RhoA‐stimulated intra‐capillary morphology switch facilitates the arrest of individual circulating tumor cells

Abstract

Metastasis is the primary cause of death for most cancer patients. Hematogenous arrest of circulating tumor cells (CTCs) is an essential prerequisite for metastases formation. Using transparent transgenic zebrafish (kdrl:eGFP; Casper), together with resonant laser scanning confocal microscopy, we tracked the fate of CTCs in vivo in the blood circulation for days. We found the intra-capillary morphology-switch (ICMS) of individual CTCs from strip to sphere was necessary for their intravascular arrests. Further genetic and pharmacological inhibition experiments indicated that the RhoA signaling was necessary for ICMS and the arrest of CTCs. At last, we demonstrated that early treatment by a clinically approved RhoA/ROCK inhibitor, Fasudil, could efficiently inhibit the initial arrest of individual CTCs and reduce the incidence of tumor metastasis in both zebrafish and mouse models. These results together indicate that RhoA-stimulated ICMS represents a mechanism for the arrest of individual CTCs, providing a potential target for future treatments of hematogenous metastatic disease.

Rho GTPases Signaling in Zebrafish Development and Disease

“…This was a major breakthrough that highlighted a rather unappreciated role for RhoA in liver tumorigenesis in regards to several previous contradictory in vitro studies [99][100][101][102], underscoring the importance of in vivo models to better understand the role of RhoA signaling in health and disease. Further evidence revealed by Xi Huang and his colleagues linked RhoA activation to the arrest of circulating tumor cells in vivo [103]. Altogether, RhoA stimulation might represent a mechanism for reducing liver tumorigenesis and for halting the circulation of tumor cells in hematogenous metastatic disease.…”

Squeezing through the microcirculation: survival adaptations of circulating tumour cells to seed metastasis

“…This cellular rounding facilitated the entrapment of CTCs and their subsequent extravasation. 25 Accordingly, treatment with the ROCK inhibitor fasudil decreased the arrest of individual CTCs and the incidence of tumour metastasis in mice. Moreover, treatment with the myosin II inhibitor blebbistatin over a short period of time (3-6 h) delayed the formation of metastasis and the number of foci.…”

“…16 Other factors, such as the adhesion potential of CTCs, 17 the local blood pressure of the organ, its blood flow and the cell deformability, can also influence whether CTCs become arrested. 16,25 Indeed, Follain et al 16 demonstrated in vivo that low blood flow profiles can promote cell arrest without the need for the cells to be physically occluded, suggesting that active adhesion between CTCs and the endothelium is required for a successful extravasation. Other factors, such as the adhesion potential of CTCs 17 and the mechanical constraints of the capillary, 3,16 may also determine CTC arrest.…”

“…44 Mechanical squeezing of cells in both microfluidic and animal model capillaries, such as in mice and zebrafish, has been reported to increase the activity of RhoA, resulting in the recruitment and activation of ROCK. 25,45 When CTCs became lodged in capillaries, the subsequent activation of the RhoA-ROCK-myosin II axis upregulated contractility, causing CTCs to change from an elongated to a spherical phenotype. This cellular rounding facilitated the entrapment of CTCs and their subsequent extravasation.…”

“…When a CTC encounters a capillary bed, two situations can take place: it can get arrested and initiates extravasation, or the CTC can transit through the capillary and escape from it, enabling its migration to a more distant capillary bed. During transit and/or arrest, CTCs undergo intense cell deformation, which can induce changes in the mechanotransduction of signalling pathways, such as RhoA-ROCK and YAP/TAZ, 7,25,[47][48][49] resulting in an increase of cell invasiveness and survival (see inset "Signalling changes"). Upon activation, YAP/TAZ gets translocated to the nucleus, inducing changes in the transcription activity.…”