Tesis sobre el tema "Glioma, microenvironment, tumor associated macrophages"

Glioblastoma (GBM) es el tumor más agresivo dentro de la clasificación de gliomas y actualmente no tiene cura. Dado que hasta el 30% de las células en GBM son microglia y macrófagos (GAMMs desde ahora, por sus siglas en ingles), decidimos estudiarlos para ayudar en el diseño de posibles futuras inmunoterapias. El primer gran descubrimiento de esta tesis es la presencia de GAMM en las pseudo-empalizadas (PPs, por sus siglas en inglés) in GBM humano. Estas estructuras se cree que pueden contribuir a la capacidad de invasión del tumor, por lo que el papel de los GAMM en este microambiente puede ser crucial. En concreto, describimos que los GAMMs viajan a través de las PPs hacia su foco necrótico, de forma inversa a las células tumorales. Además, los GAMMs parece que ganan persistencia celular, es decir que viajan de forma directa, bajo las condiciones hipóxicas de las PPs y se desplazan de manera haptotáctica, siendo las fibras GFAP+ su sustrato para avanzar. Al llegar al área necrótica, cambian su fenotipo y fagocitan material del tumor, incluyendo fragmentos GFAP+ y núcleos celulares. En segundo lugar, en cultivos celulares hemos inducido la translocación de NF-κB p65 para aumentar la facogitosis de células de glioma (C6) por parte de microglia primaria de rata. Además, por medio de líneas celulares inmortalizadas (BV-2 y GL261) hemos descrito una serie de supuestos pasos que pueden ser importantes en el proceso de fagocitosis, así como la distribución de determinados receptores (CD11b y CD16/32) durante estas etapas. Asimismo, describimos la distribución de Iba-1 en los procesos de interacción fagocítica en modelos animales de glioma. Por último, hemos probado dos estrategias de inmunoterapia en un modelo murino de GBM (ratones C57/BL6 inyectados con células GL261 intracranealmente), descubriendo que estas inmunoterapias tienen efectos distintos: mientras la neutralización de CD47 pareció no ser efectiva, neutralizar SIRP1α parece ser beneficiosa. Así, los animales del grupo denominado CD47 no mostraban un aumento de su supervivencia comparados con los ratones control, incluso mostraban pérdida de peso, indicador de que la terapia pueda producir efectos secundarios sistémicos. Por otro lado, el bloqueo de SIRP permitió el incremento del peso corporal de los animales durante el experimento, y, de hecho, disminuyó el número de células en el centro del tumor incrementando la capacidad fagocítica de los GAMMs en las zonas periféricas de invasión, sin interferir en su capacidad de infiltrar en el tumor. En conclusión, esta tesis contribuye a una mejor comprensión de la función de los GAMMs en GBM y su capacidad fagocítica intrínseca, posiblemente ayudando al desarrollo de nuevas herramientas terapéuticas para luchar contra este tumor.
