Study On The Role Of Rac1 And RhoA In Invasion And Migration Of Glioma Based On 3D Model

Objective: Gliomas are the most common primary tumors in the central nervous system(CNS).Glioblastomas(GBMs)are the most malignant among gliomas.GBM,which is characterized with invasion,is difficult to be removed completely,and thus has the high recurrence rate.Inhibiting the invasion and migration of tumor cell is an important strategy for glioma treatment.Rac1 and Rho A are the key members of the Rho subfamily.They are critical in regulating invasion and migration of tumor cells.Recent studies have shown that inhibiting Rac1 activity reduces the cell migration ability through the scratch test,but little is known regarding the function of Rac1 and Rho A in cell migration in 3D matrix.This study established the 3D hydrogel model providing the three-dimensional environment to investigate the mechanism of Rac1 and Rho A in GBM invasion and migration.We tested the response of the mesenchymal and amoeboid-like cell movement and the cell invasion and migration abilities against inhibiting Rac1 and Rho A signaling activity,respectively.Simultaneous Rac1 and Rho A activity inhibition induced the conversion of cell movement types and decreased invasion ability,thus providing the theoretical basis for anti-invasion treatment against GBM.Methods: We chose French database to analyze the correlation between the overall survival of glioma patients and the protein expression of Rac1 and Rho A.TCGA database was introduced for the enrichment analysis between low grade glioma and high-grade glioma against Rac1 and Rho A protein expression.Immunohistochemistry staining was applied to detect Rac1 and Rho A expression in the non-neoplastic brain and human brain glioma tissue slides;We investigated the migration ability of cells treated by NSC23766 and Y27632 through scratch test.The glioma cells cultured in 3D hydrogels were treated with NSC23766 and Y27632 respectively,and then the cell 3D morphology and the movement form conversion were observed by laser confocal living cell workstation.Immunofluorescence was used to observe the alternation of Integrin expression with different treatments.We recorded the cell chemotaxis effect through the living cell workstation and calculated the cell movement velocity and distance.We also observed the change of the invasion and migration ability of the cells with different treatments.Results: The expression of Rac1 and Rho A were negatively correlated with the overall survival of glioma patients(p < 0.0001)and positively correlated with tumor grades(p < 0.05)by French and TCGA database analysis.Immunohistochemistry showed the Rac1 and Rho A expression was positively correlated with pathological grade of glioma.The combination treatment of NSC23766 and Y27632 significantly decreased the glioma cell migration capacity through the scratch assay.In the 3D hydrogel-based cell culture model,we observed round circle-like amoeboid cell movement and spindle-like mesenchymal cell movement.We also showed that some cells could transform from spindle-like to rounded-like form with obvious vesicles,called mesenchymal-amoeboid transition(MAT).Some cells could change from round-like to spindle-like form,called amoeboid-mesenchymal transition(AMT).The integrin expression was decreased in MAT and increased in AMT.In the cell chemotaxis experiment,glioma cells treated with NSC23766 harbored increased moving velocity and distance(p<0.05).The NSC23766-treated cells exhibited enhanced cell movement ability,indicating that the cells change to the faster movement form.Cells treated with Y27632 showed decreased movement velocity and distance(p<0.05).Surprisingly,cells treated with both Y27632 and NSC23766 presented significantly decreased movement velocity and distance(p<0.05).The ability of cell motility was obviously compromised,and no morphological change was observed.Conclusion: 1.Rac1 and Rho A expression were negatively correlated with the overall survival of glioma patients.2.Suppressing Rac1 activity decreased the migration ability of U87 glioma cells through the scratch test.However,it increased the cell invasion and migration ability in the 3D chemotaxis experiment.The cells underwent MAT in the 3D hydrogel.3.The combination of inhibiting both Rac1 activity and Rho A signaling pathway effectively reduced the invasion and migration ability,thus providing a theoretical basis for the anti-invasion treatment against glioma.