Identify The Functional Heterogeneity Of Tumor-Associated Fibroblasts In Oral Squamous Cell Carcinoma And Screen The Target Gene

Objective:Oral squamous cell carcinoma(OSCC)is a group of squamous cell carcinoma originating from the tongue,buccal mucosa,oral floor,the posterior triangle of molars,alveolar ridge,hard palate,and medial lip.It accounts for 90%of head and neck malignant tumors.Although improvement has been made in treatment,the 5-year survival rate of OSCC patients is still low,which might be related to many factors such as late diagnosis,high metastasis rate,and the treatment methods that need to be improved.To find new therapeutic targets and develop new therapeutic strategies to reduce mortality,it is important to study the mechanism of local recurrence and distant metastasis of OSCC.Tumor progression is determined by the complex tumor microenvironment(TME)where the tumor stroma plays an important role in the regulation of tumor cell proliferation and invasion.TME consists of the extracellular matrix(ECM),fibroblasts,and blood and lymphatic vessels.Fibroblasts are the main cellular components in TME,but cancer-associated fibroblasts(CAFs)are highly heterogeneous.Some subtypes of CAFs mainly secrete ECM and maintain the structural framework of tissues.Some subtypes secrete a variety of cytokines and chemokines to participate in the regulation of tumor cells and immune cells.With the rapid development of single-cell sequencing,more and more subtypes of CAFs have been found.However,The function of these different subtypes of CAFs in tumor progression is largely unknown.OSCC is a heterogeneous malignancy,while the stable cell lines cultured in two dimensions can not represent its heterogeneous traits in vitro.Using 3D culture technology,cancer stem cells(CSCs)can be cultured to form organoid tissues,which shows tumor heterogeneity in vitro.In this study,we cultured patient-derived organoids(PDOs)from normal epithelial tissues and tumor tissues of OSCC patients.Fibroblasts are isolated from OSCC and established a library of patient-derived fibroblasts(PDBs),so as to study the role of PDBs in OSCC progression in vitro and in vivo.GATA6 is selected as a candidate gene of patient-derived fibroblasts by RNA-sequencing and gene screening analysis of fibroblasts isolated from OSCC.The correlation of GATA6 with enrichment,gene changes,clinical features,prognostic value,immune infiltration and immune-related genes is analyzed.In addition,TCGA,GTEx,CCLE,TIMER,Immu Cell AI and c Bio Portal data sets,are used to evaluate GATA6-targeted drugs and IC50,and further explore whether GATA6 can be used as a therapeutic target in pan carcinomas.Methods:1.Extraction of primary cells:Tumor tissues,adjacent tissues and normal tissues were collected from patients in Stomatological Hospital of China Medical University.2.Isolation and culture of patient-derived fibroblasts(PDBs):about0.3-0.5cm~3tissues were trimmed and washed with PBS containing penicillin-streptomycin,then digested with 1?Dispase II for about 10min,and the digested cells were isolated and cultured.3.PDBs were identified by immunofluorescence staining.4.Isolation and culture of patient-derived organoids(PDOs):about 0.3-0.5cm~3tissues were trimmed,washed with PBS containing penicillin-streptomycin,digested with 1?Dispase II,and digested cells were cultured in Matrigel.5.PDOs were identified by immunofluorescence staining.6.The PDBs were sequenced and grouped,namely tumor-associated fibroblasts(CAFs),inflammatory tumor-associated fibroblasts(AFs/i CAFs)and normal fibroblasts(NFs).7.Characterization of PDBs by q RT-PCR.8.Co-culture of PDBs with PDOs from the same patient,and the effect of PDBs on PDOs was observed.9.In vitro and in vivo experiments were used to detect the effects of different subtypes of PDBs on tumor.10.The effects of PDBs on tumor cell migration and invasion were observed by scratch and Transwell invasion assay.11.Immunohistochemical staining was used to detect the infiltration of immune cells in the tumors obtained from in vivo experiments.12.Statistical analysis was performed to show the average±standard deviation,p<0.05indicates the significant difference.13.TCGA database and GEO database were used to analyze the expression level,copy number variation,mutation,and survival correlation of GATA6 in 33 different cancer types.14.Download