Glioblastoma (GBM) is the most aggressive form of glioma and currently has no cure. Given that around 30% of the cells within the tumor are microglia/macrophages (glioma- associated microglia/macrophages or GAMM from now on), we decided to study them in order to shed light in possible future immunotherapies. The first discovery during this investigation was the presence of GAMM in pseudopalisades (PPs) of human GBM. These structures are thought to be very important in the contribution of the tumor invasiveness, therefore the knowledge of the role of GAMMs here might be crucial. Particularly, GAMMs were found to be traveling through the PPs towards the necrotic focus, contrasting with the tumor cells. Moreover, the myeloid cells seem to gain cellular persistence with the hypoxic gradient and travel in a haptotactic manner using the gradient of glioma cells as a cue. When they reach the necrotic focus, they shift their phenotype and phagocytose tumor material, including GFAP+ fragments and nuclei. Secondly, by means of cell cultures we achieved to translocate p65 NF-κB and promote phagocytosis of tumor glioma cells (C6) by primary microglia. Moreover, using cell lines (BV-2 and GL261) we described the putative steps of phagocytosis and the distribution of some receptors (CD11b and CD16/32) involved in the process of phagocytosis. Importantly, the distribution of Iba-1 in interacting GAMMs was also defined in the animal models. Finally, we tested two immunotherapy strategies in a immunocompetent GBM animal model (C57/BL6 intracraneally inoculated with GL261 cells), and discovered that both immunotherapies have different outcomes: while CD47 neutralizing antibody seemed to be non-effective, neutralizing SIRP1α had a beneficial outcome. This way, anti-CD47 treated animals did not have any increase on survival rate than control groups; and they showed decreased bodyweight throughout the experiment, suggesting that the therapy had some systemic side effects. On the other hand, blocking SIRP1α allowed the increase of the bodyweight of the animals throughout the experiment, and decreased the cellularity of the tumor core by increasing the phagocytic activity of GAMM at the peripheral area of tumor invasion without interfering in their infiltration capacity. In all, this thesis contributes to a better understanding of the role of GAMMs in GBM and the intrinsic phagocytic capacity they can play, possibly helping in the development of immunotherapeutic tools to fight this fatal tumor.

BACKGROUND: Prostate cancer is the most common cancer among Swedish men. For patients with metastatic prostate cancer the standard therapy is castration, a treatment that initially provides symptomatic relief but unfortunately is not curative. New therapeutic targets for advanced prostate cancer are therefore needed.  Prostate cancers are composed of tumor epithelial cells as well as many non-epithelial cells such as cancer associated fibroblasts, blood vessels and inflammatory cells.  Many components of the tumor microenvironment such as tumor associated macrophages and angiogenesis have been shown to stimulate tumor progression. This thesis aims to explore mechanisms by which the local environment influences prostate tumor growth and how such mechanisms could be targeted for treatment. MATERIALS AND METHODS: We have used animal models of prostate cancer, in vitro cell culture systems and clinical materials from untreated prostate cancer patients with long follow up. Experiments were evaluated with stereological techniques, immunohistochemistry, western blotting, quantitative real-time PCR, PCR arrays and laser micro dissection. RESULTS: We found that the presence of a tumor induces adaptive changes in the surrounding non-malignant prostate tissue, and that androgen receptor negative prostate tumor cells respond to castration treatment with temporarily reduced growth when surrounded by normal castration-responsive prostate tissue. Further, we show that macrophages are important for prostate tumor growth and angiogenesis in the tumor and in the surrounding non-malignant tissue. In addition, the angiogenesis inhibitor Pigment epithelium-derived factor (PEDF) was found  to be down-regulated in metastatic rat and human prostate tumors. Over-expression of PEDF inhibited experimental prostate tumor growth, angiogenesis and metastatic growth and stimulated macrophage tumor infiltration and lymphangiogenesis. PEDF was found to be down-regulated by the prostate microenvironment and tumor necrosis factor (TNF) α. CONCLUSIONS: Our studies indicate that not only the nearby tumor microenvironment but also the surrounding non-malignant prostate tissue are important for prostate tumor growth. Both the tumor and the surrounding non-malignant prostate were characterized by increased angiogenesis and inflammatory cell infiltration. Targeting the surrounding prostate tissue with castration, targeting tumor associated macrophages, or targeting the vasculature directly using inhibitors like PEDF were all shown to repress prostate tumor growth and could prove beneficial for patients with advanced prostate cancer.

"Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancer, with very high mortality rate. Tumor-associated macrophages (TAMs) can represent up to 70% of ATC’s tumor mass, making them an interesting target for novel therapies. In this thesis, the aim was to further understand the crosstalk between ATC and TAMs. To achieve that, cell surface proteomics of two ATC cell lines (C3948 and T235), in mono- or co-culture with THP-1-derived macrophage-like cells, was performed. The protein Spry-4, an inhibitor of the MAPK-ERK pathway, was found to be downregulated in C3948 cells upon co-culture. Spry-4 protein levels were further evaluated by Western blot; only T235 in co-culture showed a trend for downregulation, although without statistical significance.(...)"
