Cell Deformability Controls Metastasis Of Malignant Tumor-repopulating Cells In Zebrafish

Cancer metastasis is the deadliest stage of cancer.Although significant efforts have been devoted to studying the process of metastasis over the past decades,it is still unclear why only a very small fraction of cancer cells can form micrometastases and metastatic colonization.Recently,by culturing mouse melanoma cells(B16-F1)in three-dimensional(3D)soft fibrin gels,we have selected a subpopulation(?5-10%)with high tumorigenic potency.We defined these cells as tumor repopulating cells(TRCs)as they expressed high self-renewal gene Sox2 and low differentiation gene Mitf,since they were different from cancer stem cells isolated using the conventional stem cell markers.TRCs were high tumorigenic,as we had shown that 10 TRCs could form metastases in syngeneic and non-syngeneic immunocompetent mice.However,mouse tissue are opaque thus are not suitable to visualize the metastatic dynamics.During the recent years,zebrafish has been used as a useful vertebrate model to study metastatic processes of tumors because of its tissue transparency.Here,using the zebrafish model,we have studied cancer metastasis and focused on dynamics of extravasation,a critical step in metastasis.In our study,transparently transgenic zebrafish Tg(fli1:EGFP)(green color)or Tg(kdr1:mCherry)(red color)were used.After injecting red fluorescent B16-F10 into the yolk sac of fish at 2 days post fertilization(dpf),we show that TRCs could grow better and form more micrometastases at secondary sites than control melanoma cells(Cont)that were cultured on two-dimensional(2D)rigid plastic.The metastasis of TRCs was dependent on the presence of Sox2,a self-renewal gene,and silencing Sox2 led to the inhibition of TRC metastasis.After injecting tumor cells into the pericardial cavity,we found that injected tumor cells could quickly metastasize to the tail of fish via blood vessels within 1 hour.High-resolution of 3D confocal images of the TRCs at the secondary sites show that extravasation and formation of micrometastases by TRCs were more efficient than by the control cells.Remarkably,efficient extravasation of TRCs in vivo and transmigration in vitro were determined by TRC deformability,as a result of low Cdc42 and high Sox2:High Sox2 expression downregulated Cdc42 expression in melanoma cells,resulting in less filamentous actin(F-actin)and a more deformable and invasive tumor cell phenotype,so TRCs were more deformable to extravasate and form more micrometastases in zebrafish.In summary,our findings suggested that tumor cell deformability was a key factor in controlling extravasation dynamics of tumor-repopulating cells during metastasis.