The Hard Fibrin Gel Mediates Tumor Regenerative Cells Into A Dormant State Through The Cdc42-Tet2 Signaling Pathway

Tumor dormancy is emerging as a key event for tumor long-term survival from escaping intrinsic(immune surveillance)and extrinsic(toxic drugs)attacks.Although quiescent tumorigenic cells may stay in a dormant status for years based on clinical observations,how this dormancy process is achieved and maintained in cancer patients remains an enigma.Binding of extracellular matrix(ECM)proteins such as collagen and fibrin to integrins leads to mechanotransduction along clustered integrins to focal adhesions.Thus,outside mechanical signals are sensed at focal adhesions and can be converted into biochemical signals inside the cells.In line with this concept,cell dormancy can be postulated to be regulated by the stiffness of ECM.To date,whether and how stiffness regulates the dormancy of tumorigenic cells that can repopulate tumors(tumor-repopulating cells or TRCs)is unknown.Previously,we used a soft 3D fibrin gel culture system to generate TRCs.This fibrin gel corresponds to 90 Pa in elastic stiffness and the TRCs were trapped individually in the gel for colony formation.Those TRCs grew into spheroid-like morphological shapes resembling stem-like cells.Importantly,as few as 10 TRCs,after intravenous injection via the tail vein,are able to grow tumors in the lungs of immunocompetent mice.Here,we have further used this unique 3D fibrin gel system to examine the possibility of matrix stiffness-driven TRC dormancy.First,In this studywe chose 90,450 and 1050 Pa as soft,medium and stiff fibrin gels,respectively,to study the influence of matrix stiffness on TRC growth.We found that although TRCs grew rapidly and formed spheroids effectively in 90 Pa gels,their growth was retarded in 450 Pa gels and almost completely inhibited in 1050 Pa gels。And TRCs with Ki67 negative staining were found to express COUP-TF1 or DEC2 in 1050 Pa fibrin gels.Then we found that TRCs indeed underwent G0/G1 cell cycle arrest,downregulated the expression of the PCNA gene(a cell proliferation marker)and decreased glucose consumption.In addition,TRCs did not undergo senescence as evaluated by β-galactosidase assay.Dormant tumor cells may decrease their response to xenobiotics,including chemotherapeutic drugs.Interestingly,our data showed that TRCs in 1050 Pa fibrin gels were more resistant to methotrexate and paclitaxel than those in 90 Pa gels.Together,these data suggest that increasing 3D matrix stiffness alone is enough to induce TRCs into a dormant state.We then put 5 B16 TRCs into 1050 Pa stiff fibrin gels,and embedded this stiff gel under the skin of mice.Surprisingly,no visible melanoma was formed.While,even though no macroscopic melanoma was formed,micro-tumor nodules were stained by melanoma-specific S100 antibody and confirmed by H&E staining.Such micro-tumor nodules showed a COUP-TF1+Ki67-dormant phenotype,which was also evidenced by unchanged sizes on day 30,50 or 70,suggesting that the stiffness of matrix induces TRCs into dormancy in vivo.Next,we investigated the molecular mechanism through which nuclear Cdc42 regulates TRC dormancy in stiff fibrin gels.In the present study,we found that small G protein Cdc42 in TRCs was changed based on its environmental stiffness.In 90 Pa 3D fibrin gels,Cdc42 exists in its cytoplasmic form,but is translocated to the nucleus in 1050 Pa stiff gels.This translocation alters the role of Cdc42 from a mechanotransduction mediator to a nuclear transcription regulator that upregulates the expression of Tet2,an epigenetic modifier gene.Subsequently,5-hmC is generated in cell cycle inhibitor genes via Tet2-catalyzed hydroxymethylation,leading to the upregulation of p21 and p27 followed by cell cycle arrest and cellular dormancy.Such mechanical signals by matrix stiffness not only induce TRCs into dormancy but also maintain this dormant state through a feedback-regulated integrin β3 pathway.Finally,in order to demonstrate 3D matrix stiffness also can induce TRCs into a dormant state,we isolated primary human melanoma(PHM)cells and seeded them in 90 or 1050 Pa 3D fibrin gels for 5 days culture.Consistently,PHM TRCs were also induced into dormancy by the 1050 Pa stiffness,which was evidenced by cell cycle arrest without senescence,lower glucose consumption and highly expressing COUP-TF1+Ki67-dormant markers.Together,these data suggest that increasing 3D matrix stiffness also can induce PHM TRCs into a dormant state.In summary,our finding that a stiff 3D matrix can initiate and maintain the dormancy of TRCs via a Cdc42-Tet2 epigenetic program would stimulate more research in the area of tumor dormancy and dormancy-related tumor recurrence and metastasis.