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Macrophages are pivotal orchestrators of tumor-promoting inflammation and promising targets for new anti-cancer therapies. To identify new molecular players underlying their pro-tumoral activities, we analyzed the phosphoproteoma of tumor associated macrophages (TAMs) isolated from murine fibrosarcoma. We identified the protein TRIM28, a pleiotropic molecule that is known to be involved in the dynamic organization of chromatin, and we characterized the signaling pathway driving its phosphorylation in response to inflammatory signals and its impact on LPS-induced gene expression. We explored in vivo the functional relevance of TRIM28 and found a significant reduction of colitis associated cancer lesions in mice lacking TRIM28 in intestinal epithelial cells. Single cell RNAseq analysis pointed out alterations of both immune and intestinal cell populations during the transition from colitis to cancer, that are dependent on TRIM28. Overall, these results identify TRIM28 as a new molecular target at the crossroads between inflammation and cancer. Beyond contributing to tumorigenesis, TAMs can profoundly affect the response to anti-cancer therapies. We investigated their impact on EPZ-6438, an inhibitor of the histone methyltransferase EZH2 that has recently entered in clinical trials due to the anti-proliferative effects shown on malignant pleural mesothelioma cells (MPM). We generated an MPM spheroid model that recapitulates in vitro, both monocyte recruitment in tumor and their functional differentiation towards a TAM-like phenotype (Mo-TAMs) capable of promoting tumor cell proliferation and spreading. Prolonged treatment of MPM spheroids with EPZ-6438 enhances both Mo-TAMs recruitment and pro-tumor phenotype expression, thereby limiting the anti-proliferative effects due to EZH2 inhibition in MPM cells. These findings indicate that strategies of TAM depletion should be combined with EPZ-6438 to improve the therapeutic efficacy of pharmacological EZH2 inhibition.

Tumour microenvironment is a complex network of cells, including immune cells, with a role in tumorigenesis and metastatic spread. Among the immune cells, M2-polarized Tumour-Associated-Macrophages (M2-TAMs) show immunosuppressive activities by expressing inflammatory molecules, thus promoting tumorigenesis. Triple-Negative Breast Cancer (TNBC), lacking both hormone receptors (i.e. Progesterone Receptor [PgR] and Estrogen Receptor [ER]) and Human Epidermal growth factor Receptor 2 (HER2), is associated with poor prognosis and high probability of distant metastases. In TNBC, a large number of infiltrating M2-TAMs is positively correlated to higher risk of metastases and lower rates of Disease-Free Survival” (EFS) and Overall Survival (OS). Prune-1 belongs to DHH (Asp-His-His) phosphoesterase superfamily with an exopolyphosphatase activity. The overexpression of Prune-1 is correlated with metastases and poor prognosis in several tumours including Breast Cancer (BC). Prune-1 was also found to induce Epithelial-Mesenchimal-Transition (EMT) and metastatic dissemination throught the enhancement of canonical TGF-β signaling by counterbalancing its inhibition operated by NDPK-A. Further evidences suggested that lung cancer progression is driven by Prune-1 through the canonical WNT signaling pathway in both autocrine and paracrine manner via Wnt3a cytokine secretion, thus suggesting a potential role for Prune-1 also in extracellular environment. Here, we identified Prune-1 protein as overexpressed in TNBC patients and positively correlated to lynphnode metastases, inflammatory pathways and infiltrating M2-TAMs (CD68+/CD163+). Furthermore, we developed a Genetically Engineered Mouse Model (GEMM) of metastatic TNBC over-expressing both Prune-1 and Wnt-1 in mammary glands (MMTV-Prune1/Wnt1). We found Prune-1 to recruit TAMs and to enhance their polarization toward a pro-tumorigenic M2-phenotype in the tumour microenvironment, thus promoting lung metastases in this GEMM. We also show that Prune-1 takes part to the communication between TNBC cells and TAMs through intracellular pathways activation (i.e., TGF-b and NF-kB), but also in a paracrine manner by inducing the secretion of inflammatory cytokines (e.g., IL-17F and IL-28) and modulating the exosome protein contents. Finally, we found an anti-Prune-1 drug (AA7.1) with the ability to reduce the primary tumour growth by reducing the percentage of M2-TAMs in orthotopic xenograft immunocompetent models of TNBC. In conclusion, our GEMM of metastatic TNBC driven by Prune-1 will be a useful source for future immunotherapy pre-clinical trials targeting M2-polarized TAMs within the tumour microenvironment to inhibit distant metastases in TNBC with poor prognosis.

