Dissertations / Theses on the topic 'Preclinical oncology'

This thesis comprises studies on murine and human models of bladder carcinoma with the aim to develop novel immunogene therapies. On the basis of the results presented in this thesis, a clinical trial is underway. The potential of activating the immune system to combat cancer has long intrigued immunologists. Research has now been intensified and clinically effective treatments are beginning to materialize. We evaluated the induction of anti-tumor responses by inserting immunomodulating genes into tumor cells with adenovectors. Human biopsies and cell lines were positive for adenovirus attachment receptors, and cell lines were easily transduced. CD40L modified cells efficiently induced maturation of dendritic cell (DC). Phenotypical changes of AdCD40L transduced cells, such as increased apoptotic rate, upregulated MHC-I, Fas and TNFR may further strengthen the anti-tumor response. CD40L modified murine bladder cancer cells activated systemic immunity upon vaccination and in situ injections of AdCD40L inhibited tumor progression. Cytotoxic assays revealed the presence of cytotoxic T cells (CTLs) in vaccinated mice. Many tumors have developed ways to evade the immune system. Bladder carcinoma is associated with immune escape mechanisms like IL10 production. We demonstrated that immunosuppression by IL10 inhibited CTL function and that IL10 suppression may be reverted by AdCD40L therapy. In conclusion, AdCD40L therapy induces systemic immunity and inhibits tumor progression in murine models. The immunological mechanisms involve maturation of nearby DCs and CTL induction. AdCD40L therapy is effective despite immune escape mechanisms, e.g. IL10 secretion. The thesis argues for using AdCD40L immunogene therapy as a treatment of bladder carcinoma.

The aim of this body of work was to characterise a novel cisplatin drug delivery system and to develop new tools based on biophotonic imaging that could be used to enhance studies of drug delivery in vivo. Cucurbiturils (CB) are macrocycles which are formed by acid catalysed condensation of glycoluril and formaldehyde. The internal cavity of CB[7] encapsulates a single molecule of cisplatin and the hypothesis was that encapsulation would reduce thiol degradation of the drug. Drug sensitivity studies in vitro with the cisplatin-sensitive human ovarian cancer cell line, A2780, and a cisplatin-resistant derivative, A2780/cp70, showed that the CB[7] encapsulated cisplatin retained activity but that this encapsulation drug delivery system was not able to overcome resistance to platinum. However, when these cell lines were grown as subcutaneous xenografts in nu/nu mice, the encapsulated cisplatin was able to reduce the growth of A2780/cp70 tumours which are resistant to the maximum tolerated dose of cisplatin in vivo. One possible explanation of this observation is that encapsulation might alter the pharmacokinetics of cisplatin and a method for the detection of platinum in biological samples by ICP-MS was established and validated. This assay was sufficiently sensitive to detect the low levels of platinum present in mouse plasma 24 hours after administration of either free or encapsulated cisplatin. Plasma and tissue pharmacokinetics show that encapsulation had no effect on the peak plasma concentration of cisplatin but did reduce the rate at which cisplatin was cleared from the plasma. The increased plasma AUC of cisplatin resulted in a non-selective increase in the delivery of cisplatin to both tumour and normal tissues. However, there was no apparent increase in toxicity which could be explained by the fact that encapsulation, unlike an increase in the dose of free cisplatin, had no effect on the peak plasma concentration. Subcutaneous xenografts lack critical features of human tumours. The development of more complex models for use in drug development has been limited due to lack of a method for monitoring tumour growth. Biophotonic imaging was, therefore, investigated to determine whether it is sufficiently sensitive and reproducible to be able to evaluate growth of disseminated tumours in mice. The bioluminescent signal is dependent on the metabolism of luciferin by luciferase. Subcutaneous injection of luciferin was shown to produce a consistent signal in all injected mice. The bioluminescent signal was transient but reached a maximum intensity 6 minutes after injection and remained stable for about 4 minutes which defined the window during which measurements were taken. Sensitivity was shown to be dependent on the level of expression of luciferase by the cells. Injection of commercially available HCT116Luc cells, where the luciferase gene was inserted by a lentiviral system, was shown to allow detection of 10,000 cells in the lungs of mice. This sensitivity was about 10 fold greater than was obtained by lipofectamine based gene transfection. When HCT116Luc cells were grown as subcutaneous xenografts in mice, an exponential growth pattern was easily detected by bioluminescence imaging and the reproducibility between mice was comparable to that routinely obtained by calliper measurements. Activity of encapsulated cisplatin was determined in a model of disseminated ovarian cancer. Rab25, a member of the RAS oncoprotein superfamily, is up-regulated in around 80% of ovarian cancer samples compared to normal ovarian epithelium. Rab25 contributes to tumour progression by enabling the tumour cells to invade the extracellular matrix by altering the trafficking of integrin. Transfection of Rab25 into A2780 cells results in cells that can grow in the peritoneal cavity of mice. A2780-Rab25 cells were 4 fold resistant to cisplatin in vitro which confirms a previous observation that Rab25 expression in A2780 makes them less sensitive to the induction of apoptosis in response to stress. A2780-Rab25 cells that express the luciferase gene (A2780-Rab25Luc) were injected into the peritoneal cavity of mice and growth was measured by biophotonic imaging. Exponential growth was clearly apparent at a stage at which no obvious abdominal distension was apparent. The disseminated A2780-Rab25Luc tumour xenografts were less sensitive to cisplatin than are subcutaneous xenografts of A2780. This is the first study that suggests that Rab25 over-expression results in reduced drug sensitivity in vivo. In contrast, a very significant growth inhibition was observed when mice were treated with an equivalent dose of encapsulated cisplatin regardless of whether it was administered by the intraperitoneal or subcutaneous route. These results are very encouraging since they confirm the enhanced activity of encapsulated cisplatin and also demonstrate the value of biophotonic imaging for measurement of tumour growth in vivo. Pharmacodynamic measures of drug activity in vivo in animal models are often based either on measures of surrogate tissue response or on single measures on tumour tissue removed at the end of the experiment. Biophotonic imaging in vivo allows the translation of reporter assays used in cell lines in vitro to studies of tumour response in vivo. A plasmid was prepared that links the p53 transcriptional response element to the luciferase gene and it was then transfected in to A2780 cells which express wild type p53. Stable transfectants of A2780p53Luc were treated with cisplatin, doxorubicin and paclitaxel and induction of p53 determined by bioluminescence and confirmed by Western blotting. A very low bioluminescent signal was present in untreated cells and a clear dose dependent increase in bioluminescence was seen in response to all three drugs. When A2780p53Luc cells were grown as subcutaneous xenografts the bioluminescent signal was significant in untreated tumours but was markedly increased 24 hours after treatment of the mice with cisplatin. Induction of p53 in the tumours was confirmed by immunohistochemistry and this also confirmed significant expression of p53 in untreated tumours. The possible implications of these findings for the improved delivery of cisplatin are discussed.

