Periodic Reporting for period 4 - HOLDING-HANDS (Holding hands: cell-cell junctions in breast cancer metastasis and resistance to therapy) Reporting period: 2020-09-01 to 2021-02-28 Summary of the context and overall objectives of the project (1) What is the problem/issue being addressed?The development of cancer metastasis accounts for more than 90% of cancer-related deaths. This project has been focused on the issue of cancer metastasis and on the identification of new treatment to suppress it. Specifically, we recently found that clusters of circulating tumor cells (CTC-clusters) - multicellular aggregates that detach from the primary tumor and enter the bloodstream, on their way to seed metastasis elsewhere - are highly efficient metastatic effectors in breast cancer. Our project has been focused on finding methods to disrupt CTC-clusters and as a consequence, suppress the development of metastasis.(2) Why is it important for society?The development of cancer metastasis from various cancer types is responsible for more than 7 million deaths per year worldwide. Targeting CTC-clusters might reduce or suppress metastasis formation and be beneficial to a large number of patients with metastatic cancers.(3) What are the overall objectives?Our project has been designed to identify and target cell-cell junction components that are required for the formation and maintenance of CTC-clusters. We also aimed at identifying FDA-approved compounds whose net effect is to disrupt CTC-clusters. Overall, our objectives are focused on the identification of a CTC-cluster-suppressing therapy. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The project has been very successfully completed and it has led to major publications (Krol et al., British Journal of Cancer, in press; Donato et al., Cell Rep, 2020; Gkountela et al., Cell, 2019; Szczerba et al., Nature, 2019) and patents filing. Specifically, we have found that intra-tumor hypoxia leads to cell-cell junction upregulation, including overexpression of NDRG1. In turn, NDRG1-positive CTC clusters lead to distant metastasis formation (Donato et al., Cell Rep, 2020). Previously, we also found that CTC clusters rely on specific cell-cell junction components such as CLDN3 and CLDN4 for maintaining their multicellular structure in circulation, and that their dissociation with FDA-approved compounds or knockout of CLDN3 and CLDN4 leads to DNA methylation remodelling at binding sites for stemness- and proliferation-associated transcription factors (Gkountela et al., Cell, 2019). This study has already led to the clinical testing of approved NaK-ATPase inhibitors for the treatment of patients with cancer. Further, we also found that CTCs can cluster with immune cells such as neutrophils, and that this association is mediated by the cell-cell junction component VCAM1. Interaction of CTCs with neutrophils leads to increased proliferation of CTCs within the bloodstream, and a higher ability to initiate metastasis (Szczerba et al., Nature, 2019). More recently, we could also determine that CTC clusters are not only present at late disease stage is patients, but they manifest early, i.e. before primary tumor surgery (Krol et al., British Journal of Cancer, in press). Altogether, the HOLDING-HANDS project has achieved very important results, some of which already translated into novel clinical trials and new approaches to suppress the metastatic spread of cancer. These results have been disseminated in a number of conferences, on social media and publications. Exploitation of the results in underway, as we plan to generate a spinoff company that will develop anti-cluster therapeutics for patients with cancer. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The project has been successfully completed and allowed us to gain very important insights into the biology and vulnerabilities of CTC clusters. Among these, we discover fundamental biological features of CTC cluster and their key cell-cell junction components. We also found FDA-approved NaK-ATPase inhibitors with activity against CTC clusters, and these are now being tested in clinical trials. Compared to the state-of-the-art, our project allowed to achieve three main steps forward: (1) identification of key cell-cell junction components and their biological function in CTC clusters and metastasis, (2) understanding of the molecular features involved in CTC cluster formation and maintenance, and (3) identification of novel and unconventional therapeutic approaches to suppress the metastatic spread of cancer. In the long term, we expect that these results will translate into novel treatment opportunities for patients with aggressive cancers, and will enrich and stimulate the metastasis research field.