Cell competition is a fundamental mechanism of tissue quality control that enables the selective elimination of less fit, mis-specified, diseased or aged cells. By shaping tissue composition, it plays a critical role in development, organismal health and a wide range of physiological and pathological contexts, including cancer. As its biological significance continues to grow, elucidating the molecular mechanisms underlying cell competition is essential for advancing our understanding of tissue biology, disease progression and future therapeutic strategies. In this review, we highlight recently identified, evolutionarily conserved pathways that govern cell competition through metabolites and systemic signals, proteostasis and mechanical exchange. By integrating findings across species and pathways, we reveal how these distinct mechanisms may intersect and coordinate to determine competitive outcomes, providing a conceptual framework to inform and guide future research.

Normal tissues actively employ a phenomenon called cell competition to drive the elimination and replacement of less fit loser cells by fitter winner cells. This quality control mechanism promotes tissue health by favoring the selective expansion of fitter cells. Indeed, through cell competition many mutant cells are eliminated from tissues by fitter normal cells. However, some oncogenic mutations can turn cells into supercompetitors that outcompete normal cells, promoting tumorigenic growth and metastasis. Several cellular stresses have been associated with the loser status such as oxidative stress, DNA damage responses, unfolded protein response, and mitochondrial dysfunction. By affecting these pathways, metabolism and dietary choices can regulate cellular fitness and cell competition. However, how these pathways affect competitive interactions in vivo, during the early establishment of mutant clones, is relatively little understood. Recent work from Hemalatha and colleagues introduces real-time fluorescence ratio metric imaging of NAD(P)H and FAD to investigate cellular redox status-live and over time, at a single-cell level-as cells compete in the mouse epidermis. Their work demonstrates that redox status changes dynamically during competition between cells carrying oncogenic mutations. It further shows that drugs that modulate mitochondrial metabolism and cellular redox are strong modulators of cell competition. The introduction of live redox imaging will prove a powerful tool to further dissect how metabolic states affect cell competition in normal physiology and in tumorigenesis.

Abstract TP53 Mutations confer clonal advantage in several tissues contributing to tumorigenesis. It has traditionally been proposed that heightened resistance to apoptosis/cell cycle arrest can account for clonal advantage. P53 mediated clonal dominance has also been observed in cultured embryonic stem (ES) cells, though it is not clear how this is established. Using live cell imaging we show that human ES cells (hESCs) mutant for TP53 induce death of hES-WT cells and outcompete them. Competition is mediated by apoptosis induction and requires direct cell contact, as in co-cultures of hES-P53KO and hES-WT cells where contact is prevented competition is not observed. To ask if hES-P53KO competition is mediated by mechanical interactions, we cultured cells on deformable membranes to measure their resistance to cell compaction. Interestingly, while hES-WT cells displayed apoptosis induction upon cell compaction, hES-P53KO cells were resistant to compaction-induced apoptosis. We suggest that differential sensitivity to compaction in crowded cultures/tissues can drive p53-induced competitive cell elimination. Identifying the mechanisms by which p53 confers resistance to cell compaction could lead to novel actionable targets to prevent the expansion of p53 mutant stem cells in cancerous and pre-cancerous lesions. Citation Format: Eugenia Piddini. p53-mediated stem cell competition: Insights into mechanisms of clonal dominance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY07-01.

The ability to optically interact with cells on both an individual and collective level has applications from wound healing to cancer treatment. Building systems that can facilitate both localised light illumination and visualisation of cells can, however, be challenging and costly. This work takes the Dynamic Optical MicroEnvironment (DOME), an existing platform for the closed-loop optical control of microscale agents, and adapts the design to support live-cell imaging. Through modifications made to the imaging and projection systems within the DOME, a significantly higher resolution, alternative imaging channels and the ability to customise light wavelengths are achieved (Bio-DOME). This is accompanied by an interactive calibration procedure that is robust to changes in the hardware configuration and provides fluorescence imaging (Fluoro-DOME). These alterations to the fundamental design allow for long-term use of the DOME in an environment of higher temperature and humidity. Thus, long-term imaging of living cells in a wound, with closed-loop control of real-time frontier illumination via projected light patterns, is facilitated.