O carcinoma espinocelular de boca (CEB) é a neoplasia maligna mais comum da cavidade oral, correspondendo à aproximadamente 94% dos casos dessa região. Apesar dos diversos estudos moleculares e celulares do CEB, a taxa de sobrevida dos pacientes é de aproximadamente 50%, devido principalmente ao tamanho do tumor, metástase em linfonodos regionais, grau de diferenciação das células e sítio anatômico. O microambiente tumoral do CEB, é extremamente complexo e diversificado, tendo como característica principal um estado inflamatório crônico imunossupressivo. Este microambiente é sustentado pela liberação de diferentes citocinas inflamatórias, como IL-6, TNF- - atividades exercidas tanto pelas células tumorais quanto pelas estromais. Dentre essas atividades, tem sido relatado na literatura que as citocinas inflamatórias são capazes de aumentar a migração e a capacidade de invasão das células tumorais. Entre as células estromais, os macrófagos são as mais abundantes e participam da manutenção do microambiente tumoral. De acordo com o estímulo, podem ser polarizados M1, com papel pró-inflamatório e antitumoral, e M2, com papel anti-inflamatório e pró-tumoral. O objetivo desta tese foi compreender o papel dos macrófagos no prognóstico de CEB e das citocinas inflamatórias IL-6, TNF- - linhagens celulares de CEB. Para verificar o papel dos macrófagos no prognóstico, foi realizada uma revisão sistemática na qual foram incluídos apenas os estudos que utilizavam amostra de pacientes com CEB e avaliavam o prognóstico com marcadores para macrófagos. Foi observado que maiores concentrações de macrófagos CD68+ e CD163+ estavam relacionados com pior prognóstico de pacientes com CEB, embora não tenha sido possível concluir qual região tumoral a presença destas células seja mais importante 7 para o desfecho. Para analisar o papel das citocinas inflamatórias IL-6, TNFILensaios in vitro utilizando duas linhagens celulares, SCC25 e Cal27, em condições promotoras de migração sob a influência dessas citocinas. Foi observado que a citocina IL-6 foi capaz de aumentar a velocidade de migração e a direcionalidade tanto da SCC25 quanto da Cal 27 e que esta melhora na capacidade migratória ocorreu através de um crosstalk entre a via de sinalização relacionada a IL6 (STAT3) e a via reguladora de migração celular, Rho GTPase Rac1. Estes dados reforçam o papel do microambiente tumoral no processo de progressão tumoral e sugerem potenciais alvos terapêuticos como a modulação do perfil da população de macrófagos e o papel de interleucinas no controle de invasão tecidual e metástase.
Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the oral cavity, corresponding to approximately 94% of the cases in this region. Despite the diverse molecular and cellular studies of OSCC, the patient survival rate is approximately 50%, mainly due to tumor size, regional lymph node metastasis, cell differentiation and anatomic site. The OSCC tumor microenvironment is extremely complex and diverse, with the main characteristic being an immunosuppressive chronic inflammatory state. This microenvironment is supported by the release of different inflammatory cytokines, such as IL-6, TNF- - and enhance the activities of both tumor and stromal cells. Among these activities, it has been reported in the literature that inflammatory cytokines are capable of increasing migration and invasiveness of tumor cells. Among stromal cells, macrophages are the most abundant and participate in the maintenance of the tumor microenvironment. According to the stimulus, macrophages can be polarized in M1, with pro-inflammatory and anti-tumoral role, and M2, with antiinflammatory and pro-tumoral role. Thus, the aim of this thesis was to evaluate the role of macrophages in the prognosis of OSCC and the influence of inflammatory cytokines IL-6, TNF- - OSCC cell lines. To assess the role of macrophages in the prognosis, a systematic review was conducted in which only studies using a sample of OSCC patients were evaluated and the prognosis was evaluated with macrophage markers. It was observed that higher concentrations of CD68 + and CD163 + macrophages were related to worse prognosis in patients with OSCC, although it was not possible to conclude which tumor region the presence of these cells is more important for the outcome. In order to analyze the role of the inflammatory cytokines IL-6, TNF- - atory 9 behavior of OSCC cells, in vitro assays using two cell lines, SCC25 and Cal27, were performed in migration-promoting conditions under the influence of these cytokines. It was observed that IL-6 was able to increase the speed migration and directionality of both SCC25 and Cal 27 and that this improvement in migratory capacity occurred through a crosstalk between the IL6-related signaling pathway (STAT3) and cell migration-related pathway, RhoGTPase Rac1. These data reinforce the role of the tumor microenvironment in the tumor progression process and suggest potential therapeutic targets such as the modulation of the profile of the macrophages population and the role of interleukins in the control of tissue invasion and metastasis.

Introdução: Indivíduos sobreviventes à sepse apresentam maior mortalidade à longo prazo e maior risco de apresentar infecções oportunistas. Existem evidências clínicas e experimentais de desregulação imune no estado pós-sepse. Essas alterações apresentam semelhança com aquelas encontradas no microambiente tumoral, estando relacionadas à imunossupressão. O presente trabalho avaliou o papel de macrófagos associados ao tumor (TAM) em modelo de progressão tumoral em camundongos sobreviventes à sepse. Materiais e Métodos: Camundongos C57/BL6 foram submetidos a ligadura e punção cecal (CLP) e tratados com ertapenem (20 mg/kg, i.p., 6 horas após CLP e 12/12 h por 3 dias). Os animais sobreviventes de sepse eram inoculados com células de melanoma B16-F10 (30 mil, s.c., 15 dias após a CLP). Animais naïve foram usados como controle. Foram avaliadas a progressão tumoral, sobrevida e formação de metástases espontâneas à distância. No D+14, animais foram sacrificados para mensuração do acúmulo de TAM por citometria de fluxo (CD45+F4/80+CD206+) e de citocinas no soro e no tumor por ELISA (IFN-?, IL-10, TNF-?, TGF-?, CCL2, CXCL12). Macrófagos derivados de medula óssea de animais pós-CLP ou naïve foram coinoculados com células B16 para avaliação de progressão tumoral e sobrevida. TAM de animais naïve ou pós-CLP foram isolados através de gradiente de Percoll seguido de adesão seletiva e o RNA foi isolado para análise diferencial de expressão gênica por microarray. Para avaliação da participação da via CXCL12/CXCR4 foi realizada sua inibição com o AMD3100, antagonista de CXCR4 (5 mg/kg, i.p., D+10 e D+14). Foi avaliada a progressão tumoral, sobrevida, acúmulo de TAM e proliferação extramedular de TAM no D+14. Resultados: Animais sobreviventes de sepse apresentaram aumento de progressão tumoral (após 15, 30 e 60 dias da CLP), aumento da carga de metástases (após 15 dias da CLP) e redução de sobrevida. Foi detectado o aumento de TAM nos animais pós-CLP, associado a maior marcação de Ki67, em comparação com animais naïve no D+14. Verificamos aumento das concentrações séricas de TGF-?, CXCL12, CCL2 e TNF-?. Camundongos naïve que coinoculados com macrófagos derivados de medula óssea de animais pós-CLP apresentaram aumento de progressão tumoral e redução de sobrevida em comparação com o grupo controle. TAM de animais pós-CLP apresentaram menor expressão de genes relacionados ao MHC-II e genes relacionados à ativação leucocitária. A inibição de CXCL12/CXCR4 preveniu a progressão tumoral induzida por sepse, com menor acúmulo de TAM e menor presença de TAM Ki67+. Conclusões: O estado pós-sepse promove a progressão tumoral de melanoma B16 em camundongos, o qual foi associado a aumento de 12 TAM. A via CXCL12/CXCR4 participa do processo de acúmulo de TAM nesse modelo experimental.