Sarcomas are a group of malignancies characterized by their bad prognosis and the absence of effective treatments. Median survival of advanced sarcoma patients is only one year in spite of treatment. Therefore, it is mandatory to identify new therapeutic strategies. Our hypothesis is that the inhibition of angiogenesis and the mTOR pathway in sarcomas in combination with active cytotoxic agents enhances each strategy alone without increase in toxicity. To confirm such hypothesis, we conducted two phase I trials with associated preclinical studies which have been published in international scientific journals. Article 1: Phase I trial of sorafenib in combination with ifosfamide in patients with advanced sarcoma: a Spanish group for research on sarcomas (GEIS) study. Background: This phase I trial assessed safety, pharmacokinetics (PK), dose limiting toxicity (DLT), maximum tolerated dose and recommended dose (RD) of the combination of sorafenib plus ifosfamide in patients with advanced sarcoma. Methods: Twelve sarcoma patients (9 soft-tissue, 3 bone sarcoma) were treated with sorafenib plus ifosfamide (starting doses 200 mg bid and 6 g/m(2) respectively). A 3 + 3 dose escalation design with cohorts of 3-6 patients was used. A study to assess the in vitro efficacy of the combination was also conducted. Results: Three DLTs were observed: fatigue grade 4 with sorafenib 400 mg bid plus ifosfamide 6 g/m(2) and encephalopathy and emesis grade 3 with sorafenib 400 mg bid plus ifosfamide 7.5 g/m(2). Other toxicities included diarrhea, hand-foot syndrome, mucositis, neutropenia, skin rash and thrombocytopenia. There were no relevant effects on PK of sorafenib but an increase in ifosfamide active metabolite 4-hydroxy-ifosfamide was observed. Eight patients achieved stable disease lasting more than 12 weeks. An additive effect was observed in vitro. Conclusions: RD was sorafenib 400 mg bid plus ifosfamide 6 g/m(2), allowing administration of active doses of both agents. Limited preliminary antitumor activity was also observed. A phase II study is currently ongoing.
Los sarcomas son un grupo de tumores caracterizados por su mal pronóstico y la ausencia de tratamientos efectivos. La mediana de supervivencia de los pacientes afectos de sarcoma avanzado es de tan solo 1 año a pesar de recibir tratamiento. Por lo tanto, es necesario encontrar nuevas estrategias terapéuticas efectivas. Nuestra hipótesis es que la inhibición de la angiogénesis y de la vía de mTOR en sarcomas en combinación con agentes citotóxicos activos potencia la actividad anti tumoral de cada una de las estrategias terapéuticas por separado sin toxicidad significativa. Para confirmar dicha hipótesis realizamos dos ensayos clínicos fase I con experimentos preclínicos asociados que han sido publicados en revistas científicas internacionales. Artículo 1: Ensayo clínico fase I de sorafenib en combinación con ifosfamida en pacientes con sarcoma avanzado: un estudio del Grupo Español de Investigación en Sarcomas (GEIS). Este ensayo clínico fase I evaluó la seguridad, la farmacocinética, la toxicidad limitante de dosis, la dosis máxima tolerada y la dosis recomendada de la combinación de sorafenib más ifosfamida en pacientes con sarcoma avanzado. La dosis recomendada fue sorafenib 400 mg bid más ifosfamida 6 g/m2, un esquema que permite la administración de dosis activas de ambos fármacos. También se observaron signos preliminares de actividad antitumoral. Artículo 2: Ensayo clínico fase I y evaluación de la eficacia preclínica del inhibidor de mTOR sirolimus más gemcitabina en pacientes con tumores sólidos avanzados Llevamos a cabo un ensayo clínico fase I en pacientes con tumores sólidos avanzados para identificar la dosis recomendada, evaluar la PK, la actividad farmacodinámica y la eficacia antitumoral preclínica de la combinación de sirolimus y gemcitabina. La dosis recomendada fue sirolimus 5 mg al día más gemcitabina 800 mg/m2. Además, se observó actividad antitumoral en los modelos preclínicos de sarcoma, así como inhibición de la vía de mTOR.

Biology
Ph.D.
Hepatitis B virus encoded X protein (HBx) contributes centrally to the pathogenesis of hepatocellular carcinoma (HCC). Aberrant activation of the Hedgehog (Hh) pathway has been linked to cancer. Thus, experiments were designed to test the hypothesis that HBx contributes to HCC via activation of Hh signaling. HBx expression correlated with up-regulation of Hh markers in human liver cancer cell lines, in HBx transgenic mice that developed HCC and in liver samples from HBV infected patients with HCC. The findings in human samples provide clinical validation of those in the HBx transgenic mice (HBxTg), and underscore the relevance of these transgenic mice to disease pathogenesis. Further, blockade of Hh signaling inhibited HBx stimulation of cell migration, anchorage independent growth, HCC tumorigenesis in HBx transgenic mice and tumor growth in xenograft model. These results suggest that the ability of HBx to promote cancer is at least partially dependent upon Hh activation and that activation of Hh signaling appears to be important for the development of HBx associated HCC. HBx also activates pathways that stimulate downstream Hh signaling, such as PI3K/AKT and Ras/Raf/MEK, also referred as non-canonical Hh signaling. Upon canonical Hh inhibition, compensatory activation of these pathways was seen in the presence of HBx in liver cancer cell lines and in HBxTg mice. Individual inhibition of these pathways also down-regulated Gli2 expression in HBx positive cell lines. These data suggests that in addition to canonical Hh signaling, activation of PI3K/AKT and ERK pathways by HBx leads to up-regulation of Gli2 expression in HBV-mediated HCC. This work identifies Hh pathway inhibition as a therapeutic strategy to slow tumor development and this work could lead to combination therapies that target Hh, AKT and ERK pathways, which may prevent or delay the appearance/progression of HCC.
Temple University--Theses

Increased understanding of the molecular mechanisms underlying cancer development has shifted drug discovery towards target driven drug development the last decades, but the development of effective cancer drugs has been hampered by the lack of predictive preclinical models. 3-D cultures, considered to more accurately reflect solid tumors in vivo, have been proposed as one way to increase the predictability of clinical efficacy in cancer drug discovery and development. The aims of this thesis were to improve preclinical models for cancer drug development, with focus on colorectal cancer (CRC) and use of multicellular tumor spheroids (MCTS), and also to mechanistically characterize some potentially new anticancer drugs (papers I – IV). The most important technical improvement was the development of direct measurement of green fluorescent protein (GFP) marked cells in spheroids, simplifying live collection of viability data and enabling high-throughput screening (HTS) in the MCTS model (paper I). In paper III and IV, the 3-D model was adapted to enable studies on the interaction between drugs and radiation. Two potentially new anticancer drugs, VLX50 and VLX60, were mechanistically characterized. VLX60, a novel copper containing thiosemicarbazone, induced reactive oxygen species (ROS) formation, was selectively active against BRAF mutated colon cancer cells and exhibited anticancer activity in vivo (paper II). Furthermore, two potentially new anticancer drugs were found suitable for further development for use in combination with radiation (papers III and IV). In paper III, synergy with radiation in spheroids compared to monolayer cultured colon cancer cells was shown with the novel iron-chelating inhibitor of oxidative phosphorylation, VLX600. In paper IV, the antiprotozoal drug nitazoxanide was shown to sensitize quiescent clonogenic colon cancer cells to radiation. In conclusion, introduction of measurement of fluorescence of GFP marked cells in spheroids makes clinically relevant 3-D models feasible for HTS experiments and characterization of candidate drugs and radiosensitizers in early cancer drug discovery and development. VLX60 has several characteristics suitable for further development into a cancer drug, notably against BRAF mutated colorectal cancer cells. VLX600 and nitazoxanide show radiosensitizing properties making them promising for further development for use as cancer drugs in combination with radiation.

Constitutive activation of the chemokine receptor CXCR4 is associated with tumor progression, invasion and resistance to treatment. Overexpression of CXCR4 in diffuse large B-cell lymphoma (DLBCL) confers a reduced prognosis. However, the biological relevance of this receptor in DLBCL progression remains underexplored. In this thesis, the new CXCR4 inhibitor IQS-01.01 has been preclinicaly evaluated in in vitro and in vivo models of DLBCL. It has been concluded that 1) inhibition of CXCR4 presents antitumor properties in DLBCL, 2) that IQS-01.01RS holds better pharmacological properties than the reference CXCR4 inhibitor, AMD3100 3) that treatment with IQS- 01.01RS reduces the levels of the oncogene MYC and 4) that the combinantion of IQS- 01.01RS with the BET-bromodomain inhibitor, CPI203, is synergistic in DLBCL. The results of this doctoral thesis unravel a cooperation between CXCR4 and MYC in DLBCL, and indicate that CXCR4 inhibition in combination with inhibition of MYC is a promising novel therapeutic approach in DLBCL.
La activación constitutiva del receptor de quemocinas CXCR4 está asociada a la progresión tumoral, invasión y resistencia al tratamiento. En el linfoma difuso de células grandes (LDCG) la sobreexpresión de CXCR4 concede un peor pronóstico, pero la relevancia biológica de este receptor no se ha estudiado en profundidad. En esta tesis se ha evaluado un nuevo inhibidor de CXCR4 (IQS-01.01) en modelos preclínicos de LDCG. Usando tanto modelos in vitro como in vivo de LDCG se ha concluido 1) que la inhibición de CXCR4 en LDCG tiene un efecto antitumoral, 2) que IQS-01.01RS tiene mayores propiedades farmacológicas que el inhibidor de referencia, AMD3100 3) que el tratamiento con IQS-01.01RS reduce los niveles del oncogén MYC y 4) que la combinación de IQS-01.01 RS con el inhibido de BET, CPI203, confiere un efecto antitumoral sinérgico. Los resultados de esta tesis doctoral ponen en evidencia una cooperación entre MYC y CXCR4 en LDCG e indican que la inhibición de CXCR4 en combinación con un inhibidor de MYC es una terapia prometedora contra el LDCG.

Allogeneic stem cell transplantation (SCT) is the only curative treatment option for many haematological malignancies. Adenovirus (Ad) infections are a significant cause of morbidity and mortality post SCT. Lack of effective anti-viral treatment for Ad disease has led to the development of adoptive immunotherapy of Ad-specific T-cells as a promising therapeutic option for patients in this setting. The aim of this project was to establish preclinical criteria for the development of a clinical trial comparing two T-cell enrichment methods- multimer selection and cytokine secretion selection to enrich Ad-specific T-cells for the purposes of adoptive transfer directly without the need for in vitro culture. Eight pHLA tetramers containing HLA class I restricted Ad epitopes were generated and their ability to identify and enrich Ad-specific T-cells investigated. HLA A*01 TDL tetramer consistently detected T-cells in all (13/13) healthy adult donors screened. Frequency and enrichement of Ad-specific T-cells by cytokine secretion and selection was also investigated. Despite the low frequency of Ad-specific T-cells, clinical grade enrichment was feasible by both methods. T-cells selected by both methods were then characterised for homing and proliferative potential. Ad-specific T-cells identified by either method had a high proliferative potential, possessed a novel minimally differentiated memory phenotype, were cytotoxic towards Ad species responsible for infections in SCT recipients and capable of limiting virus replication. In conclusion, Ad-specific T-cells enriched by multimer selection or cytokine secretion selection are suitable for adoptive transfer to patients with Ad infection following HSCT. Both methods also allow the monitoring of Ad-specific immune reconstitution after adoptive transfer.

