including expanding the repertoire of accurate disease models, the development of novel therapies and the initiation of clinical trials, and an improvement in the understanding of the disease pathogenesis. The purpose of this thesis was to contribute to the repertoire of animal models, assist in identifying novel therapeutic approaches to treatment, and to advance the knowledge of the medulloblastoma immune microenvironment. We did so by aiming to (1) develop more accurate, immune-competent animal models of MYC- or NMYC-amplified medulloblastoma, (2) testing a novel treatment that combines conventional chemotherapies with a cell cycle checkpoint kinase inhibitor, and (3) investigating the effects of clinically used chemotherapies on immune cell populations in the brains of medulloblastoma-bearing mice. Currently, the limited availability of preclinical mouse models that accurately represent subgroup-specific medulloblastoma has hindered the development of therapies that succeed in the clinical setting. In addition, the distinct lack of immunologically competent models has prevented the advancement of immunotherapy drugs in treating medulloblastoma. The models of medulloblastoma developed here were designed to aid in preclinical studies, and to contribute specifically to the repertoire of immune competent mouse models for studies to clarify the role of the immune system in medulloblastoma. Here, we utilised mutated human variants of CMYC, NMYC, and P53, to transform mouse neural stem cells into tumour forming cells. Tumours were established in C57Bl/6 mice and characterised by histology and RNAseq analysis. Whilst these animal models could not be confirmed to accurately represent Group 3 or Group 4 medulloblastomas, this study demonstrated that mouse neural stem cells could be transformed using human genes and could generate brain tumours within immune competent hosts. As we were unable to conclusively define the exact nature of the tumours that were produced here, existing mouse models were used for subsequent chapters in this thesis. Prior to implementing new therapeutic agents into clinical trial, these are evaluated in a pre-clinical setting. The data presented in chapter two contributed to a larger scale pre-clinical testing pipeline that led to the identification and evaluation of multiple kinase inhibitors for the treatment of medulloblastoma. Here, I examined the combination of the cytotoxic agent gemcitabine (GEM) with the cell cycle checkpoint kinase inhibitor (LY2606368, prexasertib) as a novel approach in the treatment of Group 3 medulloblastoma. Combination GEM/LY2606368 treatment improved the survival of mice with aggressive medulloblastoma. Using immunoblotting and flow cytometry I showed that mechanistically LY2606368 enhanced GEM-induced cytotoxicity by impairing DNA damage response pathway activity, which promoted the accumulation of DNA damage leading to increased apoptosis. Together, these data formed part of the preclinical evidence that supported the establishment of the SJ-ELiOT clinical trial, targeted towards improving outcomes for patients who experience recurrent or relapsed disease following standard-of-care therapy. There is evidence to suggest that inhibiting the DNA damage response pathway can stimulate the immune system, and aid in tumour elimination. This provides strong rationale for implementing immunotherapies for patients who are predicted to have a poor response to conventional treatments. Unfortunately, to date all clinical trials investigating current popular immune-based therapies have failed in medulloblastoma, likely due to a poor understanding of the immune microenvironment in this disease. This presented an opportunity to improve our understanding of the effects of treatment on the immune system in brain. Here I characterised the immune cell populations in the brains of mice with Group 3 medulloblastoma treated with vehicle or two clinically-used chemotherapies – cyclophosphamide (CPA) and GEM. I revealed that CPA and GEM differentially alter immune cells within the brain in a manner similar to that observed outside of the central nervous system. I also demonstrate that the lack of an adaptive immune system (using mice deficient in Rag1) does not influence the anti-cancer effects of these drugs. This information provides a rationale for exploring alternative avenues when considering the use of cancer-targeting immunotherapies in combination with conventional medulloblastoma treatments. Collectively, these studies demonstrate the complicated nature of modelling high-risk medulloblastoma in the lab. I have improved upon current preclinical tools for medulloblastoma by the development of accurate immune competent models and advanced our knowledge of disease pathogenesis by elucidating the way medulloblastomas interact with the immune system in the brain. Furthermore, I highlight the translational value of preclinical models in the evaluation of new drug combinations ahead of clinical trial. Improving on the current tools available and accurately evaluating new therapies will ideally lead to improved clinical outcomes for patients with medulloblastoma.

