Intensive actin filaments assembly

Intensive actin filaments assembly is associated with ICMS and arrest of CTCsAs cytoskeleton is critical for global architecture and shape of the cells, we next labeled the F‐actin in living CTCs by pCMV LifeAct‐GFP to investigate dynamics of F‐actin filaments in the ICMS19 (Fig. 3a). First, to prove the LifeAct is a qualified living tracer for the F‐actin filaments, after transfected with LifeAct‐GFP, tumor cells were either treated with lysophosphatidic acid (specifically activates Rho subfamily and induce the F‐actin filaments formation) (LPA, 10 μM)20 or Cytochalasin B (efficiently depolymerize the F‐actin) (CB, 1 μM). Eight hours after drugs treatment, confocal imaging was applied and the results have confirmed that the LifeAct‐GFP actually worked as expected (Fig. 3b).Then, we injected B16‐LifeAct‐GFP cells into the zebrafish circulation as previously described. A total of 19 CTCs were tracked by confocal (Zeiss Airyscan) for 12 hr till the rounding and arrest of the CTCs. Interestingly, intensive F‐actin filaments formation was detected in rounding CTCs, while in those roaming CTCs, GFP signals were still diffusive (∼4–8 hr) (Fig. 3c).Next, to test the necessity of F‐actin filaments formation in the ICMS and arrest of CTCs, we then used Cytochalasin B (CB, 1 μM for 4 hr) pretreatment to depolymerize the F‐actin of TCs before cell injection.21 As expected, for the DMSO control groups, about 8–10 hr post‐cell injection, most CTCs underwent ICMS and were arrested in the host capillaries, occasionally, extravasation was followed (Fig. 3d). Interestingly, for the tumor cells that pretreated with CB, most of the cells failed to undergo ICMS. They were greatly elongated to various degrees depending on the shape of capillaries they entered (Fig. 3d). CB‐pretreated tumor cells generally kept roaming in the blood, suffering from hydrodynamic shear forces then torn apart (Fig. 3e). Further in vitro and in vivo transplantation experiments showed that CB (1 μM for 4 hr) treatment has little or no effect on the growth of B16 cells or CT26 cells either in dish or in zebrafish perivitelline space (Supporting Information Fig. S7).Similar experiments were also performed in mouse experimental metastasis model by injecting CB or DMSO pretreated B16 cells through tail vein. Fluorescent imaging of the intact mouse lungs showed that CTCs efficiently entered into pulmonary capillaries in both groups (Fig. 3f, 20‐min panel). For the CB pretreated group, CTCs were generally in a loaf shape at 8 hpi (Fig. 3f, arrows), while the CTCs in the control group were not. In addition, serial images and counting results (at 20 min, 8 hr, 24 hr and 10 days, n = 3 mice for each time point) indicated a higher rate of CTC loss in the CB pretreated group, which is similar to our observation in zebrafish (Figs. 3e, 3g and 3h). Thus, these experimental data suggested that F‐actin assembly was essential for the ICMS and the intravascular arrest of CTCs.