Radioactive substances are used in vivo to localize and characterize malignant tumours, generally by scintigraphic methods. In this context positron emission tomography (PET) in combination with radiolabelled monoclonal antibodies (mAbs) may provide a sensitive and specific method for detection of cancer. Individual dose calculations, based on such PET measurements, may be carried out to predict the possible use of mAbs labelled with therapeutic nuclides. The positron emitter 76Br, with a half-life of 16 h, is a well-suited candidate for radiolabelling and PET imaging. One drawback of radiobromine is that bromide, the ultimate catabolite after degradation of brominated mAb, is only tardily excreted from the body and is evenly distributed throughout the extracellular space, thereby increasing the background radioactivity. The aim of this work was to produce 76Br-mAb preparations with high accumulation and retention in tumour tissue together with a quick clearance of 76Br-labelled catabolites. Furthermore, the possibility to use brominated or iodinated mAbs in combination with PET to predict 211At-mAb dosimetry was evaluated. Monoclonal Abs directed against colorectal cancer were labelled with 76Br using the direct Chloramine-T-method or indirectly by labelling the precursor molecule N-succinimidyl para-(tri-methylstannyl) benzoate with 76Br, which was subsequently conjugated to the mAbs. Monoclonal Ab A33 labelled with 76Br using the two labelling protocols was characterized in vitro and in vivo in a rat tumour xenograft model. The mAb A33 was also labelled with 125I for comparison. In addition, mAb A33 was labelled with 211At, 125I and 76Br using the indirect labelling protocol and the mAb pharmacokinetics was studied in normal rats in order to estimate if data from brominated or iodinated mAb could be used for dosimetry of 211At in healthy organs and tissue. In conclusion, both direct and indirect labelling resulted in high yields and mAbs with preserved immunoreactivity. In vivo characterization of 76Br-brominated mAb A33 showed that the indirect labelling method makes 76Br-brominated mAb A33 a promising candidate for tumour imaging with PET due to the faster excretion of radiolabelled catabolites compared with direct bromination. Finally, mAb A33 labelled with 76Br and 124/125I can be used to predict the 211At dose of astatinated mAb A33 in most organs given that a correction factor is applied for organs with varying uptake.

L'imagerie par bioluminescence (BLI) est une technologie d'imagerie optique dans laquelle un organisme ou cellule vivant émet de la lumière à travers une réaction biologique substrat/enzyme sans aucune excitation lumineuse. Cette technologie, utilisée en oncologie préclinique afin de quantifier l'état des tumeurs de manière non invasive, est encore assez récente et, pour l'instant, les biologistes manquent d'outils de traitement automatisé pour améliorer la quantification des images. De plus, certains protocoles expérimentaux nécessitent l'extraction du flux de photons de plusieurs tumeurs situées sur le côté de l'animal. Cela peut être difficile et peut introduire des erreurs et des biais car la BLI souffre d'un manque de robustesse en raison d'une variabilité dans la vascularisation, ou des zones hypoxiques et nécrotiques au sein des tumeurs. Dans ce travail, nous proposons l'utilisation de la factorisation en matrices non négatives pour séparer le flux de photons de différentes tumeurs au sein de la même image de bioluminescence en tirant parti des différents patterns temporels pixel par pixel. Un tel démélange spatio-temporel présente plusieurs importants défis que nous avons relevés. Dans une première contribution, nous utilisons des connaissances préalables sur l'apparence des tumeurs et montrons l'importance de pénaliser la norme des coefficients d'ondelettes correspondant aux sources estimées pendant le processus d'optimisation afin d'obtenir une forte cohérence spatiale des tumeurs démêlées. Dans une deuxième contribution, nous traitons les fortes hétérogénéités au sein des tumeurs corrompant la séparation en présentant une chaîne de traitement dédiée pour pré-aligner le flux de photons des différents pixels. Nous montrons que la méthode résultante est capable d'extraire avec précision le flux de photons de différentes tumeurs présentes dans une seule image de bioluminescence. Ces algorithmes ont été testés et validés sur deux ensembles de données réelles de BLI et sur un ensemble de données synthétiques généré avec un simulateur d'image de bioluminescence que nous avons conçu et développé. Dans une troisième contribution, nous proposons un modèle de pharmacocinétique pour calibrer le flux de photons de la tumeur en fonction du signal de bioluminescence émis par un muscle. Cela nous permet d'extraire des paramètres physiologiques significatifs de l'image comme les taux d'échange de substrat. Nous montrons que ces paramètres représentent des caractéristiques significatives de l'état de la tumeur et peuvent être utilisés pour améliorer la quantification des images de bioluminescence
Bioluminescence imaging (BLI) is an optical imaging technology in which a living organism or cell emits light through a biological substrate/enzyme reaction without any light excitation.This technology, used in preclinical oncology in order to quantify the tumor status in a non-invasive way, is still quite recent and for now biologists lack automated processing tools to improve the quantification of images. In addition, some experimental protocols require to extract the photon flux of multiple tumors on the same side of the animal. This can be difficult and can introduce errors and biases as BLI suffers from a lack of robustness because of a variability in vascularization, or hypoxic and necrotic zones within the tumors. In this work, we propose the use of Non-Negative Matrix Factorization to separate the photon flux of different tumors within the same bioluminescence image by leveraging the different pixel-wise temporal patterns. Such spatio-temporal unmixing yields several important challenges that we have tackled. In a first contribution, we use prior knowledge on the appearance of the tumors and show the importance of penalizing the norm of the wavelet coefficients corresponding to the sources estimated during the optimization process to obtain a high spatial consistency of unmixed tumors. In a second contribution we deal with strong heterogeneities within tumors corrupting the separation by presenting a dedicated pipeline for pre-aligning the photon flux of the different pixels. We show that the resulting method is capable of accurately extracting the photon flux of different tumors present within a single bioluminescence image. These algorithms were tested and validated on two real BLI datasets and on one synthetic dataset generated with a bioluminescence image simulator we designed and developed. In a third contribution, we propose a pharmacokinetics model to calibrate the tumor photon flux based on the bioluminescence signal emitted by a muscle. This allows us to extract meaningful physiological parameters from the image like substrate exchange rates. We show that these parameters represent significant features of the tumor state and can be used to improve the quantification of bioluminescence images

A significant number of patients requiring adoptive T-cell therapy (ATCT) need to resort to third party donors; we aimed to find ways to optimise ATCT from third party donors in EBV-associated diseases. Firstly, we evaluated the T-cell response to 29 EBV-restricted peptides in a cohort of 100 healthy donors. For each peptide we found at least one high-responding donor. Also, we compared the efficacy of different separation techniques. These results support the setting up of a registry of third party donors, to provide fresh EBV-specific T cells for ATCT. Secondly, we investigated the mechanisms generating T memory stem cells (T_SCM), which are considered most suitable for ATCT. We demonstrated that homeostatic cytokines revert recently differentiated CD8⁺ memory T cells from cord blood (CB) to cells with a T_N-like phenotype (T_Nrev) and T_SCM-like characteristics. Finally, we compared phenotype and function of CD8⁺ T cells from peripheral blood and CB, after transduction of an EBV-specific TCR. Transduction efficiency, growth kinetics and cytolytic activity were comparable. However, TCR-transduced CB T cells showed less differentiated phenotype, increased multi-cytokine expression, and lacked expression of the senescence marker CD57. These data suggest that survival of engineered T cells in vivo is likely to be improved by using cells from CB.

Radioactive substances are used in vivo to localize and characterize malignant tumours, generally by scintigraphic methods. In this context positron emission tomography (PET) in combination with radiolabelled monoclonal antibodies (mAbs) may provide a sensitive and specific method for detection of cancer. Individual dose calculations, based on such PET measurements, may be carried out to predict the possible use of mAbs labelled with therapeutic nuclides. The positron emitter ⁷⁶Br, with a half-life of 16 h, is a well-suited candidate for radiolabelling and PET imaging. One drawback of radiobromine is that bromide, the ultimate catabolite after degradation of brominated mAb, is only tardily excreted from the body and is evenly distributed throughout the extracellular space, thereby increasing the background radioactivity. The aim of this work was to produce ⁷⁶Br-mAb preparations with high accumulation and retention in tumour tissue together with a quick clearance of ⁷⁶Br-labelled catabolites. Furthermore, the possibility to use brominated or iodinated mAbs in combination with PET to predict ²¹¹At-mAb dosimetry was evaluated.

