Multi-omics Investigations Revealed Underlying Molecular Mechanisms Associated With Tumor Stiffness And Identified Potential Drugs For Suppressing Stiffness In Pituitary Adenomas

Objective:Pituitary adenomas(PAs)are the second most common intracranial tumor.The mass effect and secondary hypopituitarism,and the multisystem complications caused by excessive secretion of hormones seriously reduce the quality of life and increase the mortality of PA patients.Transsphenoidal surgery is the first-line therapy for PAs,and total tumor resection is extremely important for curing PAs.However,only 66-78%of patients can achieve total resection as reported in the literature.Tumor stiffness has gradually become one of the most critical factors affecting resection rate of PAs.This study aimed to investigate the molecular mechanisms for tumor stiffening and explore novel medications to reduce stiffness for improving remission rates in PA patients.Methods:Twenty-two patients with PAs confirmed by postoperative pathology in the Department of Neurosurgery in Peking Union Medical College Hospital were prospectively enrolled.The tumor stiffness was evaluated by the neurosurgeons during the operation.Transcriptomic and whole-genome bisulfite sequencing(WGBS)were used to identify key genes,molecular pathways and tumor microenvironment components related to tumor stiffness,and to find potential drugs that can reduce tumor stiffness.Finally,to verify the efficacy of sunitinib,the GH3 cell line was first treated with different concentrations of the drug to analyze its effect on cell viability.Then,PA xenograft model in Wistar rat was constructed to analyze the effect of the drug on tumor growth,and atomic force microscopy(AFM)was used to detect tumor stiffness.Results:Eleven patients with stiff tumors and 11 patients with soft tumors were finally enrolled for multi-omics analyses.There was no significant difference in baseline clinical parameters such as age and gender between the two groups(P>0.05).Tumor microenvironment analysis and immunofluorescence staining showed that endothelial cells(ECs)and tumor-associated fibroblasts(CAFs)were the most abundant in stiff tumors(P<0.05).Weighted gene co-expression network analysis identified the most critical stiffness-related gene(SRG)modules.KEGG analysis indicated that SRGs could regulate PA stiffness by regulating the development,differentiation,and apoptosis of ECs and CAFs.The promoter methylation of stiffness-related genes C5orf66-AS1 and CAVIN3 was strongly negatively correlated with mRNA expression,suggesting that abnormal DNA methylation of SRGs may be involved in tumor stiffness.Additionally,m6A RNA methylation has a bidirectional regulatory effect on pituitary tumor stiffness.Sunitinib was finally identified as a potential drug to reduce tumor stiffness based on the Genomics of Drug Sensitivity in Cancer(GDSC)database.In vitro experiments found that the survival rate of GH3 cells decreased with the increase of sunitinib concentration,indicating that sunitinib can inhibit cell viability.In vivo experiment found that compared with the control group,tumor growth was significantly inhibited in the sunitinib treatment group(P<0.05).AFM showed that the Young’s modulus of the tumors in the drug treatment group was significantly lower than that in the control group(P<0.0001),indicating that sunitinib could significantly reduce the stiffness of PAs.Conclusions:This study comprehensively assessed the molecular landscape of PA stiffness through multi-omics analysis,and uncovered unique features of the tumor microenvironment and regulation patterns in stiff tumors.In vitro and in vivo experiments showed that sunitinib can inhibit tumor growth and reduce tumor stiffness.In conclusion,this study provides an important theoretical basis for drug therapy to reduce the stiffness of PAs,and provides a reliable drug choice for future clinical trials.