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Anna, François. „Développement d'une immunothérapie anti-tumorale basée sur un récepteur antigénique chimérique (CAR) ciblant le point de contrôle immunitaire HLA-G : implications pour les tumeurs et leur microenvironnement“. Thesis, Université de Paris (2019-....), 2019. https://wo.app.u-paris.fr/cgi-bin/WebObjects/TheseWeb.woa/wa/show?t=4021&f=26655.

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Au cours de la dernière décennie, l’avènement des immunothérapies antitumorales a été une vraie percée dans le monde de la cancérologie avec le succès clinique des inhibiteurs de point de contrôle immunitaire (ICP) ou des thérapies cellulaires basées sur l’utilisation de récepteurs antigéniques chimériques (CAR). Cependant, aujourd’hui elles montrent leurs limites contre les tumeurs solides, notamment à cause d’un manque d’antigène spécifique et d’un microenvironnement tumoral complexe capable de moduler la réponse immune au profit de l’expansion des cellules cancéreuses. La molécule HLA-G est une protéine immunosuppressive participant exclusivement à la tolérance foeto-maternelle en contexte normal mais dont la fonction a été détournée par les tumeurs pour inhiber la réponse effectrice à son encontre. HLA-G est donc identifié comme un excellent antigène associé aux tumeurs et son inhibition est un élément crucial pour restaurer la réponse immunitaire anti-tumorale. Cependant, aucune stratégie immunothérapeutique ciblant HLA-G n’existait jusqu’à aujourd’hui.L’absence de traitement efficace contre ou ciblant HLA-G provient de l’incapacité à générer efficacement des anticorps contre cette protéine complexe du fait de la présence de nombreuses isoformes et de leurs caractères immunotolérogéniques. Dans la première partie de ces travaux, grâce à une méthode d’immunisation originale basée sur l’utilisation de vecteurs lentiviraux, nous avons pu générer des anticorps capables de reconnaitre le site d’interaction de HLA-G avec ses récepteurs et donc de potentiellement bloquer la fonction ICP de HLA-G. La deuxième partie de cette étude décrit la génération d’une thérapie CAR ciblant HLA-G en tant qu’antigène spécifique aux tumeurs. Tout d’abord, nous nous sommes préalablement concentrés sur la régulation et l’expression de la chaine CAR au niveau transcriptionnel. Cette approche visait à limiter les multiples effets secondaires liés à une thérapie CAR tels que l’activation continue des cellules CAR-T ou l’élimination de cellules saines exprimant l’antigène ciblé. Nous avons ensuite généré deux nouveaux CAR de 3ème génération capable de reconnaitre spécifiquement les isoformes majoritaires de HLA-G en contexte tumoral, et nous avons démontré leur efficacité à éliminer des tumeurs exprimant HLA-G in vitro et in vivo. Enfin, une série d’optimisations ont été réalisées sur la structure des protéines CAR pour augmenter leur capacité cytotoxique et permettre leur régulation par l’introduction du gène suicide iC9 pour les modèles précliniques. Nous démontrons pour la première fois que la thérapie CAR anti-HLA-G est extrêmement efficace in vitro et in vivo.Enfin, nous discutons du potentiel des immunothérapies anti-HLA-G aussi bien au travers des anticorps monoclonaux bloquants que des cellules CAR-T dans le contexte des tumeurs solides, de leurs implications sur le microenvironnement et de leur possible combinaison avec d’autres immunothérapies
Over the last decade, anti-tumor immunotherapies have been a breakthrough in the oncology field following the clinical successes obtained with immune checkpoint inhibitors (ICPs) or chimeric antigenic receptors (CAR) based therapies. However, they are less effective against solid tumors, especially because of the lack of tumor specific antigen and of a tumor microenvironment capable of inhibiting the immune response favoring the tumor expansion. The HLA-G molecule is an immunosuppressive protein originally exclusively demonstrated to be involved in maternal-fetal tolerance but whose function has been hijacked by tumors to inhibit and escape from immune responses. HLA-G is now identified as an exquisite tumor associated antigen and its inhibition is crucial to restore the anti-tumor immune responses. Yet, no immunotherapy directed against HLA-G has been developed to date.The lack of effective treatment against or targeting HLA-G is related to the inefficiency to induce antibodies against this complex protein since HLA-G could be expressed through several isoforms that are immunosuppressives. In the first part of this study, thanks to an original immunization method based on the use of lentiviral vectors, we demonstrate the possibility to generate antibodies which are capable to recognize the HLA-G interaction domain with its receptors and are expected to inhibit the ICP function of HLA-G. The second part describes a CAR-T cell immunotherapy targeting HLA-G for its TAA properties. We first focused on the regulation and on the expression of the CAR chain at the transcriptional level. This approach was meant to limit the side effects caused by CAR therapies such as continuous activation of the CAR-T cells or elimination of healthy cells expressing the targeted antigen. We then generated two new 3rd generation CARs demonstrated to specifically recognize major HLA-G isoforms expressed by tumor cells and to eradicate HLA-G expressing tumor cells in vitro and in vivo. Several optimizations were carried out on the CAR chain structure to increase CAR-T cells cytotoxic function and to control their persistence through the insertion of the iC9 suicide gene. Given the results presented here, we provide the first vitro and vivo proofs of concept that a CAR therapy directly targeting HLA-G, and more generally an ICP is strikingly efficient.Finally, we discussed the potential for both anti-HLA-G blocking monoclonal antibodies and CAR-T cells immunotherapies against solid tumors and its implication against the tumor microenvironment and possible combinations with other immunotherapies

