Thèse Régulation du Microenvironnement Tumoral par les Différentes Modalités de Mort Cellulaire Régulées H/F - Doctorat.Gouv.Fr

Établissement : Université Claude Bernard Lyon 1 École doctorale : CanBioS - Cancérologie, Biologie, Santé de Lyon Laboratoire de recherche : CRCL - CENTRE DE RECHERCHE EN CANCÉROLOGIE DE LYON Direction de la thèse : Olivier MEURETTE ORCID 0000000210875981 Début de la thèse : 2026-10-01 Date limite de candidature : 2026-05-11T23:59:59 Malgré le développement récent des thérapies ciblées et des immunothérapies, la réduction de la masse tumorale par l'induction de la mort cellulaire apoptotique demeure la pierre angulaire des traitements anticancéreux. Cependant, l'apoptose peut avoir des conséquences inattendues à long terme en renforçant l'agressivité des cellules résistantes aux traitements. La capacité des cellules apoptotiques à stimuler la prolifération des cellules voisines est reconnue de longue date dans différents contextes, mais il est désormais établi que les cellules cancéreuses peuvent mourir par de multiples modalités, susceptibles d'affecter différemment le microenvironnement tumoral et la progression tumorale. Par exemple, notre groupe a montré que la mort cellulaire indépendante des caspases n'a pas le même effet sur la prolifération cellulaire que l'apoptose.

Alors que l'apoptose est généralement considérée comme tolérogène, certaines chimiothérapies induisent une mort cellulaire immunogène, incluant des formes de mort indépendantes des caspases, la nécroptose et la mort cellulaire associée à l'autophagie, qui peuvent dans des contextes spécifiques stimuler le système immunitaire. Bien que les différences entre les diverses modalités de mort cellulaire régulée (RCD, pour regulated cell death) et leurs effets secondaires potentiellement protumoraux soient désormais bien décrites, les études actuelles restent difficiles à comparer pour définir des stratégies thérapeutiques claires.

Plusieurs limites expliquent cette difficulté : les inducteurs des différentes modalités de RCD présentent souvent des effets hors cible ; les soustypes de RCD ne sont pas induits dans les mêmes types cellulaires, ce qui complique les comparaisons ; enfin, une phénotypage contrôlé et systématique des conséquences de la mort cellulaire sur le microenvironnement tumoral fait encore défaut. De plus, les aspects protumoraux de la mort cellulaire ont rarement été explorés de manière systématique en interrogeant simultanément plusieurs dimensions de la progression tumorale (prolifération, migration, caractère stemlike, potentiel métastatique) au sein du microenvironnement tumoral, et en comparant directement l'apoptose aux autres formes de mort cellulaire régulée dans des conditions contrôlées.

Afin d'apporter de nouvelles connaissances sur ces mécanismes, nous avons développé une plateforme cellulaire permettant l'induction contrôlée et spécifique de plusieurs modalités de RCD au moyen de systèmes optogénétiques (optoRCD, pour regulated cell death induite par optogénétique ; Figure 1A), nous permettant de déclencher l'apoptose (optocaspase9), la nécroptose (earlyoptoRIPK3 et lateoptoMLKL) et la pyroptose (OptoC1 pour la caspase1), ainsi que des modèles additionnels de mort cellulaire associée à l'autophagie.