EDI allies congregated to highlight initiatives intended to address barriers to research opportunities and support that could promote recruitment and retention of diverse talent, encourage collaborative research, improve community engagement, and cultivate public trust in research.

The rapidly evolving stem cell field puts much stress on developing educational resources. The ISSCR Education Committee has created a flexible stem cell syllabus rooted in core concepts to facilitate stem cell literacy. The free syllabus will be updated regularly to maintain accuracy and relevance.

The DOME is a powerful and adaptable platform that facilitates the study of light-reactive systems at the microscale. While the projection module of the DOME can produce light patterns with high spatial and temporal resolution, the maximum irradiance (incident electromagnetic energy per unit area) that can be generated by its native LEDs is limited. Increasing the irradiance is crucial to enabling new biomedical applications such as inducing DNA damage. In this paper, we present a modular solution to allow general light sources to be used with the DOME. By switching to a high-powered near-UV light source, we show that DNA damage can be caused by the Epi-DOME's projection system at a targeted location.

Investigating organ biology often requires methodologies to induce genetically distinct clones within a living tissue. However, the 3D nature of clones makes sample image analysis challenging and slow, limiting the amount of information that can be extracted manually. Here we develop PECAn, a pipeline for image processing and statistical data analysis of complex multi-genotype 3D images. PECAn includes data handling, machine-learning-enabled segmentation, multivariant statistical analysis, and graph generation. This enables researchers to perform rigorous analyses rapidly and at scale, without requiring programming skills. We demonstrate the power of this pipeline by applying it to the study of Minute cell competition. We find an unappreciated sexual dimorphism in Minute cell growth in competing wing discs and identify, by statistical regression analysis, tissue parameters that model and correlate with competitive death. Furthermore, using PECAn, we identify several genes with a role in cell competition by conducting an RNAi-based screen.

Epithelial cells migrate across wounds to repair injured tissue. Leader cells at the front of migrating sheets often drive this process. However, it is unclear how leaders emerge from an apparently homogeneous epithelial cell population. We characterized leaders emerging from epithelial monolayers in cell culture and found that they activated the stress sensor p53, which was sufficient to initiate leader cell behavior. p53 activated the cell cycle inhibitor p21 WAF1/CIP1 , which in turn induced leader behavior through inhibition of cyclin-dependent kinase activity. p53 also induced crowding hypersensitivity in leader cells such that, upon epithelial closure, they were eliminated by cell competition. Thus, mechanically induced p53 directs emergence of a transient population of leader cells that drive migration and ensures their clearance upon epithelial repair.

Gustatory Receptor 64 (Gr64) genes are a cluster of 6 neuronally expressed receptors involved in sweet taste sensation in Drosophila melanogaster. Gr64s modulate calcium signalling and excitatory responses to several different sugars. Here, we discover an unexpected nonneuronal function of Gr64 receptors and show that they promote proteostasis in epithelial cells affected by proteotoxic stress. Using heterozygous mutations in ribosome proteins (Rp), which have recently been shown to induce proteotoxic stress and protein aggregates in cells, we show that Rp/+ cells in Drosophila imaginal discs up-regulate expression of the entire Gr64 cluster and depend on these receptors for survival. We further show that loss of Gr64 in Rp/+ cells exacerbates stress pathway activation and proteotoxic stress by negatively affecting autophagy and proteasome function. This work identifies a noncanonical role in proteostasis maintenance for a family of gustatory receptors known for their function in neuronal sensation.

