Although fibroblast heterogeneity is recognized in primary tumors, both its characterization in and its impact on metastases remain unknown. Here, combining flow cytometry, immunohistochemistry and RNA-sequencing on breast cancer samples, we identify four Cancer-Associated Fibroblast (CAF) subpopulations in metastatic lymph nodes (LN). Two myofibroblastic subsets, CAF-S1 and CAF-S4, accumulate in LN and correlate with cancer cell invasion. By developing functional assays on primary cultures, we demonstrate that these subsets promote metastasis through distinct functions. While CAF-S1 stimulate cancer cell migration and initiate an epithelial-to-mesenchymal transition through CXCL12 and TGFβ pathways, highly contractile CAF-S4 induce cancer cell invasion in 3-dimensions via NOTCH signaling. Patients with high levels of CAFs, particularly CAF-S4, in LN at diagnosis are prone to develop late distant metastases. Our findings suggest that CAF subset accumulation in LN is a prognostic marker, suggesting that CAF subsets could be examined in axillary LN at diagnosis.

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.

We describe a novel method to achieve a universal, massive, and fully automated analysis of cell motility behaviours, starting from time-lapse microscopy images. The approach was inspired by the recent successes in application of machine learning for style recognition in paintings and artistic style transfer. The originality of the method relies i) on the generation of atlas from the collection of single-cell trajectories in order to visually encode the multiple descriptors of cell motility, and ii) on the application of pre-trained Deep Learning Convolutional Neural Network architecture in order to extract relevant features to be used for classification tasks from this visual atlas. Validation tests were conducted on two different cell motility scenarios: 1) a 3D biomimetic gels of immune cells, co-cultured with breast cancer cells in organ-on-chip devices, upon treatment with an immunotherapy drug; 2) Petri dishes of clustered prostate cancer cells, upon treatment with a chemotherapy drug. For each scenario, single-cell trajectories are very accurately classified according to the presence or not of the drugs. This original approach demonstrates the existence of universal features in cell motility (a so called “motility style”) which are identified by the DL approach in the rationale of discovering the unknown message in cell trajectories.

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.

There is a compelling need for approaches to predict the efficacy of immunotherapy drugs. Tumor-on-chip technology exploits microfluidics to generate 3D cell co-cultures embedded in hydrogels that recapitulate simplified tumor ecosystems. Here, we present the development and validation of lung tumor-on-chip platforms to quickly and precisely measure ex vivo the effects of immune checkpoint inhibitors on T cell-mediated cancer cell death by exploiting the power of live imaging and advanced image analysis algorithms. The integration of autologous immunosuppressive FAP⁺ cancer-associated fibroblasts impaired the response to anti-PD-1, indicating that tumors-on-chips are capable of recapitulating stroma-dependent mechanisms of immunotherapy resistance. For a small cohort of non-small cell lung cancer patients, we generated personalized tumors-on-chips with their autologous primary cells isolated from fresh tumor samples, and we measured the responses to anti-PD-1 treatment. These results support the power of tumor-on-chip technology in immuno-oncology research and open a path to future clinical validations.

Cell migration is central to many developmental, physiologic and pathological processes, including cancer progression. The Ral GTPases (RalA and RalB) which act down-stream the Ras oncogenes, are key players in the coordination between membrane trafficking and actin polymerization. A major direct effector of Ral, the exocyst complex, works in polarized exocytosis and is at the center of multiple protein-protein interactions that support cell migration by promoting protrusion formation, front-rear polarization, and extra-cellular matrix degradation. In this review we describe the recent advancements in deciphering the molecular mechanisms underlying this role of Ral via exocyst on cell migration. Among others, we will discuss the recently identified cross-talk between Ral and Rac1 pathways: exocyst binds to a negative regulator (the RacGAP SH3BP1) and to the major effector (the Wave Regulatory Complex, WRC) of Rac1, the master regulator of protrusions. Next challenge will be to better characterize the dynamics in space and in time of these molecular interplays, to better understand the pleiotropic functions of Ral in both normal and cancer cells.