Monoclonal Abs directed against colorectal cancer were labelled with ⁷⁶Br using the direct Chloramine-T-method or indirectly by labelling the precursor molecule N-succinimidyl para-(tri-methylstannyl) benzoate with ⁷⁶Br, which was subsequently conjugated to the mAbs. Monoclonal Ab A33 labelled with ⁷⁶Br using the two labelling protocols was characterized in vitro and in vivo in a rat tumour xenograft model. The mAb A33 was also labelled with ¹²⁵I for comparison. In addition, mAb A33 was labelled with ²¹¹At, ¹²⁵I and ⁷⁶Br using the indirect labelling protocol and the mAb pharmacokinetics was studied in normal rats in order to estimate if data from brominated or iodinated mAb could be used for dosimetry of ²¹¹At in healthy organs and tissue.

In conclusion, both direct and indirect labelling resulted in high yields and mAbs with preserved immunoreactivity. In vivo characterization of ⁷⁶Br-brominated mAb A33 showed that the indirect labelling method makes ⁷⁶Br-brominated mAb A33 a promising candidate for tumour imaging with PET due to the faster excretion of radiolabelled catabolites compared with direct bromination. Finally, mAb A33 labelled with ⁷⁶Br and ^124/125I can be used to predict the ²¹¹At dose of astatinated mAb A33 in most organs given that a correction factor is applied for organs with varying uptake.

Apoptosis is an important process in normal mammary gland physiology and evasion of apoptosis has also been identified as a hallmark of cancer. In breast cancer cells apoptotic resistance is an acquired feature that can promote tumour growth and progression. Induction of apoptosis by the extrinsic death ligand TRAIL has been shown to be a promising clinical therapy targeting a number of different cancer cells whilst sparing normal cells. Unfortunately most breast cancers are inherently resistant to TRAIL treatment. Herein it is shown that by reducing the expression of the downstream TRAIL inhibitor c-FLIP, a range of different breast cancer subtypes can be sensitised to TRAIL treatment resulting in significant cancer cell death. Significantly, suppression of c-FLIP in combination with TRAIL (FLIPi/TRAIL) ablated the tumour-initiating breast cancer stem cell (bCSC) subset, as defined by mammosphere formation assay, within cell lines. This selective killing of bCSCs translated to reduced tumour initiation and metastasis in animal transplant models. However, continued culture of FLIPi/TRAIL treated cell lines in adherent conditions resulted in bCSC re-acquisition suggesting a phenotypic plasticity of non-bCSC cells. Re-acquired bCSCs also demonstrated sensitivity to repeated FLIPi/TRAIL treatment and maintaining reduced c-FLIP expression prevented bCSC re-acquisition. These results substantiate the importance of resistance to apoptosis in tumour initiation and metastasis and identify the targeting of c-FLIP proteins as a promising anti-cancer therapeutic approach. Acquired resistance to existing mainstay therapies such as antiestrogens (AEs) (tamoxifen and Faslodex) and aromatase inhibitors (AIs) is an ongoing obstacle in treatment of a large number of breast cancer patients. AE-resistant models of breast cancer and a multiple endocrine-resistant patient sample demonstrated hypersensitivity to TRAIL. This sensitivity was observed in both in vitro and in vivo models of AE resistance and cell death was prevalent in both bulk tumour cells and bCSCs. Sensitisation was not attributed to combination AE/TRAIL treatment suggesting cellular changes during the acquisition of AE resistance are responsible for TRAIL sensitivity in these models. Further investigation suggested that the mechanism of AE-resistant cell sensitivity to TRAIL was not dependant on functional estrogen receptor signalling and is most likely dependant on the AE agent that the cancer cells have acquired resistance to. Interestingly tamoxifen-resistant MCF-7 cells were shown to have reduced c-FLIP protein expression compared to parental cells, further supporting c-FLIP’s potential in cancer therapy. Recent success in the use non-MHC-restricted γδ T cells as a targeted immunotherapy in clinical trials has identified this therapeutic methodology as desirable. Here it is shown that TRAIL is readily expressed by this subset of T cells that also demonstrate cytotoxicity to breast cancer cell lines. Neither the secretion of TRAIL or surface expression of TRAIL appeared to contribute significantly towards γδ T cell cytotoxicity and the majority of breast cancer cell death induced by γδ T cells would seem to be perforin-mediated. The suppression of c-FLIP in target cells increased γδ T cell cytotoxicity but again not via TRAIL. Preliminary results also indicated that the bCSCs of some cell lines were exquisitely sensitive to γδ T cell treatment. In summary these results indicate that targeting c-FLIP and TRAIL can be therapeutically beneficial in a range of different breast cancer subtypes by certain therapeutic strategies.

L’imagerie préclinique est devenue une ressource incontournable pour l’évaluation de paramètres physiopathologiques, pour le suivi du développement tumoral ainsi que pour le développement de thérapies anticancéreuses. Les évolutions technologiques apparues ces dernières années ont conduit au développement de nouvelles modalités d’imagerie ayant un fort potentiel de translation vers la clinique. Ce manuscrit présente diverses approches par imagerie échographique, photoacoustique et de fluorescence dans le proche infrarouge pour le suivi de la pathologie cancéreuse. Dans un premier temps, nous nous sommes intéressés à la caractérisation de l’hypoxie et son suivi au cours du temps dans différents modèles de cancers humains. Différentes stratégies d’imagerie multimodale ont ensuite été mises en oeuvre pour évaluer l’efficacité d’une nouvelle prodrogue thérapeutique permettant la libération d’une molécule active dans le proche environnement tumoral sur des modèles humains de tumeurs pancréatiques, mammaires, pulmonaires. Enfin, dans un contexte de recherche translationnelle, nous avons exploré le potentiel de l’imagerie photoacoustique et de la fluorescence infrarouge pour la mise en évidence de l’invasion ganglionnaire tumorale en mettant en oeuvre des modèles de ganglions sentinelles minimalement envahis. Au cours de ce travail, nous avons montré l’intérêt du suivi de l’hypoxie tumorale en onco-pharmacologie et mis en évidence le fort potentiel de l’imagerie PA pour les approches translationnelles en oncologie. La principale limitation correspond à la profondeur relativement faible des régions explorables, mais ce point suscite actuellement de nombreux développements technologiques. Les études de faisabilité réalisées ainsi que la validation de protocoles de preuves de concept permettront l’exploitation en routine de ces nouvelles modalités d’imagerie
Preclinical imaging has become an unavoidable step for pathophysiological parameters assessments, for the follow up of tumor growth and for the anticancer therapies development. Technological improvements have emerged in recent years, allowing the emergence of new imaging modalities with a high potential for translation into clinical practice. This manuscript presents several approaches by ultrasound imaging, photoacoustics and near infrared fluorescence in order to monitor the cancer pathology. Initially, we focused on the characterization of hypoxia and its longitudinal assessment in various preclinical models of human cancers. Various multimodal imaging strategies were implemented to assess the efficacy of a new therapeutic prodrug allowing the release of an active molecule in the tumor microenvironment on human models of pancreatic, breast and lung tumors. Finally, in a context of translational research, we explored the potential of photoacoustic and near infrared fluorescence imaging to highlight the lymph node invasion by cancer cells implementing minimally invaded sentinel lymph node models. In this work, we have shown the interest in monitoring the tumor hypoxia in onco-pharmacology and highlighted the high potential of photoacoustic imaging for oncology translational approaches. The main limitation is the relatively shallow depth of regions that we can explore, but this point is currently subject to many technological developments. Feasibility studies performed and validation of proof of concept protocols will enable routine exploitation of these new imaging modalities

Radionuclide molecular imaging is an emerging multidisciplinary technique that is used in modern medicine to visualise diseases at cellular and molecular levels. This thesis is based on five papers (I-V) and focuses on the development of site-specific radiolabelled recombinant anti-HER2 Affibody molecules and preclinical evaluations in vitro and in vivo of the labelled conjugates. This work is part of a preclinical development of an Affibody molecule-based tracer for molecular imaging of HER2 expressing tumours. Papers I and II report the evaluation of the Affibody molecule ZHER2:2395-C, site-specifically labelled with the radiometals 111In (for SPECT) and 57Co (as a surrogate for 55Co, suitable for PET applications) using a thiol reactive DOTA derivative as a chelator. Both conjugates demonstrated very suitable biodistribution properties, enabling high contrast imaging just a few hours after injection. Papers III and IV report the development and optimization of a technique for site-specific labelling of ZHER2:2395-C with 99mTc using an N3S chelating peptide sequence. 99mTc-ZHER2:2395-C demonstrated high and specific tumour uptake and rapid clearance of non-bound tracer from the blood, resulting in high tumour-to-non-tumour ratios shortly after injection, enabling high contrast imaging. In addition, in the study described in paper IV, freeze-dried kits previously developed for 99mTc-labelling were optimised, resulting in the development of a kit in which all the reagents and protein needed for labelling of ZHER2:2395-C with 99mTc were contained in a single vial. Paper V reports the evaluation of an anti-HER2 Affibody molecule, ABY-025, with a fundamentally re-engineered scaffold. Despite the profound re-engineering, the biodistribution pattern of 111In-ABY-025 was very similar to that of two variants of the parental molecule. It seems reasonable to believe that these results will also be applicable to Affibody molecules towards other targets. Hopefully, this work will also be helpful in the development of other small proteinaceous tracers.