The effective resolution of wounds is crucial to minimise the risk that lesions pose to the organism. Wound healing is a complex process that achieves the impressive goal of sealing the breach in the tissue and reinstating its integrity via the activation of a multifaceted biological response involving hemostasis, inflammation, cell migration, cell proliferation and tissue remodelling. A fundamental step in tissue repair is the activation of a collective migration program that ensures rapid closure of the breach by the cells at the edge of the wound before cell proliferation can restore the initial architecture. The mechanics of collective migration in epithelial wound healing have been dissected by a number of studies, many of which make use of simplified in vitro models of cultured epithelial monolayers injured by removal of a strip of cells.1, 2 Such models make it possible to investigate the dynamics and mechanisms of cell migration in tissue repair in a tractable system, where epithelial migration is isolated from the complex injury response that takes place in vivo. The collective migration that enables epithelial repair is often initiated by a few specialized cells at the leading edge of migrating cell sheets that become migratory and guide cell migration. For this reason, these are called ‘leader cells’. Leader cells acquire a characteristic flattened morphology and activate migratory signaling cascades (such as elevation of integrin β1 and phosphoinositide 3-kinase and increased activity of Rac1—Ras-related C3 botulinum toxin substrate 1), which endow them with the capacity to drive migration of their neighbours.2, 3 How only a few cells from the initially homogeneous injured epithelial population are specified into leaders remained a question in the field. In a recent study, we identified an unexpected role for the tumour suppressor p53 in leader cell specification.4 We found that p53 levels are elevated in leader cells and, remarkably, that induction of p53 was able to instruct leader behaviour.4 Downstream of p53, its target p21 (also known as cyclin-dependent kinase inhibitor 1A, CDKN1A) is also elevated in leader cells, and both p53 and p21 are sufficient and necessary to promote the leader function.4 Mechanistically, we could determine that p21-dependent CDK inhibition and, consequently, cell-cycle delay instruct leader cell specification.4 In fact, an asymmetry in CDK activity levels between leaders and follower is necessary for the initiation of directed migration.4 Accordingly, inhibiting p53 or p21 reduces the speed of migration and epithelial repair, whereas elevating p53 and p21, via irradiation-induced DNA damage, accelerates migration and repair of injured epithelial monolayers.4 The discovery of the role of p53 in leader cell specification moves the question one step further: What induces p53 elevation in epithelial injury, triggering the commitment to leader fate? Using a fluorescent reporter of p53 activity, we showed that this is increased in cells at the edge of a scratched epithelium, compared to their undamaged neighbours.4 Such elevation can be impaired by inhibition of p38, which has been previously reported to activate p53 upon mechanical stress.5 We therefore propose that mechanical damage, upon epithelial injury, induces p53 levels and consequently determines the specification of leader cells. We then asked: What happens to leaders when the epithelium is repaired? Tracking leader cells as the epithelial gap closed, we could observe their elimination via cell competition,4 as previously reported for cells, which elevate p53.5 p53 has therefore a dual role in epithelial repair: It accelerates collective migration, by instructing leader behaviour in damaged cells, and it elicits leader cell elimination once the epithelium is repaired, ensuring that a physiological epithelial tissue architecture is reinstated. The novel involvement of the p53-p21-CDK signaling axis in epithelial healing raises the question of whether such mechanism is relevant for tissue repair in vivo. Previous studies seem to support this idea. For example, the presence of a non-proliferative migrating leading edge has been observed in wounds in vivo,6, 7 and tight spatiotemporal regulation of the balance between migration and proliferation is crucial for effective wound healing.6, 7 Analogously, during the regeneration of epicardial tissue, collective migration is led by a population of cytokinesis-impaired cells,8 which are likely to elevate p53/p21 in response to incomplete cell division. Consistent with our own observation that leader cells are removed by cell competition on wound closure, some of these studies also reported that the specialised cells acting as leaders are transient, as cells carrying the leader signature are not observed once the wound has been repaired.7, 8 The presence of cell-cycle arrested cells at the front of migrating cell populations is not limited to the context of tissue repair. During placenta formation, cytotrophoblasts penetrate the uterine wall to form the floating villi that mediate mother-embryo exchanges.9 It has been reported that, as they commit to the invasive phenotype, cytotrophoblasts accumulate chromosomal abnormalities, often acquire an aneuploid karyotype and stop proliferating,10 all suggestive of p53/p21 activation. Similarly, angiogenesis in the developing mouse retina occurs by extension of vascular sprouts that invade the tissue following an actively migrating non-proliferative tip cell.11 Given the established role of p53 in preventing neoplastic transformation and the importance of collective migration in tumour invasion and metastasis, it is tempting to question if p53-mediated leader specification plays a role in cancer dissemination. Most human cancers carry mutations that abolish the normal function of p53. However, the tissue surrounding the tumour expresses functional p53, susceptible to be elevated by radiation and chemotherapy. It is thus possible that asymmetric expression of p53 or CDK activity in adjoining tumour–non-tumour cell populations could result in leader-follower behaviour, promoting tumour cell migration. This effect would explain the observation that chemotherapy-induced senescent cells in tumour-adjacent tissue promote metastasis12 and the enhanced metastasis potential observed as a side effect of radiotherapy.13 The discovery of p53 function in leader cell specification, if confirmed by in vivo studies, could therefore have broad implications in physiological and pathological activation of collective migration and, potentially, inform future therapeutic approaches. Figure 1. The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

