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Study of the mechanical cues driving cell competition and its role in pretumoral cell expansion

Periodic Reporting for period 1 - MechDeath (Study of the mechanical cues driving cell competition and its role in pretumoral cell expansion)

Reporting period: 2019-06-01 to 2021-05-31

The healthy function of our tissues is achieved by the adjustment of single cell behavior to tissue scale parameters. This adjustment, through the local cell proliferation-disapearance equilibrium is necessary for organ morphogenesis and adult tissue size regulation. Most studies have been focusing on cell growth and cell division while survival rate and cell death (apoptosis) has drawn little attention. Cell competition is the process by which cells with small proliferating rate and survival properties are forced to die by the surrounding cells. For example, cell competition ables the elimination of suboptimal healthy cells to limit the appearance of errors in our organs during embryogenesis. However, genetic mutation, usually found in tumor, can create cells with high growth and survival rate, called «super competitor », which will force the elimination of surrounding healthy cells.

Using the Drosophila pupal notum, Dr. Levayer – the experienced researcher’s supervisor – recently proved that local compaction of WT cells by fast-growing and apoptosis resistant RasV12 clones can induce cell death in vivo. The initial working hypothesis of this project is that clonal overgrowth forces crowding in the clone and at its periphery. This will activate caspase preferentially in the cells sensitive to apoptosis (the WT cells), hence promoting the expansion of the fast growing clones. In this project, the experienced researcher (ER) aim to characterise the mechanical conditions allowing cell elimination and define where and when mechanical competition contribute to cell competition and if it can significantly support pretumoral cell expansion. This project is divided in three objectives: Work Package 1 (WP1): Building a predictive framework linking single cell deformation, local mechanical cues and the probability to trigger apoptosis. WP2: Quantitatively characterise the contribution of mechanical cues to cell elimination in pretumoral competition scenarios. WP3: Exploring the parameters modulating mechanical cell competition and pretumoral clone expansion in silico and in vivo.

First, in this project, the ER correlated mechanics with activity of the EGFR/ERK signaling pathways in physiological context as well as in pre-tumoral contexts. He especially observed that tissue stretching/compaction triggers an increase in cell survival/death probability. The ER proved that dying cell stretch their neighbours which activate in turn the EGFR/ERK prosurvival pathway and protect these cells for about one hour. This process is especially important to keep epithelial integrity safe. Third, the ER carefully analysed the behavior of mutant clones in multiple genetic condition associated to perturbation of cell growth, cell proliferation, cell death and cell forces. Along these quantifications, the ER managed to extract two independent mechanical conditions which are sufficient to generate the phenotype of mechanical cell competition: growth versus cell tension.
WP1. The experienced researcher (ER) has characterized the correlation/causality between cell mechanics and the probability to engage in apoptosis at the mesoscale level (5-50um) via the activity of EGFR/ERK pathway. He especially observed that tissue stretching/compaction triggers an increase/decrease in ERK activity. At a cellular scale, he quantified that more than 70% of cell death is associated to a sudden decrease in ERK activity. This has been published in a research article (Moreno, Valon et al. Current Biology, 2019). This work is at the origin of a technical paper and of the creation of an open source FiJi plugin (Herbert*, Valon* et al., BMC Biology, accepted 2021; gitlab.pasteur.fr/iah-public/localzprojector).

Beyond but related to WP1, the quantification of ERK activity brought some extra and related information. The ER especially demonstrated that spatiotemporal death distribution is bias. He proved that the probability of death in the first neighbours of a dying cells is reduced by 50% via a burst of ERK activity. This in turns protects the tissue from multiple cell death at the same time which could endanger tissue sealing and integrity. Last but not least, many cues are supporting the idea that cell stretching at the cell level is the trigger of these bursts. This work has been published in a research article (Valon et al., Developmental Cell 2021) and presented in six conferences with abstract submission.

In the context of mechanical cell competition, the ER worked on the idea that it can lead to two final scenarios, either the expansion of the oncogenic population or its elimination at the first stage. WP2 was based on the description of mechanical cell competition generated at the border of RasV12 clones in the drosophila pupal notum. The ER reproduced these data and tested extensively six other genetic conditions known to have putative effect on the regulation of cell size, cell contractility, cell proliferation and cell death. While mechanical cell death triggered via the formation of RasV12 seems to be an intricate mixture of cell resistance to cell death, cell growth, cell proliferation and cell tension at the boundary, the ER managed to uncouple cell growth/proliferation from cell tension in two different genetic conditions. Thus, he obtained experimentally some sufficient conditions at the origin of mechanical cell death. The goal of WP3 was to include in in silico simulations, information extracted from experiments to establish phase diagrams of clone disappearance versus invasion as a function of their initial size, shape, and mechanical competition process in game. These experiments are currently being reproduces and analysed and will be published in a research article (in preparation, first submission planned for early 2022). This work is as well at the origin of a review article (Valon, Levayer, Biology of the Cell, 2019).
Mechanics is known to affect cell behaviour through multiple pathways. While, its contribution in vivo to cell growth and cell proliferation is well studied, very few is known about its importance in regulating cell death. Via this project based on quantifications and perturbations in the Drosophila pupal notum, the ER underlines the role of mechanics as a modifier of cell death probability from the tissue to the single cell level, in physiological and pathological-like condition.

In physiologic condition, he observed that dying cells generate a burst of ERK activity in their first neighbouring cells which inhibits heir death, keeping global tissue impermeability and integrity safe. Similar work are also just been published by O. Pertz team about a similar process in mammalian cells (Gagliardi et al., Developmental Cell 2021). Further work should elucidate if it could be one of the process involved in chronic inflammation, one major cause of worldwide mortality.

In pathological condition, the ER uncoupled the effect of cell forces and cell growth on the creation of cell pressure at the origin of mechanical cell death. First, it underlines the necessity to more systematically take into account mechanics in cell competition scenarios. Second, this should give rise to cell diagrams determining the future of groups of oncogenic cells (their invasion or their elimination), as a function of their mechanical/genetical nature as well as their initial size and shape.
The experienced researcher's work: Influence of mechanical forces on apoptosis