Highlights d Cancer cells from primary tumors are resistant to fluid shear stress (FSS) d Resistance to FSS is a physiological, mechano-adaptive response in cancer cells d Cancer cells respond to FSS by activating the RhoA-myosin II axis and formins d Myosin II activity protects CTCs from hemodynamic forces in in vivo assays Authors

Circulating tumor cells (CTCs) exist in a microenvironment quite different from the solid tumor tissue microenvironment. They are detached from matrix and exposed to the immune system and hemodynamic forces leading to the conclusion that life as a CTC is "nasty, brutish, and short." While there is much evidence to support this assertion, the mechanisms underlying this are much less clear. In this chapter we will specifically focus on biomechanical influences on CTCs in the circulation and examine in detail the question of whether CTCs are mechanically fragile, a commonly held idea that is lacking in direct evidence. We will review multiple lines of evidence indicating, perhaps counterintuitively, that viable cancer cells are mechanically robust in the face of exposures to physiologic shear stresses that would be encountered by CTCs during their passage through the circulation. Finally, we present emerging evidence that malignant epithelial cells, as opposed to their benign counterparts, possess specific mechanisms that enable them to endure these mechanical stresses.

Over 90% of cancer deaths result not from primary tumor development, but from metastatic tumors that arise after cancer cells circulate to distal sites via the circulatory system. While it is known that metastasis is an inefficient process, the effect of hemodynamic parameters such as fluid shear stress (FSS) on the viability and efficacy of metastasis is not well understood. Recent work has shown that select cancer cells may be able to survive and possibly even adapt to FSS in vitro. The current research seeks to characterize the effect of FSS on the mechanical properties of suspended cancer cells in vitro. Nontransformed prostate epithelial cells (PrEC LH) and transformed prostate cancer cells (PC-3) were used in this study. The Young’s modulus was determined using micropipette aspiration. We examined cells in suspension but not exposed to FSS (unsheared) and immediately after exposure to high (6,400 dyn/cm2) and low (510 dyn/cm2) FSS. The PrEC LH cells were ~140% stiffer than the PC-3 cells not exposed to FSS. Post-FSS exposure, there was an increase of ~77% in Young’s modulus after exposure to high FSS and a ~47% increase in Young’s modulus after exposure to low FSS for the PC-3 cells. There was no significant change in the Young’s modulus of PrEC LH cells post-FSS exposure. Our findings indicate that cancer cells adapt to FSS, with an increased Young’s modulus being one of the adaptive responses, and that this adaptation is specific only to PC-3 cells and is not seen in PrEC LH cells. Moreover, this adaptation appears to be graded in response to the magnitude of FSS experienced by the cancer cells. This is the first study investigating the effect of FSS on the mechanical properties of cancer cells in suspension, and may provide significant insights into the mechanism by which some select cancer cells may survive in the circulation, ultimately leading to metastasis at distal sites. Our findings suggest that biomechanical analysis of cancer cells could aid in identifying and diagnosing cancer in the future.

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.

Cancer cells traveling to distant tissues during metastasis must survive passing through the circulation. However, the influence of this fluid microenvironment on these cells is poorly understood. It was previously viewed that exposure to the hemodynamic shear forces within circulation was inhospitable to cancer cells, causing the cells to be destroyed. Recent evidence indicates that transformed cells are markedly more resistant to fluid shear stress when compared to non-transformed epithelial cells. Furthermore, these cells selectively adapt following exposure to fluid shear stresses and become more resistant to subsequent exposures to shear stress. The mechanisms behind this difference in phenotype and induced resistance are investigated. The elastic modulus, a measure of stiffness, may play a role in resistance and is shown to be altered upon exposure to fluid shear forces. Additionally, plasma membrane repair is a critical process in the resistance phenotype as cells sustain damage but are able to maintain viability. Cytoskeletal dynamics are also shown to play a role in resistance to fluid shear forces.