Pancreatic cancer is characterized by its desmoplastic tumor microenvironment and the infiltration of immunosuppressive cells. It is a devastating disease where most patients are diagnosed at a late stage and the treatment options are few. The development of new treatments is surly needed. One treatment option explored is the use of immunotherapy with the intent to activate the immune system and change the balance from pro-tumor to anti-tumor. This thesis presents the idea of using oncolytic adenoviruses called LOAd-viruses that are armed with immunostimulatory- and microenvironment-modulating transgenes. For effective treatment of pancreatic cancer, the virus needs to be able to be given in addition to standard therapy, the chemotherapy gemcitabine. In paper I, the immunomodulatory effect of gemcitabine was evaluated in blood from pancreatic cancer patients receiving their first 28-day cycle of treatment with infusions day 1, 8 and 15 followed by a resting period. Gemcitabine reduced the level of immunosup-pressive cells and molecules but the effect did not last throughout the resting period. On the other hand, gemcitabine did not affect the level or proliferative function of effector T cells indicating that gemcitabine could be combined with immunotherapy. The LOAd700 virus expresses a novel membrane-bound trimerized form of CD40L (TMZ-CD40L). In paper II, LOAd700 showed to be oncolytic in pancreatic cancer cell lines as well as being immunostimulatory as shown by its capacity to activate dendritic cells (DCs), myeloid cells, endothelium, and to promote expansion of antigen-specific T cells. In paper III, LOAd703 armed with both 4-1BBL and TMZ-CD40L was evaluated. LOAd703 gave a more profound effect than LOAd700 on activation of DCs and the virus was also capable of reducing factors in stellate cells connected to the desmo-plastic and immunosuppressive microenvironment. In paper IV, LOAd713 armed with TMZ-CD40L in combination with a single-chain variable fragment against IL-6R was evaluated. The virus could kill pancreatic cancer cells lines through oncolysis and could also reduce factors involved in desmoplasia in stellate cells. Most interestingly, LOAd713 could reduce the up-regulation of PD-1/PD-L1 in DCs after CD40 activation. Taken together, LOAd703 and LOAd713 seem to have interesting features with their combination of immunostimulation and microenvironment modulation. At present, LOAd703 is evaluated in a clinical trial for pancreatic cancer (NCT02705196).

Afin d'analyser les données in vitro de l'effet de l'erlotinib sur la croissance des cellules A431 suivie par impédance-métrie, nous avons développé un modèle pharmacocinétique - pharmacodynamique non linéaire décrivant simultanément la diminution de la concentration d'erlotinib et son effet sur la croissance cellulaire au cours du temps. La non-linéarité du modèle imposant le recours à des méthodes itératives pour l'estimation des paramètres, plusieurs étapes de la procédure d'identification du modèle ont été étudiées et des solutions proposées, avec des exemples d'application à des molécules utilisées en oncologie:Choix du critère d'optimisation à employer - supériorité de la relation fonctionnelle de la moyenne géométrique pour l'identification de modèles non linéaires. Application données réelles: courbe de calibration d'une expérience de dosage ELISA du bevacizumab. Choix de l'algorithme d'optimisation le plus approprié au problème d'identification du processus pharmacocinétique. Les algorithmes dérivatifs sont les plus performants. Application données réelles: estimation simultanée des paramètres du modèle pharmacocinétique du 5-fluorouracile et de son métabolite principal.Transformation de la forme différentielle initiale du modèle en temps continu vers un modèle récursif en temps discret. Par ce moyen le modèle devient linéaire en ses paramètres, ce qui permet d'estimer directement les paramètres sans utiliser d'algorithme d'optimisation. Il devient également possible de suivre les variations des paramètres au cours du temps. Application données réelles: pharmacocinétique de la fotemustine, de la mitoxantrone et du 5-fluorouracile
A nonlinear pharmacokinetic-pharmacodynamic model has been devised do simultaneously describe the loss of erlotinib and its effect on the cell growth over time, in order to analyze impedance-based data of erlotinib effect on A431 cells growth in vitro over time. The model non-linearity requiring the use of iterative methods for parameter estimation, several steps of the model identification were studied, and solutions proposed, with application examples to cancer drugs :Choice of the optimization criterion - superiotity of the geometric mean functionnal relationship for non-linear model identification. Real data application : calibration curve of a bevacizumab ELISA quantification experiment.Choice of the most appropriate algorithm for the pharmacokinetic process identification problem. The derivative algorithms perform better. Real data application : simultaneous identification of the 5-fluorouracil and of its main metabolite pharmacokinetic system.Transform of the differential initial continuous-time model in a recursive discrete time model. The transformed model becomes linear with respect to its parameters, allowing straightforward parameter estimation without using any optimization algorithm. It is then also possible to track the parameter variations over time. Real data application : pharmacokinetic model parameter estimation of fotemustine, mitoxantrone and 5-fluorouracil

