Gene expression analysis of metastases and IL-1R1 characterization in a NALP3 mouse model

Tumor microenvironment contributes strongly to tumor evolution toward metastasis, a final stage in cancer progression. It provides essential clues to promote migration of cancer cells in metastatic sites. Moreover, inflammatory cells and immunomodulatory mediators present in the microenvironment are able to polarize the host immune response, thereby potentiating primary tumor development. The reciprocal and dynamic interactions between microenvironment and tumor cells orchestrate critical steps of evolution toward metastasis. To address the role of inflammation in tumor progression, a mouse model of downregulated inflammation was used: the NALP3 gene in these mice is knocked-out, causing a impaired processing of the two inflammatory cytokines Interleukin-1[beta] and IL-18 which are essential for the recruitment and activation of immune cells. In this case, a decrease in tumor growth and metastasis was observed in previous studies, leading us to investigate the molecular and cellular interactions of tumor and immune cells in inflammatory and non-inflammatory contexts. Gene expression analysis was performed on two steps of mouse tumor cells: the B16-F10 melanoma cells seem to be more dependent on the immune cells recruitment to metastasize in vivo, while the Lewis Lung Carcinomas cells showed a quite equivalent growth in both wild-type or NALP3 knock-out mice. Different techniques were used to do so, such as standard in vitro assays and mRNA expression analyses, and in vivo tests with extraction of tumoral mRNA by Laser Capture Microdissection. Gene expression analyses of tumor cells were performed to address the influence of the inflammatory background. Comparison of these two gene expression profiles may reveal genes related to the specific reactive inflammatory response surrounding the given metastases. Another point that was investigated was the importance of the IL-1R1, receptor of interleukin-1. Since it represents the counterpart of IL-1[beta] signaling, its expression by immune and tumor cells may correlate with degree of tumor growth and invasion. In vitro and in vivo assays allowed better understanding the importance of this receptor. While a downregulation of this receptor impairs proliferation of in vitro cell cultures, at the in vivo level, the number of metastases is decreased too. These results provide potential clues indicating the important role played by the inflammatory cells, and how they can support and promote tumor progression, in particular through the expression of NALP3. The adaptation mechanisms of tumor cells to their microenvironment were also assessed, opening different insights for targeting the tumor itself as well as the inflammatory microenvironment.

New roles of the lymphatic endothelium in modulating adaptive immunity: implications for immune tolerance and cancer immunotherapy

Lambert Charles François Potin

Lymphatic vessels have traditionally been considered as passive conduits for interstitial fluid drainage and for immune cell trafficking to lymph nodes. In the last two decades, many studies have challenged this classical dogma: lymphatic endothelial cells (LECs) were shown to actively interact with immune cells by secreting chemokines and cytokines, and presenting antigens to T cells. These newly described functions pinpoint a multi-faceted role of LECs in orchestrating the immune response through both passive and active mechanisms. Yet, precise mechanisms leading to antigen-specific fine-tuning of the T cell response remain unexplored. Moreover, although lymphatics have been shown to play important roles in cancer, the contributions of LECs in regulating antitumor immunity have been largely overlooked by the biomedical community. This doctoral thesis aimed at filling these gaps. First, in order to investigate the mechanisms and implications of LEC antigen presentation to CD4+ T cells, we engineered a mouse model where LECs lacked the ability to present antigens to CD4+ T cells. In these mice, antigen-specific responses to vaccination were increased when compared to wild-type controls. Moreover, we demonstrated that LECs were poor inducers of CD4+ T cell activation in vitro, and that they could dampen CD4+ T cell activation by dendritic cells. Taken together, these findings suggest that LECs participate to the peripheral immune tolerance of CD4+ T cells. Second, we investigated the immunological consequences of the development of lymphatic vessels, called lymphangiogenesis, in mouse melanoma. Upon induction of tumor lymphangiogenesis, we observed striking tumor enrichment with immune cells, which resulted in a higher sensitivity of those tumors to immunotherapy. We subsequently demonstrated that inducing lymphangiogenesis in a mouse model of cold tumors, which lack immune infiltrates and do not respond to immunotherapies, could restore their responsiveness to immunotherapy treatments. Third, to predict which cancer types were the most likely to replicate the findings we obtained in mouse melanoma, we mined The Cancer Genome Atlas database for gene expression. We identified a set of cancers, such as colon adenocarcinoma and cholangiocarcinoma, where high expression of the lymphangiogenic growth factor was associated with a strong T cell signature. This doctoral thesis unveiled new immunomodulatory functions of LECs by which antigen-specific immune tolerance is induced. Additionally, it opened new avenues to target lymphatics therapeutically in cancer to potentiate immunotherapies. We believe this work contributes to demonstrating the immunological importance of LECs, and hope that lymphatics will be considered as an important immunomodulatory player of the tumor microenvironment when developing novel immunotherapies.

