Soft Fibrin Matrix Regulate Self-renewal Of Colon Cancer Stem Cells Through DAB2IP-Nanog Pathway

Background Colon cancer stem cells are an important cause of tumor recurrence and metastasis,with high expression of stem cell markers,strong tumorigenicity,self-renewal ability,drug resistance and other characteristics.The bottleneck of current research on colon cancer stem cells is that the existing methods of isolation and purification are difficult to meet the requirements of quantity,purity and unstimulated.Studies have shown that fibrin deposition in the extracellular matrix of tumor tissue is related to tumor progression and can affect the matrix stiffness to regulate the stemness of tumor cells.In this study,a three-dimensional enrichment system of colon cancer stem cells in vitro was established by using fibrin matrix.The key genes to maintain their self-renewal were screened,and the regulatory mechanisms were further explored.Methods Human colon cancer cell lines HT29 and HCT116 were placed in fibrin matrix with different stiffness(90Pa,420 Pa,1 050Pa)for 3D culture for 5 days.The growth of colon cancer cells was observed,and the most suitable matrix stiffness for the growth of colon cancer stem cells was selected for subsequent culture(3D).Next,through F-actin staining,the cytoskeleton change was observed between 3D cells and normal cells(2D).The tumorigenicity of two groups was compared by subcutaneous tumor formation in mice.The stem cell markers were analyzed by PCR,and the resistance to chemotherapeutic drugs induced apoptosis was detected by Flow cytometry.Western blotting was used to detect the expression of apoptosis related proteins.The key genes that maintain the self-renewal of 3D colon cancer cells were further screened,and the role of which on growth and anti-apoptotic properties of 3D colon cancer cells were analyzed by gene silencing technology.On the other hand,the expression changes of tumor suppressor DAB2IP in 3D and 2D colon cancer cells,and the colon cancer cells cultured with different stiffness were detected.The correlation between DAB2IP and the above key genes was analyzed,and the regulation effect of DAB2IP overexpression plasmid on the growth and drug resistance of 3D colon cancer cells was detected.Finally,the regulatory relationship between DAB2IP and the above key genes and specific regulatory pathways were explored,and the relevant regulatory mechanisms were verified through plasmid transfection,inhibitory signaling pathway,co-transfection and other methods.Results Some colon cancer cells grew into round colonies in fibrin gels from day 1 to day 5 and the volume of colonies formed in soft(90Pa)fibrin gel was significantly higher than that in stiffer(420Pa,1 050Pa)fibrin gel,and the number of colonies was also larger.Subsequently,90 Pa soft fibrin gel was used as a three-dimensional culture substrate(3D)and normal culture(2D)for comparison.Cytoskeletal analysis showed reduced expression of cytoskeletal molecules F-actin and β-actin in 3D colon cancer cells compared to 2D cells.Subcutaneous tumorigenesis experiments in mice showed that colon cancer cells in the 3D group had high tumorigenicity,and the tumorigenesis rate of 10 3D HT29 cells was 67%(4/6),while no tumorigenesis was found in the 2D group.Meanwhile,3D colon cancer cells expressed high expression of Nanog,CD133,CD44,OCT4,SOX2 and other stem cell markers,while low expression of CK20,CDX2 and CK7 differentiated markers.3D colon cancer cells were more resistant to chemotherapeutic drug-induced apoptosis,and the expression of anti-apoptotic protein was increased while the expression of pro-apoptotic protein was decreased.Because the stem cell factor Nanog was significantly elevated in 3D colon cancer cells,we further investigated it.The results showed that the expression of Nanog was matrix stiffness dependent,and the softer fibrin matrix could better promote the expression of Nanog.Silencing the expression of Nanog could inhibit the growth of colonies and its anti-apoptotic ability.On the other hand,the results showed that the expression of DAB2IP was down-regulated in 3D colon cancer cells and increased with the increase of matrix stiffness.Overexpression of DAB2IP could inhibit the growth of 3D colon cancer cells and its anti-apoptotic ability.The results further showed that the expression of DAB2IP in colon cancer stem cells was negatively correlated with the expression of Nanog,and DAB2IP overexpression could inhibit the expression of Nanog.PI3K/AKT signaling pathway was activated in 3D colon cancer cells,and DAB2IP overexpression could inhibit this pathway.Inhibition of PI3K/AKT signaling pathway in 3D colon cancer cells could down-regulate Nanog expression.The expression of transcription factor FOXA1 was decreased in 3D colon cancer cells,and DAB2IP overexpression promoted the expression of FOXA1.Blockage of PI3K/AKT signaling pathway in 3D colon cancer cells resulted in up-regulation of FOXA1 expression.Conclusions The three-dimensional culture system of soft fibrin matrix could promote the selection and enrichment of colon cancer stem cells in vitro,and Nanog was an important factor to maintain the self-renewal of colon cancer stem cells.DAB2IP in soft fibrin matrix maintains self-renewal of colon cancer stem cells by negatively regulating Nanog through PI3K/AKT/FOXA1 signaling axis.