Els Glioblastomes (GB) son tumors cerebrals invasius, amb mal pronòstic i resposta pobra a la teràpia. Aquesta tesi pretén millorar el tractament del GB preclínic mitjançant la immunitat relacionada amb el càncer i el seguiment no invasiu de la resposta amb Imatge Espectroscòpica de Ressonància Magnètica (IERM). El model de GB GL261 ha estat escollit per ser immunocompetent i adequat per a estudis de teràpia. S’ han investigat tres estratègies terapèutiques: a) quimioteràpia (Temozolamida, TMZ), en protocol metronòmic respectuós amb el sistema immune (Immune-Enhancing Metronomic Schedule, IMS-TMZ), b) un inhibidor de punt de control immunològic (Programmed cell death protein 1, anticòs anti-PD-1), i c) combinació de TMZ+anti-PD-1 en IMS. Diferents tipus de dany cel·lular desencadenats per la teràpia estimulen la resposta immune anti-càncer. El nostre objectiu ha estat, per una banda, induir dany cel·lular immunogènic, preservant les cèl·lules del sistema immune (IMS-TMZ). Per altra banda, contrarestar la immunosupressió al tumor (anti-PD-1). El protocol de IMS-TMZ ha millorat la supervivència dels ratolins amb GB GL261, sobrepassant resultats previs del grup. La teràpia amb anti-PD-1 ha estat efectiva en dosis elevades (500/250 μg), tot i que diferències al volum tumoral a l’ inici de la immunoteràpia impacten en la seva eficàcia. La teràpia combinada anti-PD-1+TMZ en IMS ha resultat millor que les teràpies per separat. Aquests resultats recolzen la participació del sistema immune en la resposta a la teràpia en GB. Estudis del nostre grup amb imatges nosològiques basades en IERM apunten a que els canvis al patró metabolòmic estarien relacionats amb l’ acció del sistema immune sobre els tumors, actuant com marcador vicari de resposta a la teràpia. Ens hem preguntat si els mateixos canvis metabolòmics s’ apreciarien si aquest sistema s’ apliqués a estratègies d’ immunoteràpia. S’ ha seguit amb aquest sistema l’ evolució de ratolins amb tumors GL261 tractats amb IMS-TMZ, IMS-anti-PD-1 i IMS-anti-PD-1/TMZ. El protocol de IMS-TMZ produeix oscil·lació de canvis al patró metabolòmic, amb freqüència de 6 dies. Aquest comportament s’ ha confirmat en ratolins tractats amb immunoteràpia, sola o en combinació, suggerint que els canvis al patró metabolòmic son comuns a diferents estratègies terapèutiques, sempre que hi hagi estímul i atracció del sistema immune amb atac productiu a les cèl·lules tumorals. Això obre camí per l’ ús traslacional del biomarcador basat en MRSI en la teràpia personalitzada en pacients de GB, incloent l’ immunoteràpia, per la qual encara no es disposa de biomarcadors no invasius fiables. La participació del sistema immune també es veu recolzada pel percentatge d’ animals curats observats en aquesta tesi (50-100% depenent de la teràpia), els quals han presentat memòria immune contra subsegüents intents de generació del mateix tipus de tumor. La resistència a TMZ es una de las causes del fracàs de la quimioteràpia adjuvant en el tractament de GB. Hem investigat el rol de la O6-methylguanine-DNA-methyltransferase (MGMT) y programmed death-ligand 1 (PD-L1) en la quimioresistència, utilitzant western-blot, en tumors que escapen a la teràpia IMS-TMZ després de presentar resposta transitòria. L’ expressió de PD-L1 es triplica en aquests tumors en comparació amb controls, indicant que aquests nivells podrien ser rellevants en la resistència a la TMZ in vivo, tenint la teràpia anti-PD-1 potencial per a ‘rescatar’ tumors escapant de la teràpia amb TMZ. La combinació d’ oncologia i immunologia pot obrir camí cap a una millora de l’ eficàcia de la teràpia i dels resultats obtinguts amb els pacients de GB.
Los Glioblastomas (GB) son tumores cerebrales invasivos, con mal pronóstico y respuesta pobre a la terapia. Esta tesis pretende mejorar el tratamiento del GB preclínico utilizando la inmunidad relacionada con el cáncer y el seguimiento no invasivo de la respuesta mediante Imagen Espectroscópica de Resonancia Magnética (IERM). El modelo de GB GL261 fue escogido por ser inmunocompetente y adecuado para estudios de terapia. Se investigaron tres estrategias terapéuticas: a) quimioterapia (Temozolamida, TMZ), en protocolo metronómico respetuoso con el sistema inmune (Immune-Enhancing Metronomic Schedule, IMS-TMZ), b) un inhibidor de punto de control inmunológico (Programmed cell death protein 1, anticuerpo anti-PD-1), y c) combinación de TMZ+anti-PD-1 en IMS. Diferentes tipos de daño celular desencadenados por la terapia estimulan la respuesta inmune anti-cáncer. Nuestro objetivo fue, por un lado, inducir daño celular inmunogénico, preservando las células del sistema inmune (IMS-TMZ). Por otro lado, contrarrestar la inmunosupresión en el tumor (anti-PD-1). El protocolo de IMS-TMZ ha mejorado la supervivencia de los ratones con GB GL261, sobrepasando resultados previos del grupo. La terapia con anti-PD-1 fue efectiva en doses elevadas (500/250 μg), aunque diferencias en el volumen tumoral al inicio de la inmunoterapia impactan en su eficacia. La terapia combinada anti-PD-1+TMZ en IMS ha resultado mejor que las terapias por separado. Estos resultados respaldan la participación del sistema inmune en la respuesta a la terapia en GB. Estudios de nuestro grupo con imágenes nosológicas basadas en IERM apuntan a que los cambios en el patrón metabolómico estarían relacionados con la acción del sistema inmune sobre los tumores, actuando como marcador subrogado de respuesta a la terapia. Nos hemos preguntado si los mismos cambios metabolómicos se apreciarían si este sistema fuese aplicado a estrategias de inmunoterapia. Se siguió con dicho sistema la evolución de ratones con tumores GL261 tratados con IMS-TMZ, IMS-anti-PD-1 e IMS-anti-PD-1/TMZ. El protocolo de IMS-TMZ produce oscilación de cambios en el patrón metabolómico, con una frecuencia de 6 días. Dicho comportamiento se ha confirmado en ratones tratados con inmunoterapia, sola o en combinación, sugiriendo que los cambios en el patrón metabolómico son comunes a distintas estrategias terapéuticas, siempre que haya estímulo y atracción del sistema inmune con ataque productivo a las células tumorales. Ello abre el camino para un uso traslacional del biomarcador basado en MRSI para terapia personalizada en pacientes de GB, incluyendo inmunoterapia, para la cual aún no se dispone de biomarcadores no invasivos fiables. La participación del sistema inmune también es respaldada por el porcentaje de animales curados observados en esta tesis (50-100% dependiendo de la terapia), los cuales presentaron memoria inmune contra subsecuentes intentos de generación del mismo tipo de tumor. La resistencia a TMZ es una de las causas del fracaso de la quimioterapia adyuvante en el tratamiento de GB. Hemos investigado el papel de la O6-methylguanine-DNA-methyltransferase (MGMT) y programmed death-ligand 1 (PD-L1) en la quimioresistencia, utilizando western-blot, en tumores que escapan a la terapia IMS-TMZ después de presentar respuesta transitoria. La expresión de PD-L1 se triplica en esos tumores en comparación con controles, indicando que dichos niveles podrían ser relevantes en la resistencia a la TMZ in vivo, teniendo la terapia anti-PD-1 potencial para ‘rescatar’ tumores escapando de la terapia con TMZ. La combinación de oncología e inmunología guiará el camino para una mejorar tanto la eficacia de la terapia como los resultados obtenidos con pacientes de GB.
Glioblastomas (GB) are invasive brain tumours associated with poor prognosis and limited response to therapy. This thesis focused in improving preclinical GB treatment through cancer-related immunity and Magnetic Resonance Spectroscopic Imaging (MRSI)-based non-invasive response follow-up. The GL261 GB was chosen since it is an immunocompetent preclinical model suitable for studying therapies. Three therapeutic strategies have been tested: a) chemotherapy (Temozolomide, TMZ), administered in an Immune-Enhancing Metronomic Schedule (IMS-TMZ), b) immune checkpoint inhibitor (Programmed cell death protein 1, PD-1 antibody) and c) IMS-anti-PD-1/TMZ combination therapy. Anti-tumour immune responses can be stimulated by therapies targeting different aspects of cell damage. We aimed, on one hand, to induce immunogenic tumour cell damage while sparing replicating immune system cells (with IMS-TMZ). On the other hand, we wanted to counteract the immune suppression within the tumour (anti-PD-1 immunotherapy). IMS-TMZ significantly improved survival in GL261 GB-bearing mice in comparison with standard TMZ treatment, confirming and surpassing results reported by our group. Anti-PD-1 monotherapy was effective when applied at high dose (500/250 μg), although care should be taken since results suggest that differences in tumour volume at immunotherapy starting time can have great impact in its efficacy. As expected, the IMS-anti-PD-1/TMZ combination therapy showed a great beneficial effect, with much better therapeutic outcome than monotherapies administration. These results support the fact that the host immune system is clearly involved in GB response processes. Previous studies from our group with MRSI-based nosological images pointed that the metabolomic pattern changes could be linked to host immune system local effects onto tumours, acting as a surrogate biomarker of therapy response. Accordingly, we wondered whether the application of this non-invasive MRSI approach in evaluating immunotherapeutic strategies would reflect the same type of metabolomics changes. Thus, the evolution of GL261-tumor bearing mice treated with IMS-TMZ, IMS-anti-PD-1 and IMS-anti-PD-1/TMZ was evaluated using the same MRSI-based nosological images approach. Results confirmed that the IMS-TMZ protocol consistently produced the expected oscillatory changes in the MRSI metabolomics pattern, with a frequency of ca. 6 days. This oscillatory behaviour was also confirmed in mice treated with immunotherapy both in combination with TMZ and as monotherapy, hinting that the observed spectral pattern changes observed during therapy response are shared by different therapeutic strategies, provided the host immune system is elicited and is able to productively attack tumour cells. This opens the way for a translational use of the MRSI-based biomarker for patient-tailored GB therapy, including immunotherapy, for which reliable non-invasive biomarkers are still missing. The participation of immune system is also supported by the rate of cured animals observed in this thesis (range 50 - 100 % depending on the treatment), which also held long-term immune memory against tumour cell re-challenge. Resistance to TMZ treatment is one of the main reasons for the chemotherapy failure in adjuvant treatment of GB. We investigated the relevance of O6-methylguanine-DNA-methyltransferase (MGMT) and programmed death-ligand 1 (PD-L1) content in chemoresistance, by western-blot (WB) analysis, with special focus on tumours escaping therapy after transient response. Result showed a 3-fold increase in PD-L1 expression in IMS-TMZ relapsing tumours in comparison with control tumours, indicating that PD-L1 can be involved in TMZ resistance for GL261 GB in vivo. Accordingly, anti-PD1 therapy may have potential to ‘rescue’ tumours escaping from TMZ therapy. Appropriate combination of oncology and immunology will pave the way for improving GB treatment and patient outcome.