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Seshadri, Dhruv Ramakrishna. „Immuno-nanotherapeutics to Inhibit Macrophage Polarization for Non-Small-Cell Lung Cancers“. Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case151084330337552.

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Kaewkangsadan, Viriya. „Evaluation of immune cell infiltrates and expression of cytokines/biological molecules in the microenvironment of tumours and tumour-draining axillary lymph nodes in patients with large and locally advanced breast cancers undergoing neoadjuvant chemotherapy : crucial contribution to immune-mediated tumour cell death“. Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/34155/.

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Background: Neoadjuvant chemotherapy (NAC) is being used as first line treatment in women with large and locally advanced breast cancers (LLABCs). However, the response to NAC is difficult to predict. Growing evidence suggests that these patients are immunosuppressed and that circulating immunosuppressive regulatory cells and humoral factors affect the response to NAC. We explored the possible role of the in situ tumour immune milieu in inducing and affecting the responses to NAC, and the contribution of concomitant systemic circulating regulatory cells. Methods: Paraffin-embedded breast cancers and ipsilateral axillary lymph nodes (ALNs) from pre- and post-NAC samples of a cohort of 33 women with LLABCs, 16 of whom had their blood regulatory cells previously investigated. Various immune cell infiltrations and expression of cytokines/biological molecules in the specimens were studied using appropriate monoclonal antibodies and immunohistochemistry. Statistical analysis was carried out using non-parametric tests with SPSS version 21. Results: High levels of pre-NAC tumour-infiltrating lymphocytes (TILs) (p < 0.001) and subsets of CD4⁺T cells (intratumoural, p=0.023; peritumoural, p=0.001), CD8+T cells (intratumoural, p=0.008; peritumoural, p=0.002) and CD56⁺NK cells (intratumoural, p=0.001; peritumoural, p < 0.001) were significantly associated with a pathological complete response (pCR). High levels of CD163⁺macrophages were also significantly associated with a good pathological response (p=0.004) and pCR (p=0.008). There was a positive correlation between the CD8:FOXP3 ratio and grade of pathological response. In multivariate analyses, TILs and peritumoural CD56+NK cells were found to be independent predictive factors for pCR. There was a significantly high expression of IL-10 in post-NAC breast specimens with poor responses to NAC (p < 0.001). NAC significantly reduced infiltrating T regulatory cells (Tregs) (p=0.001) and PD1⁺T cells (p=0.005), as well as expression of IL-4 (p=0.016). There was no significant difference between the percentages (%) of immune cells present in ALNs with or without metastases but there was a T helper-2 cytokine polarisation in metastatic ALNs. Metastatic ALNs with a high % of CD8+T cells (p=0.048) and low % of FOXP3+Tregs (p=0.019) were significantly associated with an ALN pCR. There was a significantly positive correlation between circulating and intratumoural infiltrating Tregs following NAC (p=0.003). Conclusions: The tumour immune microenvironment is a key factor in achieving a good pathological response with NAC. Tumour and blood immune parameters may be clinically useful in identifying women with LLABCs likely to respond to NAC. Our findings also suggest that the beneficial effects of NAC are mediated via modulation of anticancer immunity, in particular by reduction of T regulatory cells and immunosuppressive humoral factors.

