Molecular profiling and functional analysis of macrophage-derived tumour extracellular vesicles

Extracellular vesicles (EVs) are nanosized vesicles released by virtually all cell types. Increasing data suggest that EVs function in the regulation of intercellular communication both in physiological and pathological states. In cancer biology, mounting evidence suggests that cancer cell-derived EVs directly modulate several aspects of tumour progression by acting on both cancer cells and tumour-associated stromal cells. In addition to cancer cells, the tumour microenvironment (TME) also features an intertwined ensemble of recruited host-derived cells. Tumour-associated macrophages (TAMs) often infiltrate tumours in high numbers, thus representing one of the major host-derived cell populations. The involvement of TAMs in cancer progression has been extensively documented. It is well established that according to their polarisation state, TAMs can exert both pro- and anti-tumoural functions. However, under the influence of cancer cell-derived factors TAMs often acquire a phenotype that fosters tumour progression by supporting angiogenesis, metastasis and immunosuppression. While the role of secreted soluble factors contributing to the pro- and anti-tumoural functions of TAMs is well characterised, the molecular composition, properties, and effects of TAM-derived EVs (TAM-EVs) in the TME are poorly understood. In this thesis, I studied TAM-EVs produced in intact mouse tumours. We developed a methodology for the enrichment, quantification, and selective isolation of TAM-EVs produced in the TME. Furthermore, we performed comprehensive proteomic and lipidomic analyses of tumour-derived EVs and identified molecular signatures indicative of a role for TAM-EVs in immune response and lipid metabolism. Whereas source TAMs display a phenotype consistent with immunosuppressive functions, TAM-EVs have a pro-inflammatory and immune-stimulatory molecular signature. Indeed, TAM-EV-enriched preparations promoted T-cell proliferation and activation ex vivo and modulated arachidonic acid metabolism of cancer cells by inhibiting the production of the immunosuppressive PGE2 while favouring the secretion of pro-inflammatory thromboxane. Therefore, TAM-EVs might have the potential to stimulate, rather than limit, anti-tumour immunity. I also showed that while portraying some similarities with EVs of in vitro-polarised macrophages, TAM-EVs isolated from the TME present distinctive molecular profiles, suggesting that simplistic in vitro models do not faithfully recapitulate the complexity of the TME dynamics. Overall, this work emphasises the importance of studying TAM-EVs in their physiological context and identifies a potential role for TAM-EVs in favouring the development of an anti-tumoural immune microenvironment. Findings from this work extend our knowledge of the multifaceted role of TAMs in tumour biology.

Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles

Tim Beltraminelli, Chiara Cianciaruso, Michele De Palma, Romain Hamelin, Sina Nassiri, Bruno Torchia

Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity.

2019

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 ...