copy number variation data from TCGA database,and calculate the frequency of GATA6 in each cancer by CNA structural mutation,proliferation and polygenic mutation.15.Gene set variation analysis(GSVA)was used to calculate the activity of GATA6 gene expression pathway,and Pearson correlation coefficient between GATA6 and pathway activity activation was calculated.P<0.05 indicates GATA6 is closely related to this pathway.16.Analyze the correlation of GATA6 with immune-related genes by Spearman correlation coefficient,P<0.05 indicates GATA6 is closely related to this immune gene.17.Gene enrichment analysis(GSEA)in R language was used to analyze the function of GATA6 in the GO data set,KEGG data set,and Reactome data set and map it.18.Analyze the potential drug that targets GATA6 and the IC50 using GDSC2 data.Results:1.IF staining confirmed that Vimentin,β-catenin,αSMA,and FAP of PDBs were positive.CKpan and other epithelial markers were negative,which suggests that the isolated cells are fibroblasts.2.PCA analysis showed that these fibroblasts were segregated into three subgroups,namely CAFs,AFs,and NFs.Functional enrichment showed that the NFs group has the main functions of regulating cell adhesion development,embryonic organ morphogenesis,and sensory organ morphogenesis;the AFs group functions on leukocyte migration,positive regulation of chemotaxis,cell chemotaxis and leukocyte chemotaxis;the CAFs group mainly in regulating epithelial cell proliferation,keratinocyte differentiation,cell-cell adhesion and epidermal development by plasma membrane adhesion molecules.3.The results of q RT-PCR showed that the expression of IL-1B,IL-6,CXCL-5,CXCL-8 and CCL-5 in AFs were significantly higher than those in NFs and CAFs.4.When the PDBs and PDOs of the same patient were co-cultured,AFs and CAFs could significantly promote the proliferation of PDOs compared with the control group(culture with culture medium alone)and NFs.5.By Edu staining,it was found that CM from CAFs and AFs can significantly promote the proliferation of the tumor cells compared with CM from NFs.6.Scratch and invasion experiments showed that CM of CAFs and AFs significantly enhanced tumor invasion and migration compared with CM of NFs,and AFs promoted tumor invasion and migration more obviously.7.Compared with the NFs group,the growth rate of subcutaneous tumors in the AFs group and CAFs group increased significantly after 1 week,and AFs and CAFs significantly promoted the growth of subcutaneous tumors in the xenograft model.8.When the subcutaneous tumor volume of nude mice was≥200mm~3,the mice were treated by intraperitoneal injection of cisplatin.The results showed that the tumors in the AFs group and CAFs group were resistant to cisplatin treatment,and the tumor volume continued to increase,while the tumor in the NFs group and control group decreased continuously.9.Immunohistochemical staining showed there were more F4/80(+)macrophages detected in the tumors in the AFs group,while only a few macrophages were found in the CAFs and NFs groups.10.The expression of GATA6 was related to a low survival rate in certain types of cancers.11.GATA6 gene was related to the clinicopathological stage in different tumors.12.GATA6 copy number alteration(CNA),DNA methylation,gene set variation,and gene set enrichment analysis indicated that GATA6 was involved in various molecular and biological processes.13.GATA6 gene is a prognostic marker for certain types of tumors,and the high expression of GATA6 was significantly related to a low overall survival rate of KIRP,UVM,BLCA,KIRC,LUSC,and HNSC.14.GATA6 also participates in the immune response,modulates the recruitment of various immune cells and promotes the pharmacokinetics of drugs.Patients with high expression of GATA6 are predicted to be resistant to vorinostat,venetoclax,MIRA-1,daporinad,temozolomide and sabutoclax,while they are predicted to have better therapeutic effects on trametinib,SCH772984,and SB216763.Conclusion:This study confirms that different subtypes of PDBs in OSCC have distinct molecular characteristics.AFs significantly express inflammatory factors;AFs and CAFs promote the proliferation of tumor cells in vitro and in vivo;AFs recruit macrophages significantly in the tumor microenvironment,and promote the invasion and metastasis of OSCC;GATA6 might be a potential therapeutic target.