Type Ⅰ Collagen Promotes Radioresistance Via CXCL1-mediated Fibroblast Activation In Esophageal Squamous-cell Carcinoma

Background&Aims:Esophageal squamous cell carcinoma(ESCC)is one of the common malignant gastrointestinal cancers in China.Radiotherapy plays an important role in the integrative therapies of esophageal cancer,especially for unresectable esophageal cancer.Despite the development of radiotherapy technologies,the the efficacy of radiotherapy is disparate between individuals.Some patients still suffer recurrence or metastasis.Radioresistance is one of the main causes of clinical treatment failure in ESCC.In this study,we established patient-derived xenografts(PDX)model of ESCC and treated with radiotherapy.By combining transcriptomic analyses,we identified key genes related to radioresistance and explored the potiential mechanism.This study aims to elucidate the mechanism of radioresistance,search for prognostic indicators of radiotherapy efficacy and provide potential targets for combination therapies.Methods:The established PDX models of ESCC were treated by radiotherapy and divided into sensitive group and resistant group defined by relative tumor volume.The significantly differentially expressed genes between the two groups were analyzed by transcriptome sequencing.Immunohistochemistry and masson staining were used to verify differential gene expression and spatial localization of cancer-associated fibroblasts(CAFs).The effect of type Ⅰ collagen on radiosensitivity was verified by colony formation.The expression of DNA damage repair markers and integrin pathway proteins in ESCC cells were quantified by western blot.Collagen synthesis inhibitors combined with radiotherapy were used in vivo and then detected tumor volume changes.The chemotaxis of ESCC cells treated with type Ⅰ collagen to fibroblasts was detected by co-culture experiment.The supernatant of ESCC cells treated with type Ⅰ collagen was collected for cytokine arrays to screen out significantly up-regulated cytokines.Enzyme linked immunosorbent assay(ELISA)was used to quantify the cytokines in the supernatant.The expression levels of type Ⅰ collagen and other CAF markers in fibroblasts treated with recombinant protein were determined by western blot.The chemotaxis of cytokines to fibroblasts was examined by migration assay.Immunohistochemistry and immunofluorescence were used to detect the expression level and spatial localization of cytokines in tissues.Student’s t test was used to define statistical significance when two groups were compared.Results:We found that the expression of extracellular matrix related genes in the resistant group was significantly higher than sensitive group,including the α1 chain(COL1A1)and a2 chain(COL1A2)of type Ⅰ collagen.Masson staining of patient and PDX tissues demonstrated more collagen deposition in the resistant group.The number of clones in ESCC cells after irradiation was higher when coated with type Ⅰ collagen,and the magnitude and duration of DNA damage repair marker y-H2AX were remarkably reduced.Treatment with type Ⅰ collagen can activate the FAK/Src and Akt signaling pathway downstream of the integrin pathway,and promote the expression of Chkl by increasing the stability of c-Myc,thus enhancing the ability of DNA repair to promote the radioresistance of ESCC cells.In vivo,collagen synthesis inhibitors combined with radiotherapy can enhance PDX radiotherapy sensitivity.The co-culture of ESCC cells coated with type Ⅰ collagen can promote the migration of fibroblast.Cytokine array analysis showed that CXCL1 increased in the supernatant of ESCC cells treated with type Ⅰ collagen.CXCL1 recombinant protein treatment can increase the expression level of CAFs marker protein in fibroblasts and promote fibroblast migration.Cell immunofluorescence experiments showed that treatment with type Ⅰ collagen can promote the nuclear translocation of CXCL1 transcription factor NFκB to improve CXCL1 expression level.Conclusion:Type I collagen produced by CAFs is a determining factor for radiosensitivity.Type Ⅰ collagen up-regulates the expression of Chkl by activating the FAK/Src pathway through integrin β1 receptor,thereby enhancing the tolerance of cancer cells to irradiation.Meanwhile,Type Ⅰ collagen promotes the nuclear translocation of NF-κB to improve CXCL1 transcription and secretion.CXCL1 recruits fibroblasts and promotes their expression of type Ⅰ collagen,which leads to further deposition of collagen in the tumor microenvironment,initiating a positive feedback loop.Targeting synthesis of type Ⅰ collagen can enhance the sensitivity of PDX mice to radiotherapy.These results suggested the potiential of type Ⅰ collagen as a prognostic biomarker and a therapeutic target for ESCC.