Colorectal cancer (CRC) is among the most prevalent cancers worldwide with more than 1.2 million diagnoses and 600,000 deaths per year. Up to 30% of stage II patients relapse after surgery and many of them will die due to metastatic disease. Metastasis is the result of disseminated cancer cells that leave the original tumor to settle to distant organs. How cancer cells move from the primary tumor into the circulation, infiltrate distant organs and initiate metastatic growth is still poorly understood. Tumor-initiating cells (TICs) have been identified as cells that are able to drive tumor initiation, maintenance and progression. Recent literature and our own data suggest that the success of metastatic colonization is related to the capacity of tumor cells to exhibit TIC properties such as the ability to self-renew and thereby spawn large malignant growths in distant organs. Accordingly, we show that generated spheroid cultures (SC) of the metastatic cell line SW620 are more enriched in TICs compared to SC derived from the primary tumor counterpart SW480 (derived from the same patient). By comparing primary and metastatic TIC-enriched cultures, we identified a specific miRNA cluster, namely miR-371~373, that is strongly downregulated in metastatic TICs. Furthermore, metastatic TICs show an increased expression of genes involved in TGF-ß signaling. After stable overexpression of our miRNA cluster of interest, TICs show reduced expression of TGFBR2 and ID1, a master regulator of TICs, and most importantly lose their self-renewal capacity in functional TIC assays. Downregulation of the miR-371~373 cluster and resulting activation of TGF-ß signaling and expression of ID1 therefore seems to be a potential mechanism used by metastatic TICs to assure their increased tumorigenic potential and participate in metastatic colonization. The current project will focus on unraveling the exact mechanism by which miR-371~373 cluster through the TGF-ß/ID1 axis (and other identified direct targets) influences tumor initiation and metastatic colonization. We will further concentrate on the influence of cancer-associated fibroblasts with TICs and the role of the miR-371~373 cluster in these interactions. Finally, we will assess the clinical relevance of miR-371~373 cluster in CRC. This project, by addressing pertinent questions regarding tumor initiation and metastatic colonization in CRC, will generate important new fundamental insights into the role of TICs in cancer and might ultimately lead to the identification of new therapeutic targets for CRC. Importantly, this application within the FNR CORE Junior track represents a major career progression of the early career-stage researcher as it will allow the PI to consolidate her recently established independent research line.