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Live-cell microscopy routinely provides massive amounts of time-lapse images of complex cellular systems under various physiological or therapeutic conditions. However, this wealth of data remains difficult to interpret in terms of causal effects. Here, we describe CausalXtract, a flexible computational pipeline that discovers causal and possibly time-lagged effects from morphodynamic features and cell–cell interactions in live-cell imaging data. CausalXtract methodology combines network-based and information-based frameworks, which is shown to discover causal effects overlooked by classical Granger and Schreiber causality approaches. We showcase the use of CausalXtract to uncover novel causal effects in a tumor-on-chip cellular ecosystem under therapeutically relevant conditions. In particular, we find that cancer-associated fibroblasts directly inhibit cancer cell apoptosis, independently from anticancer treatment. CausalXtract uncovers also multiple antagonistic effects at different time delays. Hence, CausalXtract provides a unique computational tool to interpret live-cell imaging data for a range of fundamental and translational research applications.

Cancer associated fibroblasts are known to promote the progression of cancer. Here, the authors show that two particular subsets of cancer associated fibroblasts induce metastasis but work via distinct mechanisms including, chemokine signalling and Notch signalling.

The emerging tumor-on-chip (ToC) approaches allow to address biomedical questions out of reach with classical cell culture techniques: in biomimetic 3D hydrogels they partially reconstitute ex vivo the complexity of the tumor microenvironment and the cellular dynamics involving multiple cell types (cancer cells, immune cells, fibroblasts, etc.). However, a clear bottleneck is the extraction and interpretation of the rich biological information contained, sometime hidden, in the cell co-culture videos. In this work, we develop and apply novel video analysis algorithms to automatically measure the cytotoxic effects on human cancer cells (lung and breast) induced either by doxorubicin chemotherapy drug or by autologous tumor-infiltrating cytotoxic T lymphocytes (CTL). A live fluorescent dye (red) is used to selectively pre-stain the cancer cells before co-cultures and a live fluorescent reporter for caspase activity (green) is used to monitor apoptotic cell death. The here described open-source computational method, named STAMP (spatiotemporal apoptosis mapper), extracts the temporal kinetics and the spatial maps of cancer death, by localizing and tracking cancer cells in the red channel, and by counting the red to green transition signals, over 2-3 days. The robustness and versatility of the method is demonstrated by its application to different cell models and co-culture combinations. Noteworthy, this approach reveals the strong contribution of primary cancer-associated fibroblasts (CAFs) to breast cancer chemo-resistance, proving to be a powerful strategy to investigate intercellular cross-talks and drug resistance mechanisms. Moreover, we defined a new parameter, the 'potential of death induction', which is computed in time and in space to quantify the impact of dying cells on neighbor cells. We found that, contrary to natural death, cancer death induced by chemotherapy or by CTL is transmissible, in that it promotes the death of nearby cancer cells, suggesting the release of diffusible factors which amplify the initial cytotoxic stimulus.

Autophagy is a physiological degradation process that removes unnecessary or dysfunctional components of cells. It is important for normal cellular homeostasis and as a response to a variety of stresses, such as nutrient deprivation. Defects in autophagy have been linked to numerous human diseases, including cancers. Cancer cells require autophagy to migrate and to invade. Here, we study the intracellular topology of this interplay between autophagy and cell migration by an interdisciplinary live imaging approach which combines micro-patterning techniques and an autophagy reporter (RFP-GFP-LC3) to monitor over time, during directed migration, the back–front spatial distribution of LC3-positive compartments (autophagosomes and autolysosomes). Moreover, by exploiting a genetically controlled cell model, we assessed the impact of transformation by the Ras oncogene, one of the most frequently mutated genes in human cancers, which is known to increase both cell motility and basal autophagy. Static cells displayed an isotropic distribution of autophagy LC3-positive compartments. Directed migration globally increased autophagy and polarized both autophagosomes and autolysosomes at the front of the nucleus of migrating cells. In Ras-transformed cells, the front polarization of LC3 compartments was much less organized, spatially and temporally, as compared to normal cells. This might be a consequence of altered lysosome positioning. In conclusion, this work reveals that autophagy organelles are polarized toward the cell front during migration and that their spatial-temporal dynamics are altered in motile cancer cells that express an oncogenic Ras protein.