La médecine nucléaire a connu ces dernières années d’importantes évolutions technologiques grâce à l’arrivée de caméras dites numériques, qui reposent sur l’utilisation de détecteurs semi-conducteurs. En tomographie d’émission monophotonique (TEMP), cette technologie a d’abord été introduite sur des caméras CzT dédiées à l’exploration cardiaque. Plus récemment, des caméras CzT 360° à champ de vue étendu ont été développées pour permettre des explorations tomographiques corps entier. De même, les caméras utilisées en tomographie par émission de positon (TEP) ont connu une transition vers des technologies numériques grâce à l’utilisation de nouveaux détecteurs SiPM. Ce travail a permis d’évaluer les performances de ces caméras TEMP et TEP numériques en médecine nucléaire dans le domaine de l’oncologie clinique et préclinique. Nous avons ainsi démontré les gains en sensibilité, en résolution en énergie et en contraste image obtenus à l’aide des caméras CzT 360° par rapport aux caméras de Anger conventionnelles. Il a également été démontré la faisabilité d’utiliser ce type de caméra, initialement développée pour l’homme, pour réaliser de l’imagerie chez le petit animal. Dans un second temps, nous avons évalué sur fantôme les caméras TEP SiPM pour une utilisation en préclinique. Nous avons ainsi mesuré des performances s’approchant de celles obtenues avec des caméras microTEP dédiées, en permettant l’imagerie simultanée de quatre animaux, et montré la possibilité de réaliser de la quantification avec une précision suffisante en oncologie préclinique
In recent years, nuclear medicine has undergone significant technological advances with the introduction of digital cameras based on the use of semiconductor detectors. In single-photon emission computed tomography (SPECT), this technology was first introduced on dedicated cardiac CzT cameras. More recently, 360° CzT cameras with extended field-of-view have been developed to enable whole-body tomographic explorations. Similarly, the cameras used in positron emission tomography (PET) have undergone a transition to digital technologies thanks to the use of new SiPM-based detectors. This work has allowed for the evaluation of the performance of these digital SPECT and PET cameras in nuclear medicine, within the field of clinical and preclinical oncology. We have thus demonstrated the improvements in sensitivity, energy resolution, and image contrast achieved through the use of 360° CzT cameras compared to conventional Anger cameras. Additionally, we demonstrated the feasibility of using this type of camera developed for humans, to perform preclinical imaging in small animals. Furthermore, we have evaluated SiPM-based PET cameras using phantoms for potential preclinical applications. We have thus measured performance approaching that obtained with dedicated microPET cameras, enabling simultaneous imaging of four animals, and demonstrated the ability to perform accurate quantification in preclinical oncology

Purpose: Colorectal cancer (CRC), the third most common cancer diagnosed in both men and women shows a highly ineffective therapeutic management. In contrast, CRC is a rare pediatric tumor, representing only 1% of all pediatric malignancies, with an incidence of approximately 1 per million. In this context, an urgent needing not yet addressed is the random assignment to adjuvant chemotherapy of high-risk stage II and stage III colon cancer patients, both young and adults, without any predictive factor of efficacy. Secondly, in the field of drug discovery the critical step is the preclinical evaluation of drug cytotoxicity, efficacy, and efficiency. We purpose to develop a patient-derived 3D preclinical model useful for drug evaluation that can mimic in vitro the patient's disease. Methods: Surgically resected healthy colon mucosa and matched CRC were decellularized by a detergent-enzymatic treatment (DET). DET scaffolds were recellularized with HT29 cells. Qualitative and quantitative characterization of matched recellularized samples were evaluated through histology, immunofluorescences and DNA amount quantification. Chemosensitivity test was performed using increasing concentration of 5-Fluorouracil (5-FU), range 0.1 µM to 100 µM. In vivo studies were carried out using the zebrafish (Danio rerio) animal model. For cancer xenograft assays, HT29 cells were injected into the duct of Couvier and subsequently incubated in 96-well plates with different concentrations of 5-FU. Drug absorption and perfusion along fresh and DET tumor scaffolds were evaluated qualitative using autofluorescence of doxorubicin (doxo, 594nm) and quantitative calculated by Darcy’s law. Buffy coat-derived monocytes were cultured with DET scaffolds and macrophages lineage markers were evaluated with flow cytometry. Results: Decellularization protocol allowed the preservation of original structure and ultrastructure (SEM analysis). Five days after recellularization with HT29 cell line, the 3D CRC model exhibited reduced sensitivity to 5-FU treatments compared with conventional 2D culture. Calculated IC50 resulted in 11.5 µM and 1.3 µM of 5-FU, respectively. In the zebrafish transplantation model, HT29 extravasation was detected after 4 days post injection. Moreover, we obtained a 5-FU IC50 comparable with that observed in the 3D CRC model. Using confocal microscopy, we demonstrated that doxorubicin diffuses through the volume of 3D CRC model and co-localize with the cell nuclei which repopulate the 3D CRC scaffold. Finally, we observed that monocytes exposed to tumor decellularized ECM differentiated towards a pro-tumoral anti-inflammatory macrophage-like profile. Conclusion: 3D CRC model could be preclinical reliable tool to bridge the gap between in vitro, in vivo and ex vivo drug testing assays. The 3D CRC model, translated in the pediatric setting, could help clinicians and oncologists to identify the most suitable treatment for the patient.

Malignant melanoma is the most lethal skin cancer with no effective therapeutic treatment in its metastatic stages. RAS and PI3K pathways have been shown to play a critical role in melanoma development and progression. In this study, we assessed the in vitro and in vivo inhibition potential of a BRAF inhibitor (Sorafenib, Bayer) and a PI3K/mTOR inhibitor (PI-103, PIramed-Genentech) in primary melanoma cell lines. We used primary cell lines isolated from spontaneous melanomas obtained in the UV induced HGF transgenic melanoma mouse model.

Although PI-103 and sorafenib inhibited melanoma in vitro cell proliferation and viability, the inhibition of RAS pathway was more effective. The combination of the two drugs showed a synergistic effect inhibiting RAS and PI3K pathways and in vitro melanoma cell proliferation in a cell line dependent manner. However, the combined treatment of orthotopic xenographs in immunocompetent FVB mice did not cooperate blocking tumor growth. Surprisingly, the in vivo treatment with PI-103 enhanced tumor growth. Our results also revealed that PI-103 caused immunosuppression inducing thymus atrophy and upregulating the intratumoral transcriptional levels of inmunosuppressors. In addition, PI-103 induced the antiapoptotic BH3 family proteins Mcl-1, Bcl-2 and BclXL, which correlated with the lower apoptotic rate observed within the PI-103 treated tumors.

These data indicates that due to melanoma heterogeneity, some precautions should be taken when using these inhibitors for treatment. Moreover, these results certainly make an argument for investigating unexpected effects of rational drug combinations on immunocompetent animal models before conducting clinical studies.

In 2018, it is estimated that 55,440 Americans will be diagnosed with pancreatic cancer and this figure is expected to continue to rise with increased life expectancy. Despite some measurable progress over the past few decades, pancreatic cancer remains one of the most lethal malignancies with five-year survival rate of 8.7%. Novel therapies, and their timely translation to the clinic, are urgently needed. As part of an effort to identify and characterize novel therapeutic strategies for pancreatic ductal adenocarcinoma, we began a study of the role of Bmi1 in tumor maintenance and progression. While Bednar and colleagues showed that Bmi1 is critical for the development of pancreatic cancer, and that its pancreas-specific deletion impairs PanIN formation, we were interested in assessing its function in established tumors. During the course of this work, we acquired a novel compound, PTC596, developed by PTC Therapeutics as a post-translational inhibitor of BMI1. Treatment with PTC596 leads to hyperphosphorylated BMI1, and this modification is associated a loss of protein activity. We planned to study this compound, in vitro and in vivo, as a complement to genetic perturbations of Bmi1. Initial characterizations of the effects of PTC596 on human and murine-derived pancreatic cancer cell lines revealed a potent anti-proliferative effect, accompanied by BMI1 hyperphosphorylation, and followed by polyploidy and cell death after prolonged treatment. Further analysis showed a clear G2/M arrest and elevated levels of phospho-histone H3. Bmi1 is known to play a role the cell cycle, but its inhibition in pancreatic cancer cell lines has been shown to induce G1 arrest. We decided to further explore the mechanism of PTC596’s antiproliferative effects by carrying out RNA sequencing on Aspc1 cells treated with PTC596. We found that 8 of the ten most down-regulated genes were members of the tubulin family and began to study this compound’s effect on microtubules. Compelling results from a cell-free tubulin polymerization assay support inhibition of tubulin polymerization as the mechanism of action for PTC596. These data are further supported by evidence that PTC596 increases the fraction of free-tubulin in treated cells, as well as dramatically alters the cell’s microtubule network. Given our laboratory’s interest in identifying novel therapies for pancreatic cancer, and the fact that PTC596 has already begun clinical trials, we continued to characterize this compound in vivo. We found PTC596 to have properties favorable for in vivo administration. PTC596 is orally available, has a plasma half-life of approximately 22 hours following oral administration, and accumulates in tumor tissue where it has an expected pharmacodynamic effect. Furthermore, it is well tolerated in vivo in combination with gemcitabine. We carried out a four-arm intervention study in tumor-bearing KPC mice, examining PTC596 alone and in combination with gemcitabine. We found that PTC596 synergizes with gemcitabine to significantly reduce tumor growth rates and provide a 3-fold extension of survival as compared to vehicle. These findings are, to our knowledge, the first evidence of in vivo synergy between a microtubule-destabilizing agent and gemcitabine for the treatment of pancreatic cancer. Importantly, this study identifies an alternative mechanism for PTC596 and implicates its efficacy in a novel treatment regimen for pancreatic ductal adenocarcinoma.