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De, Vries-Brilland Manon. „Caractérisation du microenvironnement immunitaire des carcinomes papillaires du rein“. Electronic Thesis or Diss., Angers, 2023. http://www.theses.fr/2023ANGE0017.

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Article 1 : Inhibiteurs de points de contrôle dans le carcinome rénal papillaire métastatique : le carcinome papillaire à cellules rénales (pRCC) est le CCR à cellules non claires (nccRCC) le plus courant et une entité distincte, bien qu'hétérogène, associée à de mauvais pronostics. Le paysage thérapeutique du pRCC métastatique (mpRCC) reposait jusqu'à présent sur des thérapies ciblées, imitant les développements antérieurs dans le carcinome rénal métastatique à cellules claires. Cependant, les antiangiogéniques ainsi que les inhibiteurs de mTOR ne conservent qu'une activité limitée dans le mpRCC. Alors que le développement d'inhibiteurs de points de contrôle immunitaires (ICI) est actuellement en cours chez les patients atteints de mpRCC, notre objectif était de discuter des données d'activité précoces et du potentiel de futures stratégies thérapeutiques en monothérapie ou en association. L'expression de points de contrôle immunitaires tels que PD-L1 et de cellules immunitaires infiltrantes dans le pRCC pourrait fournir des informations sur leur immunogénicité potentielle, bien que celle-ci soit actuellement mal décrite. Sur la base de données rétrospectives et prospectives, l'efficacité de l'ICI en monothérapie reste limitée. Les associations avec des inhibiteurs de la tyrosine-kinase, notamment avec des inhibiteurs anti-MET, présentent des taux de réponse prometteurs et pourraient entrer dans la norme de soins chez les patients non traités. Un travail collaboratif est nécessaire pour affiner le paysage moléculaire et immunitaire du pRCC et poursuivre les efforts visant à mettre en place des essais cliniques prédictifs basés sur des biomarqueurs dans ces tumeurs rares.Article 2 : Analyses complètes du microenvironnement tumoral immunitaire dans le carcinome papillaire à cellules rénales. Contexte : le carcinome papillaire à cellules rénales (pRCC) est le CCR à cellules non claires (nccRCC) le plus courant et associé à de mauvais résultats dans le contexte métastatique. Dans cette étude, nous avions pour objectif d'évaluer de manière exhaustive le microenvironnement tumoral immunitaire (TME), largement inconnu, des patients atteints de pRCC métastatique et d'identifier des cibles thérapeutiques potentielles. Méthodes : nous avons effectué une analyse quantitative de l'expression génique du TME en utilisant la méthodologie du compteur MCP, sur 2 cohortes indépendantes de pRCC localisés (n=271 et n=98). Nous avons ensuite caractérisé le TME, en utilisant l'immunohistochimie (n = 38) et le séquençage d'ARN (RNA-seq) (n = 30) sur des pRCC métastatiques de la cohorte prospective de l'essai AXIPAP. Résultats : le clustering non supervisé a identifié 2 « sous-types de TME » dans chacune des cohortes : le « immuno-enrichi » et le « immuno-faible ». Au sein de la cohorte de l'essai AXIPAP, le cluster « immuno-enrichi » était significativement associé à un plus mauvais pronostic. selon la médiane de survie globale à 8 mois (IC95%, 6-29) versus 37 mois (IC95%, 20-NA, p=0,001). Les 2 signatures immunitaires, Teff et JAVELIN Renal 101 Immuno signature, prédictives de La réponse aux inhibiteurs de point de contrôle immunitaire (IPC) dans le ccRCC était significativement plus élevée dans le groupe « enrichi sur le plan immunitaire » (p < 0,05 ajusté). Enfin, 5 gènes différentiellement surexprimés ont été identifiés, correspondant principalement aux populations de lymphocytes B. Conclusion : pour la première fois, en utilisant RNA-seq et IHC, nous avons mis en évidence un sous-type immunitaire TME spécifique du pRCC métastatique, significativement plus infiltré par la population T et Bimmune. Ce groupe « immunoenrichi » semble avoir un pronostic plus défavorable et pourrait avoir une valeur prédictive potentielle de réponse à l’immunothérapie, justifiant la confirmation de ces résultats dans une cohorte de pRCC métastatiques traités par CPI et en association avec des thérapies ciblées
