Circulating tumor cells (CTCs) are exposed to various insults thought to reduce their survival including lack of anchorage and trophic support from the primary tumor microenvironment, immune-mediated destruction and exposure to hemodynamic forces that may mechanically destroy them. However, the relative contribution of these factors to CTC survival and their overall role in metastasis is unclear. It has recently been shown that cancer cells from many tissue origins actively resist destruction by fluid shear stress (FSS), implying that viable CTCs are not mechanically fragile as suspected. Our long-term goal is to understand the biomechanical influences on CTCs and how this contributes to metastasis. The objective of this proposal is to determine the mechanism of FSS resistance in cancer cells and its role in metastatic colonization. Our central hypothesis is that mechano-adaptation of viable CTCs to FSS promotes their survival in the circulation and “primes” them for subsequent events in metastasis. Our hypothesis is based on our previously published and preliminary data presented below as well as recently published data from others which is supportive of our hypothesis. The rationale for the proposed research is that once we understand the mechanism underlying FSS resistance; this would represent a novel therapeutic approach aimed at decreasing the survival of CTCs by enhancing their destruction due to the mechanical forces that naturally exist in the circulation. Guided by preliminary data, the central hypothesis will be tested by pursuing the following specific aims: 1) Define molecular mechanisms of FSS resistance; 2) Determine the effect of FSS exposure on metastatic colonization; 3) Determine the effectiveness of inhibiting FSS resistance as an anti-metastatic strategy. To accomplish these aims, we will employ a forward genetic screen to identify novel genes that mediate FSS resistance and determine their involvement with RhoA- actomyosin interactions. Short-term survival of CTCs will be assessed using a novel mouse model to measure entrapment of intact CTCs in the lung and destruction of CTCs measured by a plasma biomarker. We will validate novel genes identified in a forward genetic screen in similar assays. We will determine the involvement of RhoA- YAP activation by FSS in supporting the survival and extravasation of cancer cells lodged in the microcirculation. Finally, we will test the potential of clinically actionable drugs that sensitize cells to FSS as well as conditional RhoA/YAP knockdown to block productive metastatic colonization in mouse models. The proposed research is innovative because, it represents a paradigm shift from the idea that CTCs are mechanically fragile by elucidating a mechanism whereby viable CTCs actively resist destruction by hemodynamic forces and drives further events in metastasis. The proposed research is significant because defining the mechanisms and consequences of FSS resistance in CTCs will open ent...