The aim of this thesis is to investigate the role of inflammatory infiltrates and chemokines in metastasis of high-grade serous ovarian cancer, HGSC, to the omentum using human tissue biopsies and a 3- dimensional (3D) cell culture model. In ten patients with metastatic HGSC, omental tumour deposits contained a prominent leukocyte infiltrate of CD3+ T cells (9% of total cells) and CD68+ macrophages (11% of total cells). The presence of CD68+ macrophages showed a significant positive correlation with tumour cell proliferation analysed by Ki67 expression. Four ovarian cancer cell lines were co-cultured on a 3D model mimicking the microenvironment of the omentum for two weeks. The model was composed of collagen embedded human fibroblasts covered in a confluent layer of human primary mesothelial cells. The mesothelial cells in the 3D model significantly increased the growth (p = 0.002) and invasion (p = 0.0004) of the ovarian cancer cells. CXCL12 is the macrophage chemoattractant and ligand for the major chemokine receptor expressed on ovarian cancer cells. An association between CXCL12 and extracellular matrix remodelling was identified in two independent gene expression microarrays of ovarian cancer biopsies. The expression of CXCL12 in the HGSC omental metastases measured by quantitative Real Time-PCR positively correlated with decorin expression. Antibody mediated neutralisation of CXCL12 reduced growth (p = 0.012) and invasion (p = 0.029) in the 3D model. Mimicking an infiltrate of CD68+ macrophages in this multicellular 3D in vitro system also produced measurable changes in inflammatory cytokine and chemokine expression. There is currently a demand for more accurate models of HGSC and a necessity to study its metastasis that presents itself as the major clinical problem in patients. Therefore the development of this 3D model to mimic tumour-promoting inflammation in HGSC metastasis will provide researchers with an essential tool for testing novel therapeutic strategies.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20662号 / 医博第4272号 / 新制||医||1024(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 松田 道行, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The tumour microenvironment comprises the non-cancerous cells present in the tumour mass (fibroblasts, endothelial, and immune cells), as well as signalling molecules and extracellular matrix. Tumour growth, invasion, metastasis, and response to therapy are influenced by the tumour microenvironment. Therefore, characterising the cellular and molecular components of the tumour microenvironment, and understanding how they influence tumour progression, represent a crucial aim for the success of cancer therapies. High-grade serous ovarian cancer provides an excellent opportunity to systematically study the tumour microenvironment due to its clinical presentation of advanced disseminated disease and debulking surgery being standard of care. This thesis first presents a case report of a long-term survivor (>10 years) of metastatic high-grade serous ovarian cancer who exhibited concomitant regression/progression of the metastatic lesions (5 samples). We found that progressing metastases were characterized by immune cell exclusion, whereas regressing metastases were infiltrated by CD8+ and CD4+ T cells. Through a T cell - neoepitope challenge assay we demonstrated that pre- dicted neoepitopes were recognised by the CD8+ T cells obtained from blood drawn from the patient, suggesting that regressing tumours were subjected to immune attack. Immune excluded tumours presented a higher expression of immunosuppressive Wnt signalling, while infiltrated tumours showed a higher expression of the T cell chemoattractant CXCL9 and evidence of immunoediting. These findings suggest that multiple distinct tumour immune microenvironments can co-exist within a single individual and may explain in part the hetero- geneous fates of metastatic lesions often observed in the clinic post-therapy. Second, this thesis explores the prevalence of intra-patient tumour microenvironment het- erogeneity in high-grade serous ovarian cancer at diagnosis (38 samples from 8 patients), as well as the effect of chemotherapy on the tumour microenvironment (80 paired samples from 40 patients). Whole transcriptome analysis and image-based quantification of T cells from treatment-naive tumours revealed highly prevalent variability in immune signalling and distinct immune microenvironments co-existing within the same individuals at diagnosis. ConsensusTME, a method that generates consensus immune and stromal cell gene signatures by intersecting state-of-the-art deconvolution methods that predict immune cell populations using bulk RNA data was developed. ConsensusTME improved accuracy and sensitivity of T cell and leukocyte deconvolutions in ovarian cancer samples. As previously observed in the case report, Wnt signalling expression positively correlated with immune cell exclusion. To evaluate the effect of chemotherapy on the tumour microenvironment, we compared site-matched and site-unmatched tumours before and after neoadjuvant chemotherapy. Site- matched samples showed increased cytotoxic immune activation and oligoclonal expansion of T cells after chemotherapy, unlike site-unmatched samples where heterogeneity could not be accounted for. In addition, low levels of immune activation pre-chemotherapy were found to be correlated with immune activation upon chemotherapy treatment. These results cor- roborate that the tumour-immune interface in advanced high-grade serous ovarian cancer is intrinsically heterogeneous, and that chemotherapy induces an immunogenic effect mediated by cytotoxic cells. Finally, the different deconvolution methods were benchmarked along with ConsensusTME in a pan-cancer setting by comparing deconvolution scores to DNA-based purity scores, leukocyte methylation data, and tumour infiltrating lymphocyte counts from image analysis. In so far as it has been benchmarked, unlike the other methods, ConsensusTME performs consistently among the top three methods across cancer-related benchmarks. Additionally, ConsensusTME provides a dynamic and evolvable framework that can integrate newer de- convolution tools and benchmark their performance against itself, thus generating an ever updated version. Overall, this thesis presents a systematic characterisation of the tumour microenvironment of high grade serous ovarian cancer in treatment-naive and chemotherapy treated samples, and puts forward the development of an integrative computational method for the systematic analysis of the tumour microenvironment of different tumour types using bulk RNA data.