L’adénocarcinome épithélial de l’ovaire (CEO) est le cancer gynécologique le plus mortel. La recherche de nouvelles thérapies repose principalement sur des modèles précliniques 2D et in vivo avec des lignées cellulaires (LC) pour générer les évidences nécessaires à l’initiation d’essais cliniques. Ce processus requiert des fonds substantiels en recherche/santé qui est associé à un taux d’attrition élevé laissant supposer des lacunes dans le modèle actuel. Nos publications antérieures suggèrent que la sensibilité in vitro de nos LC du CEO à la chimiothérapie carboplatin varie en 2D ou 3D. Il reste à élucider lequel de ces modèles est le plus représentatif de la réponse in vivo. De ce fait, nous avons émis l’hypothèse que le modèle 3D refléterait plus étroitement la sensibilité in vivo. L’objectif de cette étude était de caractériser la réponse au carboplatin de nos LC du CEO en monocouches et en sphéroïdes (3D), puis de les comparer à leur réponse in vivo (xénogreffes). Un total de 6 LC du CEO a été injecté dans des souris qui ont reçues trois différentes concentrations de carboplatin. Leurs réponses ont été évaluées/classées selon leurs mesures de volume tumoral et l’immunofluorescence. Ces mêmes LC ont été ensemencées dans des plaques à très faible adhérence pour former des sphéroïdes et les traiter. Des analyses de cytométrie en flux ont été effectuées afin de classer les LC selon leur concentration inhibitrice médiane (CI50). Nous avons comparé le tout aux résultats 2D (CI50) précédemment publiés. Nos résultats montrent que le système 3D démontre la meilleure concordance avec le modèle in vivo. Notamment, notre LC ultra-résistance en 2D devient plus sensible en modèle murin ou encore en 3D. Inversement, une LC ultra-sensible en 2D est plus résistante en xénogreffe et en sphéroïde. Les résultats découlant de notre étude sont importants à considérer lors d’investissement de temps et de fonds dans les études de criblage et de prédiction de réponses thérapeutiques.
Epithelial ovarian adenocarcinoma (EOC) is the most lethal gynecological cancer. The drug discovery pipeline is heavily based on preclinical models. Typically, 2D cell line (CL)-based models are used to screen compounds followed by validation in animal models to generate the evidence needed to design clinical trials. This process incurs a high cost to the research pipeline and still results in high drug attrition rates. This may in part reflect the poor translation of preclinical to clinical results and points to deficiencies in modeling. Previous work from our laboratory shows that the sensitivity of our EOC CLs to carboplatin therapy varies between 2D and 3D in vitro models, however it is unclear how these differences align with the in vivo response. We hypothesize that 3D models will more closely reflect therapeutic in vivo response. The objective of this study was to characterize the carboplatin sensitivity of EOC CLs in 2D and 3D-spheroids and compare them to in vivo response using mouse xenografts. We injected mice with 6 different EOC CLs that were treated with 3 different carboplatin concentrations. Tumor volume measurements and immunofluorescence viability stains were used to categorize CLs by their sensitivity. The same CLs were seeded in low attachment plates to form, and thereafter treat, spheroids. Flow cytometry analysis was used to classify CLs by their 50% inhibitory response (IC50). The 2D response (IC50) for these CLs has previously been published. Our results show that therapeutic response changes significantly for a single CL between different systems, and the 3D model was most concordant with the in vivo model. Our ultra-resistant CL in 2D became more sensitive in 3D/mouse models. In contrast, the highly 2D sensitive CL became more resistant in our xenograft/spheroid models. The results are important to consider when investing time/funds in drug screening and therapeutic response prediction studies.

Les tumeurs des cellules de la granulosa (GCTs) sont des tumeurs avec un potentiel malin ayant tendance à récidiver, provoquant ainsi la mort dans 80% des cas de stade avancé consécutif à une rechute. Bien que les GCTs représentent 5% des tumeurs ovariennes, peu d’études ont évalué les protocoles de traitement adjuvant pour la maladie avancée ou récurrente. Notre but était d’évaluer l’efficacité de la voie de signalisation du facteur de croissance de l’endothélium vasculaire A (VEGFA) comme cible pour le traitement de la GCT utilisant le modèle murin transgénique Ptentm1Hwu/tm1Hwu; Ctnnb1tm1Mmt/+; Amhr2tm3(cre)Bhr/+ (PCA) qui reproduit le stade avancé de la maladie humaine. Un anticorps anti-VEGFA a été administré une fois par semaine par voie intrapéritonéale (IP) à partir de 3 semaines d’âge. La thérapie anti-VEGFA a permis une réduction de la taille des tumeurs à 6 semaines d’âge (p<0.05) et une prolongation de la survie des animaux traités, lorsque comparé aux animaux contrôles. L’analyse des GCTs a montré une réduction significative de la prolifération cellulaire (p<0.05) et de la densité microvasculaire (p<0.01) mais aucune différence significative n’a été détectée dans l’apoptose cellulaire. p44/p42 MAPK, un effecteur de la signalisation pour le récepteur 2 de VEGFA (VEGFR2) associé à la prolifération cellulaire, était moins activé dans les tumeurs traitées (p<0.05). Par contre, l’activation d’AKT, un effecteur impliqué dans la survie cellulaire, était similaire d’un groupe à l’autre. Ces résultats suggèrent que l’anticorps anti-VEGFA réduit la prolifération cellulaire et la densité microvasculaire chez les souris PCA par inhibition de la voie de signalisation VEGFR2-MAPK, inhibant ainsi la croissance tumorale. En conclusion, l’efficacité de la thérapie anti- VEGFA mérite d’être évaluée en essais contrôlés randomisés pour le traitement des GCTs chez l’homme.
Ovarian granulosa cell tumors (GCTs) are potentially malignant tumors that have a tendency for late recurrence and cause death in 80% of women with advanced GCT due to recurrent disease. Although GCTs represent 5% of ovarian tumors in women, few studies have evaluated adjuvant treatment protocols for advanced or recurrent disease. Our goal was to determine the potential of targeting the vascular endothelial growth factor A (VEGFA) signaling pathway for the treatment of GCT. We used a genetically engineered mouse model, Ptentm1Hwu/tm1Hwu; Ctnnb1tm1Mmt/+; Amhr2tm3(cre)Bhr/+ (PCA), which imitates the advanced human disease. A monoclonal anti-VEGFA antibody was administered by intra-peritoneal injection once a week beginning at 3 weeks of age. Anti-VEGFA therapy significantly decreased tumor weights by 6 weeks of age (p<0.05) and increased survival in treated animals in comparison to controls. Significant decreases in tumor cell proliferation (p<0.05) and microvessel density (p<0.01), but no significant difference in apoptosis was found in PCA tumors. p44/p42 MAPK, a VEGFA receptor 2 (VEGFR2) signaling effector associated with cell proliferation, was significantly less activated in anti-VEGFA-treated tumors (p<0.05). In contrast, AKT activation, a VEGFR2 signaling effector associated with cell survival was similar among all groups. These results suggest that anti-VEGFA therapy effectively reduces cell proliferation and microvessel density in PCA mice by inhibition of the VEGFR2-MAPK pathway, resulting in inhibition of GCT growth. We conclude that anti-VEGFA therapy merits further investigation in the form of controlled randomized trials for the treatment of human GCT.

Renal Cell Carcinoma (RCC) is the most common form of kidney tumor, accounting for approximately 3% of all adult malignancies. To date, RCC is still a difficult disease to diagnose and treat. Although the surgery is the standard therapy for localized tumors, one quarter of patients who underwent nephrectomy, relapse within three years. Moreover, one third of patients arrives with metastases at diagnosis. Unfortunately, the metastatic disease is generally characterized by therapy resistance and very poor outcomes. So far, the lack of valid preclinical RCC models has hampered the discovery of valuable diagnostic and prognostic biomarkers and predictive indicators of therapy response for improving patients' management. In the project, we focused our efforts on the optimization of new patient-derived preclinical models for RCC. We first isolated heterogeneous undifferentiated cell populations responsible for tumor propagation and cancer therapy resistance. By performing a phosphoproteomic analysis we identified a protein signature predictive of cancer progression that would help to select patients more likely to relapse after surgery and who may benefit of adjuvant therapy. We then established an orthotopic patient-derived xenograft (PDX) model that faithfully recapitulate grading, histology and molecular characteristics of the parental tumors. The PDX model proved to be an indicator of bad prognosis and patient tumor could be propagated for up to seventh generation in mice. These findings support the possibility to use PDXs as a platform for patient monitoring and for drug testing. Finally, we were able to establish and characterize, for the first time, long term organoid cultures from normal and tumor samples. All together, these three new models provide innovative and valuable tools for RCC research, suggesting many potential applications for reproducing disease progression models, for biomarkers discovery and drug testing.