Abstract Metastatic progression of cancer, which is responsible for 90% of patients death, results from tumor cells dissemination out of the primary tumor throughout the body. During the first step of this process -that consists in invasion of the peritumoral stroma- cancer cells can adopt 1) a single cell mode of invasion, in which cell have lost cell-cell junctions to move individually, 2) or a collective mode of invasion where cells maintain their cell-cell junctions to move as a cohort in which Leader cells at the front drag the follower cells at the rear. Although tumour histology data from cancer patients show that invasion occurs predominantly in a collective manner, this mode of cell invasion remains underinvestigated. My work aims at identifying the molecular and cellular mechanisms underlying colorectal carcinoma (CRC) collective invasion. I use 3D organotypic models of CRC: Caco-2 cell lines or organoids generated from CRC patients derived xenografts (PDX) and assess invasion in collagen-I based gels using microscopy approaches on fixed or live samples. Knowing its central role in the cytoskeleton dynamics which is the motor of cell motility, we hypothesize that RhoGTPases signaling pathways could control the collective mode of invasion. We therefore performed a siRNA based screen targeting the 98 effectors of the pathway and found ROCK to be an anti-invasive protein. Although it had been described as a proinvasive protein in the single cell mode of invasion, we confirmed using pharmacological inhibitors (Y27632 and H1152), that ROCK activity inhibition triggered collective invasion. Using a ROCK2 dominant negative mutant specifically targeting ROCK2 isoform but not ROCK1, I demonstrated that ROCK2 inhibition was sufficient to induce leader cell formation leading to collective invasion. In contrast, depletion of MyosinII -ROCK’s most common effector- was not sufficient to induce efficient protusive leader cells. However I found RAC1 to be necessary and in a 2nd siRNA based screen targeting GEFs, we identified FARP2, a GEF for Rac1, as the mediator of ROCK-RAC1 crosstalk in collective invasion. Even though the activation of FARP2 alone or RAC1 alone were not sufficient to induce leader cell formation, the concomitant inhibition of MyosinII recapitulated the collective invasion induced by ROCK inhibition. Altogether these results show a new anti-invasive role of ROCK2 kinase in collective invasion as it controls the formation of leader cells, through 1) the negative regulation of RAC1 and its GEF FARP2, and 2) the positive regulation of MyosinII. Citation Format: Fotine Libanje, Joel Raingeaud, Zoé ap Thomas, Fatiha Sangar-Mavouna, Anne Chauchereau Chauchereau, Fanny Jaulin. ROCK dependent signalling pathways contribution to collective invasion of colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 892. doi:10.1158/1538-7445.AM2017-892