The ability to optically interact with cells on both an individual and collective level has applications from wound healing to cancer treatment. Building systems which can facilitate both localised light illumination and visualisation of cells can however, be challenging and costly. This work takes the DOME, an existing platform for the closed-loop optical control of microscale agents, and adapts the design to support live-cell imaging. Through modifications made to the imaging and projection systems within the DOME, a significantly higher imaging resolution is achieved as well as the ability to customise the light projection wavelengths. Changes to the fundamental design allow for long-term use in an environment of higher temperature and humidity, facilitating the long-term imaging of live cells.

Mutations in ribosome protein ( Rp ) genes and ribosome biogenesis factors result in debilitating diseases known as ribosomopathies. Recent studies in Drosophila have shown that cells heterozygous mutant for Rp genes ( Rp/+ ) exhibit proteotoxic stress and aggregates, which drive stress pathway activation and apoptosis. Understanding how Rp/+ cells fend off proteotoxic stress could suggest mechanisms to ameliorate these and other conditions caused by proteotoxic stress. Here we find that Rp/+ epithelial cells express all six Gustatory Receptor 64 (Gr64) genes, a cluster of sugar receptors involved in taste sensation. We show that Rp/+ cells depend on Gr64 for survival and that loss of Gr64 autonomously exacerbates stress pathway activation and proteotoxic stress by negatively effecting autophagy and proteasome function in Rp/+ cells. This work identifies a non-canonical role in proteostasis maintenance for a family of gustatory receptors known for their function in neuronal sensation.

Investigating organ biology requires sophisticated methodologies to induce genetically distinct clones within a tissue. Microscopic analysis of such samples produces information-rich 3D images. However, the 3D nature and spatial anisotropy of clones makes sample analysis challenging and slow and limits the amount of information that can be extracted manually. Here we have developed a pipeline for image processing and statistical data analysis which automatically extracts sophisticated parameters from complex multi-genotype 3D images. The pipeline includes data handling, machine-learning-enabled segmentation, multivariant statistical analysis, and graph generation. This enables researchers to run rigorous analyses on images and videos at scale and in a fraction of the time, without requiring programming skills. We demonstrate the power of this pipeline by applying it to the study of Minute cell competition. We find an unappreciated sexual dimorphism in Minute competition and identify, by statistical regression analysis, tissue parameters that model and predict competitive death.