Summary: Rac1 is a small RhoGTPase switch that orchestrates actin branching in space and time and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single-molecule imaging and super-resolution microscopy, we show an additional supramolecular organization of Rac1. We find that Rac1 partitions and is immobilized into nanoclusters of 50–100 molecules each. These nanoclusters assemble because of the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs and possibly GAPs, downstream effectors, and other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms. : Rac1 is a small GTPase protein controlling the polymerization of actin at the front of migrating cells. Using super-resolution microscopy, Remorino et al. show that Rac1 forms nanoclusters of heterogeneous composition, which presumably act as discrete signaling platforms. The subcellular distribution of Rac1 nanoclusters follows its pattern of activation. Keywords: Rac1, signaling, nanoclusters, cell polarity, single molecule, optogenetics

Summary: A major challenge in cancer research is the complexity of the tumor microenvironment, which includes the host immunological setting. Inspired by the emerging technology of organ-on-chip, we achieved 3D co-cultures in microfluidic devices (integrating four cell populations: cancer, immune, endothelial, and fibroblasts) to reconstitute ex vivo a human tumor ecosystem (HER2+ breast cancer). We visualized and quantified the complex dynamics of this tumor-on-chip, in the absence or in the presence of the drug trastuzumab (Herceptin), a targeted antibody therapy directed against the HER2 receptor. We uncovered the capacity of the drug trastuzumab to specifically promote long cancer-immune interactions (>50 min), recapitulating an anti-tumoral ADCC (antibody-dependent cell-mediated cytotoxicity) immune response. Cancer-associated fibroblasts (CAFs) antagonized the effects of trastuzumab. These observations constitute a proof of concept that tumors-on-chip are powerful platforms to study ex vivo immunocompetent tumor microenvironments, to characterize ecosystem-level drug responses, and to dissect the roles of stromal components. : Inspired by the emerging technology of tumor-on-chip, Nguyen et al. reconstituted ex vivo a human tumor microenvironment (HER2+ breast cancer), characterized the ecosystem-level responses to the drug trastuzumab (Herceptin), and dissected the roles of stromal components (immune cells and fibroblasts), demonstrating the power of immunocompetent tumors-on-chip for preclinical drug studies. Keywords: tumor microenvironment, organ-on-chip, tumor-on-chip, trastuzumab, HER2+ breast cancer, cancer-associated fibroblasts, live cell imaging, microfluidics, pre-clinical models, immunotherapy

The two Ral GTPases, RalA and RalB, have crucial roles downstream Ras oncoproteins in human cancers; in particular, RalB is involved in invasion and metastasis. However, therapies targeting Ral signalling are not available yet. By a novel optogenetic approach, we found that light-controlled activation of Ral at plasma-membrane promotes the recruitment of the Wave Regulatory Complex (WRC) via its effector exocyst, with consequent induction of protrusions and invasion. We show that active Ras signals to RalB via two RalGEFs (Guanine nucleotide Exchange Factors), RGL1 and RGL2, to foster invasiveness; RalB contribution appears to be more important than that of MAPK and PI3K pathways. Moreover, on the clinical side, we uncovered a potential role of RalB in human breast cancers by determining that RalB expression at protein level increases in a manner consistent with progression toward metastasis. This work highlights the Ras-RGL1/2-RalB-exocyst-WRC axis as appealing target for novel anticancer strategies.

The human genome contains six genes coding for proteins validated in vitro as specific activators of the small GTPases "Ras-related protein Ral-A" and "Ras-related protein Ral-B", generically named Ral-guanine nucleotide exchange factors (RalGEF). Ral proteins are important contributors to Ras oncogenic signaling, and RAS oncogenes are important in human Non-Small Cell Lung Carcinoma (NSCLC). Therefore in this work, RalGEF contribution to oncogenic and non-oncogenic features of human NSCLC cell lines, as anchorage-dependent and independent growth, cell survival, and proliferation, was investigated. Among all human RalGEF, silencing of RGL1 and RALGPS1 had no detectable effect. However, silencing of either RGL2, RGL3, RALGDS or, to a larger extent, RALGPS2 inhibited cell population growth in anchorage dependent and independent conditions (up to 90 and 80%, respectively). RALGPS2 silencing also caused an increase in the number of apoptotic cells, up to 45% of the cell population in transformed bronchial BZR cells. In H1299 and A549, two NSCLC cell lines, RALGPS2 silencing caused an arrest of cells in the G0/G1-phase of cell cycle. Furthermore, it was associated with the modulation of important cell cycle regulators: the E3 Ubiquitin Protein Ligase S-phase kinase-associated protein 2 (Skp2) was strongly down-regulated (both at mRNA and protein levels), and its targets, the cell cycle inhibitors p27 and p21, were up-regulated. These molecular effects were not mimicked by silencing RALA, RALB, or both. However, RALB silencing caused a modest inhibition of cell cycle progression, which in H1299 cells was associated with Cyclin D1 regulation. In conclusion, RALGPS2 is implicated in the control of cell cycle progression and survival in the in vitro growth of NSCLC cell lines. This function is largely independent of Ral GTPases and associated with modulation of Skp2, p27 and p21 cell cycle regulators.