Purpose/Objective(s) Cancer-associated fibroblasts (CAFs), as one major component of tumor microenvironment (TME), are closely associated with tumor initiation and progression. Our previous studies have discovered that CAFs induced the resistance of esophageal squamous cell carcinoma (ESCC) cells to a variety of chemotherapeutic drugs such as cisplatin and paclitaxel. Furthermore, CAFs attenuated ionizing irradiation (IR)-induced cancer cells death by regulating DNA damage response. However, CAFs are significantly heterogeneous and different subtypes have different functions. Even some CAFs subtypes display anti-tumor activity. Our study aimed to discover the heterogeneity among ESCC CAFs and screened the subtype of CAFs which have significant pro-tumor activity. Materials/Methods By single-cell RNA-sequencing (scRNA-seq), we analyzed the heterogeneity among ESCC CAFs which had been isolated from primary ESCC patients who did not receive any therapy before surgery. Using Transwell assay, we detected the invasion ability of CAFs subtypes as well as ESCC cells. By immunofluorescence analysis, we analyzed the radiosensitivity of CAFs subtypes by detecting the expression of γ-H2AX, a marker of DNA double-stranded breaks (DSBs) following irradiation. We further detected the cells survival after irradiation using flow cytometry (FCM). Results By scRNA-seq, we found several subtypes existed among ESCC CAFs. Especially, the CAFs subtype with high expression of CXCL3 (CXCL3high CAFs) showed significant pro-tumor activity. CXCL3high CAFs significantly promoted the invasion ability and radioresistance of ESCC cells compared with CXCL3low CAFs. When CAFs were transfected with CXCL3 siRNA, their tumor-promoting activity was significantly reversed. Furthermore, down-regulation of CXCL3 expression in CAFs attenuated their invasion ability and enhanced their radiosensitivity. The co-culture of CAFs and ESCC cells induced the activation of autocrine/paracrine CXCL3 signaling. Conclusion CXCL3 signaling regulates the malignant behaviors of ESCC CAFs. Targeting CXCL3high CAFs may be a promising approach of treating ESCC.

TCF21 is a determinant of the malignant phenotype of CAFs in ESCC. Up-regulation of TCF21 expression is a promising approach of inhibiting the growth, migration and invasion, activation and radioresistance of CAFs in ESCC.

Hyperthermia significantly enhanced the radiosensitivity of PC cells by decreasing their colony formation and increasing DNA damage following IR. By qRT-PCR analysis of Wnt genes expressions, we found Wnt2B was significantly down-regulated in PC-3 cells which were treated with the combination of hyperthermia and IR compared with hyperthermia or IR alone. Functional assays showed that the expression level of Wnt2B was inversely associated with the radiosensitivity of PC-3 cells. Furthermore, we found hyperthermia inhibited the expression of DNA repair proteins such as p-BRCA1 and p-MRE11 in PC cells following IR CONCLUSION: Hyperthermia can significantly enhance the radiosensitivity of PC cells in a Wnt2B signaling-dependent manner.

Esophageal squamous cell carcinoma is a malignance with high invasiveness. Cancer-associated fibroblasts (CAFs) as one major component of tumor microenvironment (TME), promotes tumor initiation and progression. Our previous studies revealed that CAFs mediated ESCC cells significant resistance to a variety of chemotherapeutic drugs and ionizing irradiation. How to inhibit the tumor-promoting activity of CAFs is of critically important for the radical treatment of ESCC. Using high-throughput screening, 157 inhibitors of cytokines/chemokines were tested for their effect on the cell viability of CAFs, matched normal fibroblasts (NFs), ESCC cells as well as normal esophageal epithelial cell Het-1A. We also investigated the effect of the inhibitors on cell cycle distribution, ROS accumulation and cell apoptosis of CAFs. The expressions of α-SMA, a marker of CAFs activation, was detected in CAFs treated with the inhibitors. By establishment of xenograft tumors in BALB/c mice, the effect of the inhibitors on CAFs-promoted ESCC growth was studied. By high-throughput screening, GSK1838705A, an inhibitor of IGF-1R kinase, was discovered as a potent inhibitor of CAFs activity in ESCC. Compared with NFs, ESCC cells as well as Het-1A, the viability of CAFs was significantly reduced when exposure to GSK1838705A. Furthermore, GSK1838705A arrested the cell cycle of CAFs in G2/M phase, promoted cell apoptosis and ROS accumulation. The expression of α-SMA was signifcantly reduced when CAFs were treated with GSK1838705A. In vivo studies demonstrated that GSK1838705A significantly repressed the tumor-promoting activity of CAFs. Immunohistochemical analysis showed that GSK1838705A significantly reduced the percentage of CAFs in xenograft tumor tissues. GSK1838705A is a potent inhibitor of CAFs which promote ESCC progression. The combination of GSK1838705A and chemoradiotherapy may be a promising strategy against ESCC.

Radiotherapy plays a curative or palliative role in the treatment of non-small cell lung cancer (NSCLC) patients. Cancer-associated fibroblasts (CAFs) are one of the most abundant components of tumor microenvironment (TME). Our study aimed to investigate whether CAFs were involved in DNA damage response of NSCLC cells and clarify the involved mechanisms.