Cell competition allows winner cells to eliminate less fit loser cells in tissues. In Minute cell competition, cells with a heterozygous mutation in ribosome genes, such as RpS3+/− cells, are eliminated by wild-type cells. How cells are primed as losers is partially understood and it has been proposed that reduced translation underpins the loser status of ribosome mutant, or Minute, cells. Here, using Drosophila, we show that reduced translation does not cause cell competition. Instead, we identify proteotoxic stress as the underlying cause of the loser status for Minute competition and competition induced by mahjong, an unrelated loser gene. RpS3+/− cells exhibit reduced autophagic and proteasomal flux, accumulate protein aggregates and can be rescued from competition by improving their proteostasis. Conversely, inducing proteotoxic stress is sufficient to turn otherwise wild-type cells into losers. Thus, we propose that tissues may preserve their health through a proteostasis-based mechanism of cell competition and cell selection. Baumgartner et al. identify proteotoxic stress as the underlying cause of the loser status in a cell competition model caused by reduced autophagic, proteasomal flux and accumulation of protein aggregates.

Cell competition induces the elimination of less-fit "loser" cells by fitter "winner" cells. In Drosophila, cells heterozygous mutant in ribosome genes, Rp/+, known as Minutes, are outcompeted by wild-type cells. Rp/+ cells display proteotoxic stress and the oxidative stress response, which drive the loser status. Minute cell competition also requires the transcription factors Irbp18 and Xrp1, but how these contribute to the loser status is partially understood. Here we provide evidence that initial proteotoxic stress in RpS3/+ cells is Xrp1-independent. However, Xrp1 is sufficient to induce proteotoxic stress in otherwise wild-type cells and is necessary for the high levels of proteotoxic stress found in RpS3/+ cells. Surprisingly, Xrp1 is also induced downstream of proteotoxic stress, and is required for the competitive elimination of cells suffering from proteotoxic stress or overexpressing Nrf2. Our data suggests that a feed-forward loop between Xrp1, proteotoxic stress, and Nrf2 drives Minute cells to become losers.

Human induced pluripotent stem cells (hIPSCs) are an important tool, but challenges remain in optimizing their use. hIPSC cultures frequently become contaminated and overrun with cells containing genetic aberrations. In this issue of Developmental Cell, Price et al. establish that this results from cell competition between wild-type and variant cells.

ABSTRACTLive imaging can provide powerful insights into developmental and cellular processes but availability of multiplexable reporters has been limiting. Here we describe ORACLE, a cell fate reporter class in which fluorescent proteins fused with the nucleoporin POM121 are driven by promoters of transcription factors of interest. ORACLE’s nuclear rim localisation therefore enables multiplexing with conventional nuclear reporters. We applied ORACLE to investigate the dynamics of pluripotency exit at single-cell level, using human pluripotent stem cells (hPSCs) imaged by multi-day time-lapse high-content microscopy. Using an ORACLE-OCT4 pluripotency marker we reveal that G1 phase length and OCT4 level are strongly coupled and that spatial location in a colony impacts the timing of pluripotency exit. Combining ORACLE-OCT4 and an ORACLE-SOX1 early neuronal differentiation marker, we visualize in real-time the dynamics of cell fate transition between pluripotency and early neural fate, and show that pluripotency exit and differentiation onset are likely not tightly coupled in single-cells. Thus ORACLE is a powerful tool to enable quantitative studies of spatiotemporal cell fate control.

SUMMARYPredicting how stem cells become patterned and differentiated into target tissues is key for optimising human tissue design. Here, we established DEEP-MAP - for deep learning-enhanced morphological profiling - an approach that integrates single-cell, multi-day, multi-colour microscopy phenomics with deep learning and allows to robustly map and predict cell fate dynamics in real-time without a need for cell state-specific reporters. Using human pluripotent stem cells (hPSCs) engineered to co-express the histone H2B and two-colour FUCCI cell cycle reporters, we used DEEP-MAP to capture hundreds of morphological- and proliferation-associated features for hundreds of thousands of cells and used this information to map and predict spatiotemporally single-cell fate dynamics across germ layer cell fates. We show that DEEP-MAP predicts fate changes as early or earlier than transcription factor-based fate reporters, reveals the timing and existence of intermediate cell fates invisible to fixed-cell technologies, and identifies proliferative properties predictive of cell fate transitions. DEEP-MAP provides a versatile, universal strategy to map tissue evolution and organisation across many developmental and tissue engineering contexts.