Abstract PR04: Mechanisms and pathophysiologic relevance of fluid shear stress resistance in malignant cells
Authors: Krog Benjamin , Zhao Lei , Moose Devon , Burke Gretchen , Chivukula Keshav , Nauseef Jones , Milhem Mohammed , Vigmostad Sarah , Henry Michael 
Published: March 2017

Abstract
The mechanobiology of circulating tumor cells, detached from an extracellular matrix, is poorly understood. A longstanding idea in cancer biology is that during metastasis, cancer cells shed into the circulation from epithelial organs are inherently fragile and are mechanically destroyed by hemodynamic forces. To the contrary, we have recently shown that malignant cells, from diverse tissue types, are remarkably resistant to brief (millisecond) pulses of high-level fluid shear stress (FSS) that may be encountered in the microenvironment of the circulation as compared to benign cells [1]. FSS resistance is a phenotype conferred by a variety of oncogenic signaling pathways and involves both an enhanced ability of malignant cells to repair plasma membrane damage as well as to rapidly adapt to prior exposure to FSS to resist subsequent pulses of FSS. However, the mechanisms underlying the FSS resistance phenotype and whether these are relevant to cancer cells in tumor tissue or to circulating tumor cells was unknown. Here we show that cancer cells exposed to FSS exhibit an adaptive response, demonstrating an increased stiffness (Young's modulus) as measured by micropipette aspiration [2]. Increased stiffness of cancer cells is associated with reduced damage to the plasma membrane upon subsequent challenges with FSS. To examine the mechanisms underlying this response, we evaluated the Rho-dependent signaling pathway that controls cell contractility. Knockdown of RhoA, but not RhoC sensitize prostate cancer cells to FSS. Pharmacologic inhibition myosin II-based contractility also reduced prostate cancer cell resistance to FSS. To determine if cancer cells that exist in tumor tissue exhibit resistance to FSS, we isolated cell suspensions from mice bearing prostate-specific PTEN and/or p53 mutations and exposed them to repeated pulses of FSS. We found that, compared to wild-type control mice, prostate cells from tumor-bearing mice exhibited resistance to FSS characteristic of established human prostate cancer cell lines, indicating that our findings extend beyond established cell lines. Moreover, extending our findings to human tumors, we acutely isolated cells from PDX tumors from melanoma patients. 3 independently-derived tumors exhibited a characteristic biphasic response curve when exposed to multiple pulses of FSS. Interestingly, brief exposure to Vemurafenib altered the response profile of two of these isolates. To examine whether FSS impacts circulating tumor cells, we treated prostate cancer cell line acutely with blebbistatin, a myosin II inhibitor, and vehicle control and injected these differentially fluorescently-labeled cells along with 15 micron fluorescent beads. The beads lodge in the microvasculature and act as a reference for cell counts. Within one minute after injection, we euthanized the mice and isolated lungs. Labeled cells and beads were counted in frozen sections and the ratio of cells:beads was counted and compared to pre-injection ratios. ~60% of vehicle treated cancer cells survive intact in the lungs whereas only ~40% of blebbistatin-treated cells survive. This suggests that a single pass through the right mouse heart damages circulating tumor cells, but that a mechanism that depends on myosin II promotes survival of these cells. This is the first evidence that FSS resistance is a contributing factor for the survival of circulating tumor cells. This work is supported by NCI grants CA179981&CA196202.
References:
1. Barnes, J.M., J.T. Nauseef, and M.D. Henry, Resistance to fluid shear stress is a conserved biophysical property of malignant cells. PLoS One, 2012. 7(12): p. e50973.
2. Chivukula, V.K., et al., Alterations in cancer cell mechanical properties after fluid shear stress exposure: a micropipette aspiration study. Cell Health Cytoskelet, 2015. 7: p. 25-35.
This abstract is also being presented as Poster A46.
Citation Format: Benjamin Krog, Lei Zhao, Devon Moose, Gretchen Burke, Keshav Chivukula, Jones Nauseef, Mohammed Milhem, Sarah Vigmostad, Michael Henry. Mechanisms and pathophysiologic relevance of fluid shear stress resistance in malignant cells. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr PR04.