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During renewal of the intestine, cells are continuously generated by proliferation. Proliferation and differentiation must be tightly balanced, as any bias towards proliferation results in uncontrolled exponential growth. Yet, the inherently stochastic nature of cells raises the question how such fluctuations are limited. We used time-lapse microscopy to track all cells in crypts of growing mouse intestinal organoids for multiple generations, allowing full reconstruction of the underlying lineage dynamics in space and time. Proliferative behavior was highly symmetric between sister cells, with both sisters either jointly ceasing or continuing proliferation. Simulations revealed that such symmetric proliferative behavior minimizes cell number fluctuations, explaining our observation that proliferating cell number remained constant even as crypts increased in size considerably. Proliferative symmetry did not reflect positional symmetry, but rather lineage control through the mother cell. Our results indicate a concrete mechanism to balance proliferation and differentiation with minimal fluctuations, that may be broadly relevant for other tissues.

Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer, where the tumor is encapsulated by a basement membrane (BM). At the invasive phase, the BM barrier is compromised enabling tumor cells to escape into the surrounding stroma. The molecular mechanisms that establish and maintain an epithelial BM barrier in vivo are poorly understood. Myosin-X (MYO10) is a filopodia-inducing motor protein implicated in metastasis and poor clinical outcome in patients with invasive breast cancer (IBC). We compared MYO10 expression in patient-matched normal breast tissue and DCIS lesions and found elevated MYO10 expression in DCIS samples, suggesting that MYO10 might facilitate the transition from DCIS to IBC. Indeed, MYO10 promoted the formation of filopodia and cell invasion in vitro and positively regulated the dissemination of individual cancer cells from IBC lesions in vivo. However, MYO10-depleted DCIS xenografts were, unexpectedly, more invasive. In these xenografts, MYO10 depletion compromised BM formation around the lesions resulting in poorly defined tumor borders and increased cancer cell dispersal into the surrounding stroma. Moreover, MYO10-depleted tumors showed increased EMT-marker-positive cells, specifically at the tumor periphery. We also observed cancer spheroids undergoing rotational motion and recruiting BM components in a filopodia-dependent manner to generate a near-continuous extracellular matrix boundary. Taken together, our data identify a protective role for MYO10 in early-stage breast cancer, where MYO10-dependent tumor cell protrusions support BM assembly at the tumor-stroma interface to limit cancer progression, and a pro-invasive role that facilitates cancer cell dissemination at later stages.Abstract FigureHighlights-Filopodia sculpt the tumor-proximal stroma in pre-invasive ductal carcinoma in situ (DCIS).-Filopodia-dependent basement membrane (BM) assembly limits invasive transition of DCIS-like tumors in vivo.-Loss of MYO10-dependent filopodia impairs BM assembly and induces an EMT-like phenotype at the tumor-stroma interface in vivo.-MYO10 filopodia are anti-invasive in DCIS but facilitate dissemination in invasive breast cancer.

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here we show that the novel oncogene RASSF1C drives mesenchymal to amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that amoeboid cells display the cancer stem cell markers CD133, ALDH1 and the pluripotent marker Nanog; are accompanied by higher invasive potential in vitro and in vivo; and employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.

The functional activity and differentiation potential of cells is determined by their interaction with surrounding cells. Approaches that allow the unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a trade-off between spatial resolution and cell profiling depth. Here, we developed a photoswitch-based technology that allows the isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran)-cage coupled to nanobodies allowed photoswitching of cells in areas of interest with low-intensity violet light and without detectable phototoxicity. Single cell RNA sequencing of spatially defined CD8+ T cells was used to exemplify the feasibility of identifying location-dependent cell states at the single cell level. Finally, we demonstrate the efficient labeling and photoswitching of cells in live primary human tumor tissue. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples.

Significance What defines the number and dynamics of the stem cells that generate and renew biological tissues? Although several molecular markers have been described to predict stem cell potential, we propose a complementary approach that mathematically describes “stemness” as an emergent property arising from a stochastic competition for space. We predict from that competition the robust emergence of a region made of functional stem cells, as well as give simple predictions on lineage-survival probability. We test our results with data obtained from intravital live-imaging experiments in mammary gland development, existing data from kidney development, and from the self-renewal of the crypt to show that our framework can predict the number of functional stem cells and lineage-survival probability., Understanding to what extent stem cell potential is a cell-intrinsic property or an emergent behavior coming from global tissue dynamics and geometry is a key outstanding question of systems and stem cell biology. Here, we propose a theory of stem cell dynamics as a stochastic competition for access to a spatially localized niche, giving rise to a stochastic conveyor-belt model. Cell divisions produce a steady cellular stream which advects cells away from the niche, while random rearrangements enable cells away from the niche to be favorably repositioned. Importantly, even when assuming that all cells in a tissue are molecularly equivalent, we predict a common (“universal”) functional dependence of the long-term clonal survival probability on distance from the niche, as well as the emergence of a well-defined number of functional stem cells, dependent only on the rate of random movements vs. mitosis-driven advection. We test the predictions of this theory on datasets of pubertal mammary gland tips and embryonic kidney tips, as well as homeostatic intestinal crypts. Importantly, we find good agreement for the predicted functional dependency of the competition as a function of position, and thus functional stem cell number in each organ. This argues for a key role of positional fluctuations in dictating stem cell number and dynamics, and we discuss the applicability of this theory to other settings.

Stem cells maintain tissue homeostasis by driving cellular turnover and regeneration upon damage. They reside within specialized niches that provide the signals required for stem cell maintenance. Stem cells have been identified in many tissues and cancer types, but their behavior within the niche and their reaction to microenvironmental signals were inferred from limited static observations. Recent advances in live imaging techniques, such as live cell imaging and intravital microscopy, have allowed the visualization of stem cell behavior and dynamics over time in their (near) native environment. Through these recent technological advances, it is now evident that stem cells are much more dynamic than previously anticipated, resulting in a model in which stemness is a state that can be gained or lost over time. In this review, we will highlight how live imaging and intravital microscopy have unraveled previously unanticipated stem cell dynamics and plasticity during development, homeostasis, regeneration, and tumor formation.

Metastasis is a highly dynamic process during which cancer and microenvironmental cells undergo a cascade of events required for efficient dissemination throughout the body. During the metastatic cascade, tumor cells can change their state and behavior, a phenomenon commonly defined as cellular plasticity. To monitor cellular plasticity during metastasis, high-resolution intravital microscopy (IVM) techniques have been developed and allow us to visualize individual cells by repeated imaging in animal models. In this review, we summarize the latest technological advancements in the field of IVM and how they have been applied to monitor metastatic events. In particular, we highlight how longitudinal imaging in native tissues can provide new insights into the plastic physiological and developmental processes that are hijacked by cancer cells during metastasis.