EPFL2018

The role of Snail in non-small cell lung cancer

Svenja Johanna Groeneveld

The epithelial-to-mesenchymal transition (EMT) is a developmental program frequently reactivated in cancer. It plays an important role in several aspects of tumor progression, particularly in the acquisition of invasive capacities facilitating metastasis. Beyond invasion, EMT endows cancer cells with additional characteristics essential for metastatic dissemination and has been extensively studied in breast and colorectal cancer. Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide and the majority of NSCLC patients is diagnosed with metastatic disease. The expression of the key EMT transcription factor Snail is correlated with a poor prognosis in NSCLC patients, while it is unclear whether Snail contributes to disease progression by actually facilitating metastasis formation. During EMT, the metabolism of a cancer cell changes, likely to meet the environmental challenges faced during the metastatic process. In this line, we have previously reported (Masin et al, 2014) that the glucose transporter GLUT3 is upregulated during EMT in human NSCLC cell lines. Here, we demonstrate that the rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP) correlates with GLUT3 in NSCLC. This pathway produces a substrate for O-linked protein GlcNAcylations important during EMT, as they â for example â stabilize the Snail protein. Glutamine-fructose-6-phosphate amidotransferase 2 (GFPT2) was specifically upregulated during EMT, while its isoenzyme GFPT1 was unaffected. Furthermore, our results points towards a putative regulation of GLUT3 expression by GFPT2, possibly involving STAT3 signaling. We thus provide evidence for a possible reactivation of a developmental metabolic program during EMT in NSCLC. Chronic inflammation is an important disease-promoting factor during lung tumorigenesis, emphasizing the role of the immune system in this cancer type. Overexpressing or silencing Snail in the immunocompetent autochthonous KrasLSL-G12D/+;p53fl/fl mouse model of lung adenocarcinoma uncovered a substantial influence of Snail on the tumor microenvironment. An extensive analysis of tumor histology, gene and protein expression and immune infiltration revealed that while Snail did not contribute to metastasis formation, it accelerated growth and malignant progression of the lung tumors, reducing the survival time of the mice. Snail decreased B lymphocyte, while enhancing neutrophil infiltration of the tumors. Intriguingly, through the secretion of a soluble factor, Snail induced a feed-forward loop of neutrophil recruitment via Cxcl2, produced by neutrophils themselves. In our recently published collaborative work (Faget, Groeneveld et al, 2017), we identified neutrophils as main contributors to disease progression. We furthermore provided evidence for a vicious cycle formed between Snail expressing cancer cells and neutrophils in lung tumors, as neutrophils were found to impair angiogenesis, resulting in hypoxia and enhanced Snail expression. In addition, we found that Snail repressed the Dlk1-Dio3 locus, containing the genomeâs largest miRNA cluster and recently implicated in NSCLC, in tumor-infiltrating immune cells via a paracrine mechanism.

EPFL2018

Bioengineering Methods to Improve in vitro Adoptive Cell Therapy based on Tumor-Infiltrating Lymphocytes

François Réal Rivest

Cancer is one of the biggest medical challenges of our times, with a devastating impact across all social categories. Accordingly, considerable efforts are geared towards improving the standard of care. Among them, immunotherapy, which relies on the immune system's capacity to fight cancer cells, brings new hope for higher success rates and in some cases even a cure of this devastating disease. A particularly interesting approach called adoptive cell therapy, or ACT, is based on immune cells that have infiltrated tumors and have the potential to fight them. Unfortunately, these tumor-infiltrating lymphocytes (TILs) are very rare and their activity is often suppressed by components of the tumor microenvironment (TME). However, TILs can be extracted from tumor biopsies, reactivated and expanded in vitro before being reinjected into the patient. This immunotherapy strategy has shown impressive results in melanoma, where a majority of patients have seen a complete tumor remission. However, for most solid cancers (e.g. lung or colorectal cancer) that are in general less prone to mount immune responses, this approach is less effective. Other reasons for the currently limited success rate of ACT include the ineffectiveness to expand TILs in vitro, as well as the lack of functional assays to probe their potency after culture. Indeed, existing tumor models are based either on the non-physiological culture of cancer cell lines on plastic dishes or on the implantation of a patient's tumor tissue into an immune-deficient mouse, an assay that is highly complex, inefficient and expensive. Recent breakthroughs in 3D primary cell culture technology have provided paths forward in overcoming these challenges. In particular, the derivation of patient-derived miniaturized tumors, or tumoroids, has enabled the recapitulation of some key functional characteristics of real tumors. When co-cultured with fibroblasts or immune cells, tumoroids are potentially powerful models for immunotherapy applications. However, despite promising proof-of-concept demonstrations of modeling the TME with such heterotypic tumoroids, it has neither been possible to derive such complex in vitro tumor models with all key cell types isolated from the same patient's tumor, nor to maintain the numbers and phenotypes of TME-derived cells in longer-term cultures. Moreover, existing tumoroids are highly heterogeneous and unsuitable for the development of scalable and reproducible assays that are needed in diagnostic applications. This thesis focused on the bioengineering of novel in vitro tools to address some of the aforementioned obstacles in realizing the promise of TIL therapy. First, to generate heterotypic and autologous tumoroids, existing protocols were optimized to isolate and expand cancer cells, fibroblasts, and TILs from colorectal cancer (CRC) samples. A small biobank was established for each cell type as a resource for the development and application of patient-specific tumoroid assays. In the second part of this thesis, a heterotypic CRC tumoroid platform was developed for testing autologous TIL potency in combination with immunotherapeutic agents. CRC tumoroids were derived in high-throughput on a hydrogel substrate comprising an array of regularly spaced, round-bottom microcavities, a set-up that was found to highly amenable to quantitative experimentation. ... More in the full text ...

EPFL2020

New roles of the lymphatic endothelium in modulating adaptive immunity: implications for immune tolerance and cancer immunotherapy

Lambert Charles François Potin

EPFL2018

Bioengineering Methods to Improve in vitro Adoptive Cell Therapy based on Tumor-Infiltrating Lymphocytes

François Réal Rivest

EPFL2020

The role of Snail in non-small cell lung cancer

Svenja Johanna Groeneveld

EPFL2018