Background: Survivors from sepsis present higher long-term mortality and increased risk of opportunistic infections. There is clinical and experimental evidence for an immunosuppressive immune dysregulation in post-sepsis. These alterations are similar to those found in tumor microenvironment. The present work assessed the role of tumorassociated macrophage (TAM) in a model of tumor progression in sepsis-surviving mice. Materials and Methods: C57/BL6 mice were submitted to cecal ligation and puncture (CLP) and treated with ertapenem (20 mg/kg, ip. - 6 h after CLP and then each 12 h for 3 days). Sepsis surviving mice were inoculated with B16-F10 melanoma cells (30,000, sc., 15 days after CLP). Naïve mice were used as controls. Tumor progression, survival and distant spontaneous metastasis were evaluated. Mice were killed at D+14 for TAM measurement through flow cytometry (CD45+F4/80+CD206+) and for cytokines (IFN-?, IL-10, TNF-?, TGF-?, CCL2, CXCL12) quantification by ELISA. Bone marrow-derived macrophage (BMDM) were isolated and co-inoculated together with B16 melanoma cells for tumor progression and survival evaluation. TAM from naïve or post-sepsis mice were isolated through Percoll gradient (70/30) followed by selective adhesion. The RNA was isolated for gene expression analysis using microarray assay. To evaluate the role of CXCL12/CXCR4, we used the specific antagonist AMD3100 (5 mg/kg, ip., at D+10 and D+14) and assessed tumor progression, survival and TAM accumulation at D+14. Results: Sepsis-surviving mice showed increased tumor progression (15, 30 or 60 days after CLP), higher metastatic burden (15 days after CLP), and less overall survival. TAM were increased in post-sepsis mice at D+14. We found increased serum levels of TGF-?, CXCL12, CCL2 e TNF-?. Naïve mice inoculated with BMDM from post-sepsis and B16 cells showed higher tumoral progression and less survival, when compared to the control group. TAM from post-sepsis showed decreased expression of MHC-II related genes and genes related to leukocyte activation. The inhibition of CXCL12/CXCR4 prevented the post-sepsis-induced tumor progression, with less TAM accumulation and reduced expression of Ki67 in TAM. Conclusions: The post-sepsis state promotes the progression of B16 melanoma in mice, which was associated with an increase in TAM accumulation. CXCL12/CXCR4 mediates TAM accumulation in this experimental model.

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

sfondo: Il tumore associato macrofagi (TAMs) sono classificati in pro-infiammatorio M1 TAM, e anti-infiammatori M2 TAM. Le cellule staminali glioma (GSC) polarizzano TAM in M2 fenotipo che promuovono la progressione glioma. E voluto studiare l'implicazione della infiltrazione totale e differenziale TAM in gliomi a basso grado (LGG) e in gliomi a alto grado (HGG). Inoltre abbiamo studiato l'effetto di esosomi rilasciati da M1 TAM sul destino delle cellule di glioma. Metodologia: immunoistochimica è stata eseguita su 11 campioni accoppiati ottenuti da casi di progressione da LGG a HGG. iNOS è stato utilizzato come marcatore per M1 e CD163 come marcatore per M2. Negli esperimenti in vitro, abbiamo polarizzato monociti umani U937 in M1 fenotipo, poi abbiamo isolato i esosomi dal mezzo M1 condizionato con centrifugazione e filtrazione. Dopo aver aggiunto esosomi M1 di cellule di glioma U251, abbiamo studiato l'attivazione delle cellule glioma con il