Le cancer de l’ovaire (COv) est le cancer gynécologique le plus létal chez la femme et les traitements existants, chirurgie et chimiothérapie, ont peu évolué au cours des dernières décennies. Nous proposons que la compréhension des différents destins cellulaires tels que la sénescence que peuvent choisir les cellules du cancer de l’ovaire en réponse à la chimiothérapie pourrait conduire à de nouvelles opportunités thérapeutiques. La sénescence cellulaire a été largement associée à l’activité de la protéine TP53, qui est mutée dans plus de 90% des cas de cancer de l’ovaire séreux de haut grade (COv-SHG), la forme la plus commune de la maladie. Dans nos travaux, à partir d’échantillons dérivés de patientes, nous montrons que les cultures primaires du cancer de l’ovaire séreux de haut grade exposées au stress ou à des drogues utilisées en chimiothérapie entrent en senescence grâce à l’activité d’un isoforme du gène CDKN2A (p16INK4A). Dans ces cellules, nous avons évalué les caractéristiques fondamentales de la sénescence cellulaire tels que les altérations morphologiques, l’activité béta galactosidase associée à la sénescence, les dommages à l’ADN, l’arrêt du cycle cellulaire et le phénotype sécrétoire associé à la sénescence. En utilisant des micromatrices tissulaires construites à partir d’échantillons humains de COv-SHG pré- et post-chimiothérapie, accompagnées de leurs données cliniques, nous avons quantifié des marqueurs de sénescence incluant une diminution de la prolifération cellulaire quelques semaines après chimiothérapie. De façon intéressante, l’expression de p16INK4A dans les échantillons de COv-SHG prétraitement corrèle avec une survie prolongée des patientes suite au traitement. Ceci suggère ainsi pour la première fois un impact biologique bénéfique pour la présence de cellules cancéreuses qui sont capable d’activer la sénescence, particulièrement pour le traitement du cancer de l’ovaire. Dans le but de complémenter les thérapies actuelles avec des approches de manipulation pharmacologique de la sénescence, nos résultats suggèrent qu’il serait important de déterminer l’impact positif ou négatif de la sénescence induite par la thérapie sur la progression de la maladie et la survie, pour chaque type de cancer de façon indépendante. / Human ovarian cancer (OvCa) is the deadliest gynecologic malignancy and existing surgical/chemotherapeutic treatment options have been relatively static for decades. We propose that understanding OvCa cell fate decisions taken in response to chemotherapy could guide new therapeutic opportunities. Damage-induced cellular senescence is often associated with TP53 activity, which is heavily mutated in high grade serous (HGS) OvCa (>90%), the most common form of this disease. Here, using patient derived tissues, we show that primary HGS-OvCa cultures predominantly trigger CDKN2A- associated (p16INK4A isoform) senescence following exposure to stress or chemotherapy. Key senescence hallmarks including altered morphology, senescence-associated-Betagalactosidase, DNA damage, cell cycle arrest and the senescence-associated secretory phenotype were evaluated and detected in damaged cells. Using tissue microarrays built from pre- and post-treatment human HGS-OvC tissue samples with accompanying clinical data, we quantified post-treatment hallmarks of senescence including reduced cell proliferation weeks after chemotherapy. Importantly, p16INK4A expression in pretreatment HGS-OvC samples correlated with increased patients survival, suggesting for the first time that senescence-competence in human cancer cells may have a beneficial impact on treatment outcomes for patients. In order to guide the potential improvement of existing human therapies via pharmacological senescence manipulation, our results suggests that it is important to determine for many types of human cancer whether treatment-induced senescence positively or negatively impacts disease progression and patient survival.