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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - RhoSNATCH (Dissecting Rho GTPase signalling networks through acute perturbation techniques)

Teaser

Summary

Cancer is the second leading cause of death in the world, with 9 million reported deaths annually. Metastatic cancer, where secondary tumors are spread from the primary tumor into the rest of the body, still accounts for the majority of cancer-related deaths. Metastatic cancer starts with the cell migration of tumor cells from the primary tumor into the bloodstream. From the bloodstream cancer cells can extravasate to form metastatic tumors elsewhere in the body. Understanding the machinery that governs the migration and extravasation of the cells in metastatic cancer is critical for the development of new therapies to fight this deadly disease that plagues humanity.
The project RhoSNATCH aims to further our understanding of the processes underlying cell movement in healthy and diseased cells on a molecular level. RhoSNATCH aims to identify the critical molecular players in the signaling network that underlies cell motility.
Cellular motility is very strictly organized on timescales of seconds and with micrometer precision to allow for highly controlled behavior in the different tissues of the human body. The machinery that controls this cellular motility is very sensitive to perturbation from outside the cell (mechanical or chemical), which leads to adaptation of the cellular structures that govern cellular motility. Because of this reason it is difficult to study this subject, as classical experimental methods all strongly perturb the cellular structures under study. By combining state of the art genome editing and advanced fluorescent microscopy techniques RhoSNATCH can now address questions about cell migration that were inaccessible before.
By answering these novel questions RhoSNATCH contributes to the search for new molecular targets in the treatment for metastatic cancer. We identified the effects of the acute perturbation of one of the major drivers in cell migration and are currently exploring the effects of several other proteins involved in cell motility.

Work performed

The cloning and testing on several constructs targeting the genomic DNA of Rho GTPase components, using the CRISPR-Cas9 genome editing technique in several cell lines. We produced a stable knock-in cell line for endogenous RhoA and performed cell migration experiments. This is the first time that endogenous RhoA is visualized! We found that acute perturbation of RhoA leads to an increase in cell migration speed and decrease of contractility in several subcellular areas. We are now exploring these results further using super resolution microscopy and more cell migration assays. After this the results will be published in peer-reviewed open access journals. Several stable cell lines containing knock ins of other Rho GTPase signaling components are in production. We will use a similar approach to verify the genomic identity of these cell lines and perform cell migration experiments and state of the art microscopy to assess the effects of acute perturbation of these proteins. The result of this progress was disseminated on various international conferences by posters and oral presentations (International Symposium on Measuring and Modelling Cell Migration - Vienna, 2018 ; International Meeting on Optical biosensors - Gent, 2018), as well as more local conferences (LS2 Annual meeting - Zurich, 2017-2018 ; Cytomeet Bern - Bern, 2017-2019). One of the poster presentations is attached for reference.

Final results

For the first time it is possible to observe endogenous RhoA dynamics in single living cells. Concomitantly it is possible for the first time to acutely perturb RhoA in single living cells within minutes, providing insights on its function in cell motility without the interference of cellular adaptation. There is promising work in progress on several other components of the Rho GTPase signaling network. Altogether these results will provide novel insight in the function and dynamics of Rho GTPase network components in cell migration. This will provide valuable new information towards new therapies to cure metastatic cancer.