saggio MTT e abbiamo studiato l'apoptosi delle cellule glioma con la citofluorimetria. Abbiamo usato annessina V come marcatore di apoptosi precoce e ioduro di propidio come marcatore di ritardo apoptosi. Risultati: immunoistochimica ha mostrato uno squilibrio M1 / ​​M2 con la maggioranza di essere M2 sia LGG e HGG. La M2 infiltrazione superiore, la prima è stata la progressione. Gli esperimenti in vitro hanno rivelato l'effetto antitumorale di exosomes M1 che erano in grado di inibire la proliferazione e di indurre apoptosi precoce e tardiva delle cellule di glioma. Conclusione: i nostri dati confermano il ruolo di M2 TAM nella progressione di glioma ed espostano il ruolo tumoricidale di esosomi M1 contro i gliomi
Background: The tumor associated macrophages (TAMs) are classified into pro-inflammatory M1 TAMs, and anti-inflammatory M2 TAMs. Glioma stem cells (GSCs) polarize TAMs into M2 phenotype which promote glioma progression. We aimed to study the implication of the total and differential TAM infiltration in low grade glioma (LGG) and high grade glioma (HGG). Also we investigated the effect of exosomes released from M1 TAMs on the fate of glioma cells. Methodolgy: Immunohistochemistry was performed on 11 paired specimens obtained from cases progressing from LGG to HGG. iNOS was used as a marker for M1 and CD163 as a marker for M2. In the in-vitro experiments, we polarized human monocytes U937 into M1 phenotype, then we isolated the exosomes from the M1conditioned medium by centrifugation and filtration. After adding M1 exosomes to U251 glioma cells, we studied the glioma cell activation by MTT assay and we studied the glioma cell apoptosis by flow-cytometry. We used Annexin V as a marker of early apoptosis and propidium iodide as a marker of late apoptosis. Results: immunohistochemistry showed an M1/M2 imbalance with the majority being M2 in either LGG and HGG. The higher M2 infiltration, the earlier was the progression. The in-vitro experiments revealed the anti-tumor effect of M1 exosomes which were able to inhibit the proliferation and to induce early and late apoptosis of glioma cells. Conclusion: our data confirmed the role of M2 TAMs in glioma progression and exhibited the tumoricidal role of M1 exosomes against gliomas.

碩士
國立中山大學
醫學科技研究所
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Deregulated proliferation of tumor is generally associated with altered energy metabolism. A high rate of anaerobic glycolysis in solid tumors contributes to an acidification of pH to 6.7–7.2 in the tumor microenvironment due to lactate accumulation. Lactate modulates immune cell function and promotes tumor invasion and metastasis. However, a particular challenge is to clean lactate in tumor microenvironment. Here we report exploitation of tumor lactate production in designing a methylcellulose (MC) hydrogel incorporating lactate oxidase (LOX), and MC-LOX hydrogel was used controlled the degree of swelling, water uptake and subsequent degradation of the hydrogels and release rate of LOX. After 3 days, MC-LOX hydrogel was exemplified by achieving 91.7 % lactate consumption at pH 6.7, compared with control groups. Furthermore, the conversion efficiency and unique NO synthase function of tumor-supportive macrophages (M2) to tumor-suppressive macrophages (M1) was increased 16.46 % and 18.97 %, respectively. This is relevant to optimizing parameters for immune medicine applications.

