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Recent data showed that cancer cells from different tumor subtypes with distinct metastatic potential influence each other9s metastatic behavior by exchanging biomolecules through extracellular vesicles (EVs). However, it is debated how small amounts of cargo can mediate this effect, especially in tumors where all cells are from one subtype, and only subtle molecular differences drive metastatic heterogeneity. To study this, we have characterized the content of EVs shed in vivo by two clones of melanoma (B16) tumors with distinct metastatic potential. Using the Cre‐LoxP system and intravital microscopy, we show that cells from these distinct clones phenocopy their migratory behavior through EV exchange. By tandem mass spectrometry and RNA sequencing, we show that EVs shed by these clones into the tumor microenvironment contain thousands of different proteins and RNAs, and many of these biomolecules are from interconnected signaling networks involved in cellular processes such as migration. Thus, EVs contain numerous proteins and RNAs and act on recipient cells by invoking a multi‐faceted biological response including cell migration.

Introduction Tumours consist of heterogeneous populations of cancer cells with various abilities to metastasize. Recent data shows that cancer cells from different subtypes exchange biomolecules through extracellular vesicles (EVs), influencing each other’s metastatic behaviour. How EVs can mediate this effect is still largely unknown, especially in tumours from the same subtype in which molecular differences are small but instrumental drivers of metastasis. Material and methods Here, we study EVs shed by two B16 melanoma lines with different metastatic potential. Using label-free tandem mass spectrometry and RNA sequencing we profile the tumour microenvironmental EVs shed by these clones in vivo. We use the Cre-LoxP system to monitor EV transfer in vivo in combination with intravital microscopy to study phenotypic changes induced by EV exchange. Results and discussions We show that melanoma cells with different metastatic capacities functionally exchange EVs in vivo. EVs shed into the tumour microenvironment by cancer cells contain interconnected protein and RNA signalling networks involved in a variety of processes, including cell migration. Transfer of EVs from highly metastatic and migratory cells copies their phenotype to other cancer cells. Conclusion We show that cancer cells influence each other’s behaviour in the tumour microenvironment through EVs. Profiling of tumor-derived EVs suggests that this is mediated by a diverse range of EV-molecules that together amplify numerous nodes of signalling networks in recipient cells.

Cell dynamics in subcutaneous and breast tumors can be studied through conventional imaging windows with intravital microscopy. By contrast, visualization of the formation of metastasis has been hampered by the lack of long-term imaging windows for metastasis-prone organs, such as the liver. We developed an abdominal imaging window (AIW) to visualize distinct biological processes in the spleen, kidney, small intestine, pancreas, and liver. The AIW can be used to visualize processes for up to 1 month, as we demonstrate with islet cell transplantation. Furthermore, we have used the AIW to image the single steps of metastasis formation in the liver over the course of 14 days. We observed that single extravasated tumor cells proliferated to form "pre-micrometastases," in which cells lacked contact with neighboring tumor cells and were active and motile within the confined region of the growing clone. The clones then condensed into micrometastases where cell migration was strongly diminished but proliferation continued. Moreover, the metastatic load was reduced by suppressing tumor cell migration in the pre-micrometastases. We suggest that tumor cell migration within pre-micrometastases is a contributing step that can be targeted therapeutically during liver metastasis formation.

High-resolution intravital imaging (IVM) has proven to be a powerful technique to visualise dynamic processes that are important for tumour progression, such as the interplay between tumour cells and cellular components of the tumour microenvironment. The development of IVM tools, including imaging windows and photo-marking of individual cells, has led to the visualisation of dynamic processes and tracking of individual cells over a time span of days. In order to visualise these dynamic processes, several strategies have been described to develop fluorescent IVM tumour models. Genetic tools to engineer fluorescent tumour cell lines have advanced the applications of cell line-based tumour models to study, for example, changes in behaviour or transcriptional and differentiation state of individual cells in a tumour. In order to study tumour progression, fluorescent genetic mouse models have been engineered that better recapitulate human tumours. These technically challenging tumour models are key in visualising dynamic processes during cancer progression and in the translational aspects of IVM experiments.

