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Accumulating evidence suggests that dysbiosis, a state of pathologic imbalance in the human gut microbiome, is present in patients suffering from colorectal cancer (CRC). Several microbiome studies identified specific bacteria that are associated with CRC, among which Fusobacteria were shown to directly interact with cancer or immune cells of their host. However, only a limited number of CRC-associated microbes have been studied for host-microbial interactions; hence, the role of bacteria in the etiology of the disease remains unknown. Accordingly, our work aims at the development of a methodologic workflow for studying CRC-associated bacteria and their role in colon cancer tumor initiation and progression. In a first step, we identified CRC-associated bacteria that are enriched at the tumor site of CRC patients. Therefore, we used publicly available datasets and an in-house patient sample collection. Then, we predicted and optimized bacterial growth in silico by using a genome-scale metabolic reconstruction model combined with a constraint-based modeling approach. Finally, we implemented CRC-associated bacteria together with established primary CRC patient cultures into the microfluidics-based human-microbial crosstalk model (HuMiX). Our workflow allowed to analyze host-microbial interaction mechanisms of CRC-associated bacteria on a transcriptomic, proteomic, and metabolomic level. Citation Format: Dominik Ternes, Martine Schmitz, Léa Grandmougin, Mina Tsenkova, Eric Koncina, Aurélien Ginolhac, Jessica Karta, Diana Kuhn, Javier Ramiro Garcia, Kacy Greenhalgh, Paul Wilmes, Elisabeth Letellier, Serge Haan. Understanding the role of colorectal cancer-associated microbes in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A09.

The vast majority of colorectal cancer–related deaths can be attributed to metastatic spreading of the disease. Therefore, deciphering molecular mechanisms of metastatic dissemination is a key prerequisite to improve future treatment options. With this aim, we took advantage of different colorectal cancer cell lines and recently established primary cultures enriched in colon cancer stem cells, also known as tumor-initiating cells (TIC), to identify genes and miRNAs with regulatory functions in colorectal cancer progression. We show here that metastasis-derived TICs display increased capacity for self-renewal, TGFβ signaling activity, and reduced expression of the miR-371∼373 cluster compared with nonmetastatic cultures. TGFβ receptor 2 (TGFBR2) and aldehyde dehydrogenase A1 (ALDH1A1) were identified as important target genes of the miR-371∼373 cluster. In addition, TGFBR2 repression, either by direct knockdown or indirectly via overexpression of the entire miR-371∼373 cluster, decreased tumor-initiating potential of TICs. We observed significantly reduced in vitro self-renewal activity as well as lowered tumor initiation and metastatic outgrowth capacity in vivo following stable overexpression of the miR-371∼373 cluster in different colon TIC cultures. Inhibitor of DNA binding 1 (ID1) was affected by both TGFBR2 and miR-371∼373 cluster alterations. Functional sphere and tumor formation as well as metastatic dissemination assays validated the link between miR-371∼373 and ID1. Altogether, our results establish the miR-371∼373/TGFBR2/ID1 signaling axis as a novel regulatory mechanism of TIC self-renewal and metastatic colonization. Significance: These findings establish the miR-371∼373/TGFBR2/ID1 signaling axis as a novel mechanism regulating self-renewal of tumor-initiating cell and metastatic colonization, potentially opening new concepts for therapeutic targeting of cancer metastasis. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3793/F1.large.jpg. Cancer Res; 78(14); 3793–808. ©2018 AACR.