The tumor microenvironment (TME) plays an important role in tumor progression and therapy resistance. Within the TME, tumor associated macrophages (TAM) can represent up to 50% of all components, being usually associated with the most aggressive breast cancer (BC) subtypes, such as Her2 overexpressing (HER2+) and triple negative. Several reports suggest that TAM contribute to the establishment of an immunosuppressive microenvironment, which enhances tumor immune evasion and progression . In this thesis, the ultimate aim was to contribute to characterize the TAM phenotypes associated with different BC subtypes. To attain that, a cell-based BC model was implemented, based on co-cultures of BC cell lines (BT474 and HCC1954, representing the Luminal B and HER2+ subtypes, respectively) and human blood derived monocytes. Cancer cell spheroids and monocytes were co-encapsulated in alginate and cultured in stirred-tank vessels for 7 days. After this time, monocytes differentiated into macrophages, as shown by the detection of CD206 and CD163. Moreover, high detection of CD206, CD163 and low detection of CD80 suggested differentiation into M2-related phenotypes. Further, when comparing the impact of the BC cell lines on monocyte differentiation, the M2-related markers detection was higher in macrophages cultured with HCC1954, suggesting that this cell line may induce more mature M2-like populations. These differences were corroborated by distinct cytokine secretion profiles in both co-cultures: while for HCC1954, IL-4, IL-13, VEGF and CCL2 were increased, for BT474 there was higher secretion of IL-10, CCL24 and TGF- β. Finally, fibroblasts were introduced into culture, to represent the tumor stroma. In their presence, macrophages showed higher expression of M2-like markers, compared with double co-cultures. In conclusion, in this thesis a 3D cell-based model to depict BC-macrophage crosstalk was established. Moreover, the microenvironment established in the model promoted naïve monocytes differentiation into M2-like phenotypes, suggesting that this may be a useful tool to understand the induction of different TAM phenotypes in distinct BC subtypes

Head and neck cancers are etiologically associated with smoking and alcohol consumption. Part of these tumors is induced by HPV and their incidence is increasing in the last decade. Patients with virally induced tumors have better prognosis even though they are usually diagnosed with tumors in advanced stage. One of the possible explanations may be better stimulation of the immune system by viral antigens. Macrophages are cells of the innate immune system which belong to professional phagocytes. They are called TAM upon infiltration to the tumor where they represent heterogeneous group of cells. Two main phenotypes are antitumor M1 and protumor M2 macrophages. TAMs are a major component of tumor microenvironment of many types of tumors, one of them are also head and neck cancers. In my thesis I focused on the immunohistochemical detection of M1 and M2 macrophages in the head and neck tumors of viral and non-viral etiology and at the same time RT-qPCR analyses of gene expression of macrophage-associated and/or immunosuppressive genes IDO1, ARG1, CD163, NOS2 a PTGS2 was performed. My data showed that HPV- negative tumors had higher number of M2 macrophages with typical markers CD163, ARG1 and PTGS2. It is known that patients with these tumors have worse prognosis of the disease. Due to high...

Infiltration of myeloid cells in the tumor microenvironment is often associated with enhanced angiogenesis and tumor progression, resulting in poor prognosis in many types of cancer. The polypeptide chemokine PK2 (Bv8, PROK2) has been shown to regulate myeloid cell mobilization from the bone marrow, leading to activation of the angiogenic process, as well as accumulation of macrophages and neutrophils in the tumor site. Neutralizing antibodies against PK2 were shown to display potent anti-tumor efficacy, illustrating the potential of PK2-antagonists as therapeutic agents for the treatment of cancer. In this study we demonstrate the anti-tumor activity of a small molecule PK2 antagonist, PKRA7, in the context of glioblastoma and pancreatic cancer xenograft tumor models. For the highly vascularized glioblastoma, PKRA7 was associated with decreased blood vessel density and increased necrotic areas in the tumor mass. Consistent with the anti-angiogenic activity of PKRA7 in vivo, this compound effectively reduced PK2-induced microvascular endothelial cell branching in vitro. For the poorly vascularized pancreatic cancer, the primary anti-tumor effect of PKRA7 appears to be mediated by the blockage of myeloid cell migration/infiltration. At the molecular level, PKRA7 inhibits PK2-induced expression of certain pro-migratory chemokines and chemokine receptors in macrophages. Combining PKRA7 treatment with standard chemotherapeutic agents resulted in enhanced effects in xenograft models for both types of tumor. Taken together, our results indicate that the anti-tumor activity of PKRA7 can be mediated by two distinct mechanisms that are relevant to the pathological features of the specific type of cancer. This small molecule PK2 antagonist holds the promise to be further developed as an effective agent for combinational cancer therapy.
Dissertation