Article 1: Checkpoint inhibitors in metastatic papillary renal cell carcinoma : papillary Renal Cell Carcinoma (pRCC) is the most common non-clear cell RCC (nccRCC) and a distinct entity, although heterogenous, associated with poor outcomes. The treatment landscape of metastatic pRCC (mpRCC) relied so far on targeted therapies, mimicking previous developments in metastatic clear-cell renal cell carcinoma. However, antiangiogenics as well as mTOR inhibitors retain only limited activity in mpRCC. As development of immune checkpoint inhibitors (ICI) is now underway in patients with mpRCC, we aimed at discussing early activity data and potential for future therapeutic strategies in monotherapy or combination. Expression of immune checkpoints such as PD-L1 and infiltrative immune cells in pRCC could provide insights into their potential immunogenicity, although this is currently poorly described. Based on retrospective and prospective data, efficacy of ICI as single agent remains limited. Combinations with tyrosine-kinase inhibitors, notably with anti-MET inhibitors, harbor promising response rates and may enter the standard of care in untreated patients. Collaborative work is needed to refine the molecular and immune landscape of pRCC, and pursue efforts to set up predictive biomarker-driven clinical trials in these rare tumors. Article 2 : Comprehensive analyses of immune tumor microenvironment in papillary renal cell carcinoma. Background : papillary Renal CellCarcinoma (pRCC) is the most common non-clear cell RCC (nccRCC), and associated with poor outcomes in the metastatic setting. In this study, we aimed to comprehensively evaluate the immune tumor microenvironment (TME) ,largely unknown, of patients with metastatic pRCC and identify potential therapeutic targets. Methods : we performed quantitative gene expression analysis of TME using MCP-counter methodology, on 2 independent cohorts of localized pRCC (n=271 and n=98). We then characterized the TME, using immunohistochemistry (n=38) and RNA-sequencing (RNA-seq) (n=30) on metastatic pRCC from the prospective AXIPAP trial cohort. Results: unsupervised clustering identified 2 "TME subtypes", in each of the cohorts : the “immune-enriched” and the “immune-low”.Within AXIPAP trial cohort, the “immune-enriched” cluster was significantly associated with a worse prognosis according to the median overall survival to 8 months (95%CI, 6-29) versus 37 months (95%CI, 20-NA,p=0.001).The 2 immune signatures, Teff and JAVELIN Renal 101 Immuno signature, predictive of response to immune checkpoint inhibitors (CPI) in ccRCC, were significantly higher in the “immune-enriched” group (adjusted p<0.05). Finally, 5 differentially overexpressed genes were identified, corresponding mainly to B lymphocyte populations. Conclusion : for the first time, using RNA-seqand IHC, we have highlighted a specific immune TME subtype of metastatic pRCC, significantly more infiltrated with T and Bimmune population. This “immune-enriched” group appears to have a worse prognosis and could have a potential predictive value for response to immunotherapy, justifying the confirmation of these results in a cohort of metastatic pRCC treated with CPI and incombination with targeted therapies