Pour comparer les effets de ces différentes modalités de mort cellulaire dans le contexte de la progression tumorale, nous avons réalisé un séquençage d'ARNm à des temps précoces après l'induction de la mort. Ces données nous ont fourni des résultats préliminaires solides sur des gènes spécifiquement régulés aux phases initiales de l'induction de RCD (avant la disparition de la cellule), permettant aux cellules de communiquer avec leur environnement et de le remodeler activement avant leur mort. Nous nous sommes d'abord concentrés sur les protéines sécrétées et avons identifié des transcrits communs à toutes les formes de mort cellulaire, ainsi que des transcrits spécifiquement associés à l'une ou l'autre modalité de mort. Ce projet vise à disséquer la manière dont ces signatures régulatrices spécifiques modulent les « hallmarks » des cellules cancéreuses, le microenvironnement tumoral et la progression tumorale. Despite the recent development of targeted therapies and immunotherapies, reducing tumour burden by triggering apoptotic cell death remains the cornerstone of anti-cancer treatment. However, apoptosis can have unexpected long-term consequences by enhancing the aggressiveness of treatment-resistant cells 1-3. The ability of apoptotic cells to stimulate the proliferation of neighboring cells has long been recognized in different contexts 4-6, but it is now clear that cancer cells can die through multiple modalities that could differentially impact the tumor microenvironment and tumor progression 7. For example, our group has shown that caspase-independent cell death does not have the same effect on cell proliferation as apoptosis 8. While apoptosis is generally considered tolerogenic, certain chemotherapies induce immunogenic cell death, including caspase-independent cell death, necroptosis and autophagy-associated cell death, which in specific contexts can stimulate the immune system 9-11. Although the differences between regulated cell death (RCD) modalities and their potential pro-tumoral 'side-effect' are now well described, the current studies are difficult to compare for delineating clear therapeutic 6,12 13 14. This is due to several drawbacks: the triggers of RCD modalities often have off-target effects, different RCD subtypes are not induced in the same cell type making comparison difficult, while a controlled and systematic phenotyping of the effect cell death could have on the tumoral microenvironment is still lacking. Furthermore, pro-tumoral aspects of cell death have rarely been explored in a systematic manner by interrogating several dimensions of tumor progression (proliferation, migration, stemness, metastatic potential) within the tumor microenvironment and by directly comparing apoptosis to other forms of regulated cell death in controlled conditions. To bring new insights in these mechanisms, we have developed a cellular platform allowing controlled and specific induction of several RCD modalities using optogenetic systems (optoRCD, for optogenetic-induced regulated cell death; Figure 1A), enabling us to trigger apoptosis (opto-caspase-9), necroptosis (early-optoRIPK3 and late, Opto-MLKL) and pyroptosis (Opto-C1 for caspase-1), as well as additional models of autophagy-associated cell death 15. To compare the effects of these distinct cell death modalities in the context of tumor progression, we performed mRNA sequencing at early time points following death induction. These data gave us solid preliminary data on genes that are specifically regulated in early phase of RCD induction (before demise of the cell), allowing cells to communicate with and actively remodel their environment before their death. We initially focused on secreted proteins and identified transcripts that are common to all forms of cell death, as well as transcripts specifically associated with one or another death modality. This project aims to dissect how these specific regulatory signatures shape cancer cell hallmarks, the tumor microenvironment and tumor progression. Before dying, cells can communicate with their environment and secrete molecules that impact neighbor cells and foster tumor progression. Cell death modalities differently affect the microenvironment. Identifying key factors of this communication and the cell signaling involved will allow design of therapeutic approach limiting the pro-oncogenic effect of cell death.

