The Effects Of Extracellular Matrix Stiffness On The Self-renewal Of Tumor Cells And Its Mechanisms

Tumor microenvironment provides a variety of extracellular signals for tumor cells,which is significant in regulating the behavior and function of tumor cells.In the past,the most researches about tumors have focused on various biochemical signals that regulate the proliferation,invasion and migration of tumor cells.However,more and more studies have found that the mechanical properties of extracellular matrix play a critical role in the development of tumor.It has revealed that the stiffness of extracellular matrix can regulate the proliferation,migration,invasion and self-renewal of tumor cells and plays a certain role in the differentiation potential of cells.At present,the researches about the molecular mechanism of matrix stiffness regulating proliferation and migration of tumor cells have become mature,rather than cancer stem cell.Therefore we want to explore the influence of extracellular matrix stiffness on tumor cells,especially the impact of stem cell properties.We made some polyacrylamide gels with the stiffness of 7 kPa,20 kPa and 55 kPa,mimicking rigidity of connective tissue,muscle tissue and bone tissue respectively.Plastic was used as a control substrate.We investigated the influence of matrix stiffness on tumor cells.It was found that the spreading area of osteosarcoma cells increased with the increase of matrix stiffness,as well as proliferation which was detected by cell proliferation kit and Ki67 dyeing.Furthermore,we found that stiff substrate was beneficial to the migration of osteosarcoma cells.Osteosphere assay was used to inquiry the effect of matrix stiffness on the self-renewal of tumor cells,the results show that osteosarcoma cells on the soft matrix contained a higher proportion of cells capable of forming osteospheres.Additionally,osteoblastic and adipogenic differentiation ability of osteosarcoma cells was strongly reduced with the increase of matrix stiffness.Osteosarcoma cells on the soft substrates indicate more resistant to doxorubicin.qPCR and immunofluorescence experiments revealed that soft substrate accelerate the expression of stem cell markers(Sox2,Oct4,and Nanog).This indicates that with the increase of matrix stiffness,the stem cell characteristics of osteosarcoma cells decrease gradually,that is soft substrates can enhance stem properties of tumor cells.To explore the molecular mechanism of soft substrate regulating the stem characteristics of tumor cells,we test the expression of miR-371-5p and miR-29b-5p in different matrixes,theexpressions of these miRNAs on soft substrate were significantly lower than stiff substrate.However,Osteosphere assay demonstrated that only miR-29b-5p played a role in regulating self-renewal of the osteosarcoma cells.Further exploring the molecular mechanism of miR-29b-5p regulating the self-renewal of tumor cells,it was found that Spin1 was the downstream target gene of miR-29b-5p.Overexpression of miR-29b-5p inhibited the expression of PI3 K and p-Akt significantly,as well as p-stat3 and Sox2.The expression of PI3 K,p-Akt,p-Stat3 and Sox2 was also gradually decreased with the increase of the matrix stiffness.The finding uncover that cell spreading area is larger with the increase of stiffness matrix.The stiff substrate can promote the proliferation and migration of osteosarcoma cells.However,with the increase of matrix stiffness,the stem cell characteristics of osteosarcoma cells decreased obviously.Soft substrate enhanced the self-renewal,differentiation and drug resistance of osteosarcoma cells.It reveals that soft substrate can restrain the expression of mi R-29b-5p and mi R-29b-5p can inhibit PI3K/Akt/Stat3/Sox2 signal by suppressing Spin1 to regulate stem cell characteristics of tumor cells,which provides a new strategy for the treatment of osteosarcoma targeting CSCs.