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Pereira, Joana Fernandes Alberto Wilton. „Gene regulation in the tumor-immune system microenvironment crosstalk“. Doctoral thesis, 2021. https://hdl.handle.net/10216/138542.

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Huang, Wei Tzu, und 黃尉慈. „The Immune Regulation of Early Tumor Microenvironment: A Microarray Study“. Thesis, 2014. http://ndltd.ncl.edu.tw/handle/15183566451945413850.

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Young, Park Gloria Seo. „Characterization of immune cell distributions in mouse models of spontaneous breast tumors“. Thesis, 2016. https://hdl.handle.net/2144/14617.

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As immunotherapy grows in popularity as a cancer treatment option, we need to further understand how immune cells interact with the tumor microenvironment and influence tumor progression. The goal of this thesis was to characterize the different immune, cellular, and structural components within the breast tumor tissues of two orthotopic (MCaP0008 and M3C) and one spontaneous (MMTV-PyVT) murine models of immunogenic breast cancer. Identification of the tumor components in question, including CD3+ lymphocytes, CD11b+ myeloid cells, CD31+ endothelial cells, αSMA+ cancer associated fibroblasts, Ki67+ cells, cleaved caspase-3+ cells, collagen-1, and hyaluronan, were done by immunohistochemistry (IHC)-immunofluorescence (IF) staining of frozen tumor tissues with appropriate antibodies and imaging with multispectral confocal microscopy. Quantification and further data analysis were performed using a custom MATLAB program designed by Dr. Mei Rosa Ng. Gaining understanding of these stromal compositions will allow for better utilization of these breast cancer mouse models in future experiments.
2019-10-31

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„Loss of LKB1 Leads to Alteration of the Immune Microenvironment in Non-Small Cell Lung Cancer“. Master's thesis, 2015. http://hdl.handle.net/2286/R.I.29807.

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abstract: The majority of non-small cell lung cancer (NSCLC) patients (70%) are diagnosed with adenocarcinoma versus other histological subtypes. These patients often present with advanced, metastatic disease and frequently relapse after treatment. The tumor suppressor, Liver Kinase B1, is frequently inactivated in adenocarcinomas and loss of function is associated with a highly aggressive, metastatic tumor (1). Identification of the mechanisms deregulated with LKB1 inactivation could yield targeted therapeutic options for adenocarcinoma patients. Re-purposing the immune system to support tumor growth and aid in metastasis has been shown to be a feature in cancer progression (2). Tumor associated macrophages (TAMs) differentiate from monocytes, which are recruited to the tumor microenvironment via secretion of chemotaxic factors by cancer cells. We find that NSCLC cells deficient in LKB1 display increased secretion of C-C motif ligand 2 (CCL2), a chemokine involved in monocyte recruitment. To elucidate the molecular pathway regulating CCL2 up-regulation, we investigated inhibitors of substrates downstream of LKB1 signaling in A549, H23, H2030 and H838 cell lines. Noticeably, BAY-11-7082 (NF-κB inhibitor) reduced CCL2 secretion by an average 92%. We further demonstrate that a CCR2 antagonist and neutralizing CCL2 antibody substantially reduce monocyte migration to NSCLC (H23) cell line conditioned media. Using an in vivo model of NSCLC, we find that LKB1 deleted tumors demonstrate a discernible increase in CCL2 levels compared to normal lung. Moreover, tumors display an increase in the M2:M1 macrophage ratio and increase in tumor associated neutrophil (TAN) infiltrate compared to normal lung. This M2 shift was significantly reduced in mice treated with anti-CCL2 or a CCR2 antagonist and the TAN infiltrate was significantly reduced with the CCR2 antagonist. These data suggest that deregulation of the CCL2/CCR2 signaling axis could play a role in cancer progression in LKB1 deficient tumors.
Dissertation/Thesis
Masters Thesis Biology 2015

