Mechanoadaptation of circulating tumor cells to fluid shear stress during hematogenous metastasis

Introduction: To successfully generate secondary lesions, a portion of circulating tumor cells (CTCs) must hold the ability to survive all rate-limiting factors during the entire metastatic process, including fluid shear stress (FSS) experienced by CTCs in blood circulation, which is the major route of distal metastasis. However, how CTCs sense and survive FSS remains incompletely understood.

Materials and

Methods: A microfluidic system was developed to mimic blood shear flow in vitro. Various biochemical analyses were conducted to characterize the mechano-responses of suspended tumor cells to varying levels of FSS, such as cleaved caspase 3 and γ-H2AX. Nuclear strain was quantified to measure the force transmission efficiency (FTE), defined by the ratio of the resultant nuclear strain to the exerted FSS. The subcellular distribution of phosphorylated myosin light chain (p-MLC) was tested by immunofluorescence staining. Both pharmacologic and genetic approaches were adopted to modulate myosin and other mechanotransduction signaling in order to unveil the mechanisms underlying the mechanoadapatation of CTCs to different levels of FSS. When cytoplasmic p-MLC was inhibited, the influences on CTC survival and tumor metastasis were examined in the animal model in both short- and long-term.

Results, Conclusions, and Discussions: Suspended tumor cells exhibit differential survival responses to varying levels of FSS. In particular, these cells display high increase rates of cleaved caspase 3 and γ-H2AX under low FSS range, where are notably reduced under high FSS. This attenuation of mechanosensitivity is further supported by the reduction of FTE, when FSS is increased from low to high range. Interestingly, p-MLC is mainly located in cell cortex and cytoplasm under low and high FSS, respectively. Inhibiting cytoplasmic but not cortical p-MLC restores the reduced FTE to the level of control cells, which may be due to the disruptive effect of cytoplasmic p-MLC on the binding of myosin with actin. Under high FSS, Lamin A/C-mediated nuclear mechanosensing unfolds nuclear envelop and increases nuclear membrane tension, which releases calcium from endoplasmic reticulum that leads to the redistribution of p-MLC to cytoplasm. The inhibition of this mechanoadaptation induced by cytoplasmic p-MLC increases FTE and mechanosensitivity to high FSS and re-sensitizes suspended tumor cells and cancer stem cells to shear-induced DNA damage and apoptosis. Temporary inhibition of cytoplasmic p-MLC only during hematogenous dissemination effectively eradicates more CTCs in short term and suppresses tumor metastasis in long term. Therefore, these results uncover a new role of cytoplasmic myosin in regulating mechanosensitivity of CTCs to FSS, which protects them from shear-induced apoptosis during hematogenous dissemination.

Acknowledgements (Optional): The study was supported by the General Research Fund of Hong Kong Research Grant Council (PolyU 15214320, 15227523, and C5016-23G), Health and Medical Research Fund (HMRF18191421), Shenzhen Science and Technology Innovation Commission (JCYJ20220531091002006), the Hong Kong Polytechnic University (1-CDJN, 1-CD75).