Research objectives: 1. Robustly identify specific and shared secreted molecules; 2. Study the impact of these targets in cross-talk with cells from the microenvironment; 3. Study in vivo, the impact of modulating these targets in reshaping the microenvironment by dying cells.
First, we will validate the specific regulation of cytokines identified at the mRNA level and confirm their secretion into the extracellular medium. To this end, in the Cancer Cell Death team, we have established several complementary strategies to induce different forms of RCD: the optogenetic approach described above, which provides a controlled and clean system that enabled us to detect specific regulations; an inducible system based on doxycycline treatment; and chemically induced cell death. Using these approaches, we can robustly analyze the secretome from cells undergoing apoptosis, necroptosis, pyroptosis or autophagy-associated cell death. In parallel, as a contingency plan, we will perform mass spectrometry analyses of conditioned media obtained for each death modality.
Gene expression, protein production and secretion will be confirmed by RT-qPCR, western blotting and ELISA, respectively, in all systems, narrowing down the investigations on targets identified by RNAseq and validated by mass spectrometry. This strategy will allow us to define factors commonly secreted by all forms of cell death, as well as factors specific to each individual modality. A combined literature and in silico analysis (STRING and DAVID Gene Ontology) of the most robust candidates (pathway-specific and conserved across different induction systems) will guide the selection of factors most likely to impact the tumor microenvironment, including vasculature, cancer-associated fibroblasts and immune cells. This first objective will thus generate a robust set of candidate mediators validated in multiple models (optogenetic, chemical and inducible). The feasibility of this aim is supported by our preliminary data showing that necroptosis triggers the release of cytokines/chemokines that could specifically condition neutrophils within the tumor microenvironment.
Objective 2
Next, we will characterize the cells that receive signals from dying cells. For this, we will use the PUFFFIN (Positive Ultra-bright Fluorescent Fusion for identifying Neighbors) system 16 which labels cells that receive signals from neighboring sender cells. To validate the system, we introduced PUFFFIN into cells that undergo doxycycline-inducible apoptotic cell death (Figure 2A). Preliminary experiments show that these cells die by apoptosis, which can be inhibited by a pan-caspase inhibitor (QVD) (Figure 2B). When these cells are co-cultured with unlabeled cancer cells, 10-20% of receiver cells (labelled by secreted factors from dying cells) can be detected (Figure 2C). We will extend this approach to other forms of cell death by introducing PUFFFIN reporter into cells that can be driven into distinct death modalities (PUFFFIN/RCD - regulated cell death). Co-cultures with unlabeled cells will then be established, and death induction will be used to analyze specifically the characteristics of receiver cells that have sensed signals from dying cells (Figure 2D). This system will be applied to different components of the tumor microenvironment in co-culture, including vascular cells and immune cells (macrophages, neutrophils). This characterization of receiver cancer cells will include classical tests routinely used in the team (migration, proliferation, resistance to cell death). For co-culture with vascular cells, we previously developed similar approaches to study the effect of cancer cell on vascular sprouting and network establishent17. For the characterization of receiver immune cells, we have established collaboration with Ana Hennino's team (Macrophages), Yenkel Grindberg-Bleyer's team (T cells) and Marie-Cécile Michallet/Nathalie Bendriss Team (Neutrophils). In addition, candidates identified in Objective 1 will be functionally tested by blocking antibodies or CRISPR-mediated gene knockout in PUFFFIN/RCD cells. This will allow us to determine how different RCD-specific secretome influence the properties of tumor cells and of cells in the tumor microenvironment and validate the specific involvement of secreted molecules identified previously.

Figure 2. (A) The PUFFFIN system (green cytosolic fluorescence and red nuclear fluorescence) was introduced into cells with inducible apoptotic cell death. (B) Doxycycline treatment induces expression of the pro-apoptotic protein Bax, caspase-3 cleavage and PARP-1 cleavage, confirming apoptosis induction. (C) Quantification of receiver cells after doxycycline-induced cell death, in the presence or absence of a pan-caspase inhibitor (QVD) that shifts death towards caspase-independent cell death. (D) Experimental workflow to identify and phenotype cells affected by different types of cell death.
Objective 3
Finally, to precisely characterize how distinct forms of RCD remodel the tumor microenvironment, we will perform single-cell RNA sequencing in syngeneic graft models. We will be using EMT6 cells, a cell line already characterized in the team for its response to immunotherapies. First step will be to establish precise time course of tumor response to cell death induction in vivo. We will therefore study tumor regression time course for each death-induction model (Doxycycline-induced) to define the timing and extent of tumor regression. We will select the time necessary to obtain 50% regression of the tumor (after induction of RCD with Doxycyclin treatment) and study the time-course of re-growth after doxycycline withdrawal). Different approaches will be carried on to study the modification of the tumor microenvironement in response to RCD induction: 1 immunohistochemistry for vasculature and matrix architecture (SHG and collagen staining), single-cell RNA sequencing (for CD45+ enriched population and Epcam+ enriched population separately) before RCD induction, after reduction of 50% of the tumor mass and after regrowth to the original tumor size (Figure 3). With this approach we will be able to identify the modification of the microenvironment in response to cell death induction and how this affect re-growth of the tumor. This unbiased approach will provide detailed insights into how different immune cell populations within the tumor evolve in response to distinct cell death modalities, both during regression and after tumor regrowth. Next step will be to study the impact of signaling molecules between the dying cells and the microenvironment, identified in the 2 previous objectives. To do so, we will engineer CRISPR cell lines for secreted molecules identified and confirmed in the two previous objectives and study in vivo the effect of this invalidation on the restructuration of the TME after RCD induction. We will select cell lines which are not affected for the initial growth and response to RCD to assess specific effect on the microenvironment remodeling on the regrowth phase. By this strategy we hope to identify novel communication routes between dying cells and their environment that could be targeted to inhibit relapse.

Biologie cellulaire, cancérologie, culture cellulaire, travail en équipe, diplôme d'experimentation animale