Breast cancer is the second cause of death in Europe with an incidence of 464.00 new cases in 2012. Chemotherapy is frequently used to treat severe breast cancer. Current drugs like taxanes and anthracycline used alone or in combination are efficient. Moreover the use of biomarkers, like HER2 and BRCA status is now helping in the choice of the most adapted chemotherapy to the patient. De novo or acquired resistance limits, however, the clinical usefulness of drugs used in current chemotherapy. The development of new therapeutics effective against drug-resistant cancers thus still represents an important challenge. Our team has discovered a new LIM Kinases (LIMK1/2) inhibitor, “LIM-Pyr1”. LIMK1/2 are situated at a crossroads of several signaling pathways mainly activated by tyrosine kinase receptors and regulate both actin and microtubules dynamics. LIMK1/2 regulate actin dynamics through cofilin phosphorylation. Cofilin is an actin-depolymerizing factor and its phosphorylation inactivates its actin severing activity. LIMK1/2 also regulate microtubule dynamics through a mechanism unknown yet. LIMK1/2 inhibition induce microtubules stabilization (1). Moreover, LIMK1/2 are overexpressed in many invasive cancers and appear to be a relevant target for anticancer therapy (2, 3). We have shown that LIM-Pyr1 is toxic on cell lines resistant to conventional chemotherapy (4) and tested LIM-Pyr1 therapeutic activity on different breast cancer models (xenografts in mice). We found that LIM-Pyr1 shows a potent antitumor activity both on primary and secondary tumors, with no detectable undesirable side effects. The antitumor effect is effective on paclitaxel resistant xenografts. Finally, intravital microscopy analysis indicates that a LIM-Pyr1 treatment induces a strong morphological change of tumor cells inside the tumors and reduces their migration. LIM-Pyr1 and its derivatives could thus represent a pharmacological alternative to overcome resistances often observed when tumors are treated with microtubule targeting agents. (1) O. Bernard, LIM Kinases, regulators of actin dynamics, Int. Journals of Biochemistry and cell biology (2007) 1071-1076 (2) F. Manetti, Recent finding confirm LIM Domain Kinases as emerging target candidates for cancer therapy, Curr. Cancer Drug Targets (2012) 12,543-560 (3) W. Wang, R. Eddy, J. Condeelis, The cofilin pathway in breast cancer invasion and metastasis, Nat. Cancer Reviews (2007) (4) R. Prudent, E. Vassal-Stermann, C-H Nguyen et al., Pharmacological inhibition of LIM Kinases stabilizes microtubules, Cancer Research (2012) Citation Format: Chloé Prunier, Julien Vollaire, Véronique Josserand, Anoek Zomer, Amandine Hurbin, Renaud Prudent, Pascale Cohen, Jacco van Rheenen, Jean-Luc Coll, Marc Billaud, Laurence Lafanechère. Anti-cancer activity of a new LIM-Kinases inhibitor: “LIM-Pyr1”. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5399. doi:10.1158/1538-7445.AM2015-5399

Summary Most cancer cells release heterogeneous populations of extracellular vesicles (EVs) containing proteins, lipids, and nucleic acids. In vitro experiments showed that EV uptake can lead to transfer of functional mRNA and altered cellular behavior. However, similar in vivo experiments remain challenging because cells that take up EVs cannot be discriminated from non-EV-receiving cells. Here, we used the Cre-LoxP system to directly identify tumor cells that take up EVs in vivo. We show that EVs released by malignant tumor cells are taken up by less malignant tumor cells located within the same and within distant tumors and that these EVs carry mRNAs involved in migration and metastasis. By intravital imaging, we show that the less malignant tumor cells that take up EVs display enhanced migratory behavior and metastatic capacity. We postulate that tumor cells locally and systemically share molecules carried by EVs in vivo and that this affects cellular behavior., Graphical Abstract, Highlights • Visualization of extracellular vesicle exchange between tumor cells in living mice • Direct in vivo evidence of local and systemic mRNA exchange between tumor cells • The exchange of biomolecules is mediated through extracellular vesicles • Metastatic behavior can be phenocopied through extracellular vesicle exchange, Intravital imaging experiments reveal the extracellular vesicle-mediated exchange of molecules important for metastasis between tumor cells in living mice. This exchange results in metastatic properties being conferred to the receiving cell.