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Chen, Jie-Yi, und 陳傑宜. „Treating pancreatic ductal adenocarcinoma with oncolytic adenovirus and analyzing the immune properties in tumor microenvironment“. Thesis, 2019. http://ndltd.ncl.edu.tw/handle/92re8n.

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碩士
國立東華大學
生命科學系
107
Pancreatic ductal adenocarcinoma (PDAC) with an extremely low 5-year survival rate is one of the most disastrous diseases. Some advanced therapies have been developed to meet the unmet medical need. However, owing to the immunosuppressive microenvironment and chemoresistant fibrous barriers, the efficacies of treatments are limited. Because oncolytic viruses (OVs) can specifically lyse the tumor cells and trigger anti tumor immune responses, they may provide alternative strategy as the cancer-targeted and immune therapy. In this study, oncolytic adenoviruses serotype C5 and F41 (OAdV-C5 and -F41), which are specific, replicative, relatively safe and easily manipulated, were used to treat PDAC in vitro and in vivo. OAdV-C5 and -F41 were isolated and characterized using RT-qPCR and immunofluorescence assay. As infected by OAdV-C5 or -F41, the tested cancer cells, especially in pancreatic malignancy, were more prone to be killed than normal cells. Therefore, the orthotopic PDAC nude mouse model was established and used to evaluate the efficacy of intratumor injection of OAdV-C5 or -F41. It was found that the tumor size was reduced in a dose dependence, and overall survival rates of the mice were accordingly prolonged 1.5-fold. Moreover, the infiltration of monocytes, NK cells and B cells in the PDAC microenvironment was increased in a time-dependent manner by analyzing the gene expression of Mcp1 (monocytes), Ncr1 (NK cells) and Cxcl13 (B cells). It was found that the expression of PD-1 was down-regulated at 2-wk post OAdV treatment. Afterwards PD-1 level increased at 1-3 months post treatment, and it could lead to the immunosuppression and attenuate the tumor-eliminating capability of immune cells. Taken together, the OAdV has the therapeutic potential, and its combination with immune checkpoint inhibitors may be promising for PDAC treatment.

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Partlová, Simona. „Nádorové mikroprostředí a význam protinádorové imunity pro klinický průběh lidských nádorových onemocnění“. Doctoral thesis, 2017. http://www.nusl.cz/ntk/nusl-304030.

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Cancer development and progression vary depending on tumor type, localization, invasion, immunogenicity and the ability of immune system to become activated. There are frequent interactions between tumor cells and immune cells, occuring locally at the site of primary tumor or distally through paracrine signalling of various mediators and cytokines. The main subject of this PhD thesis is to study key factors and aspects of immune response in cancer patients. In the first part, we analyzed immune cells infiltrating tumor tissues of ovarian cancer patients at different stages of disease. We focused on the dynamics of immune response, primarily on frequency of individual T lymphocyte populations in peripheral blood and tumor infiltrating T lymphocytes in tumors of early and advanced stages of ovarian cancer. We found that during disease progression there is a gradual decrease of proinflammatory Th17 and Th1 immune responses and a specific recruitment of regulatory T cells to the tumor site, which results in a significant immune suppression in the tumor microenvironment. In the second part, we demonstrated that the character of immune response in HPV-positive head and neck cancer patients is very different from the patients with tumors not associated with HPV infection. In HPV-positive patients, significantly...

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