Study On The Effects Of Cancer-associated Fibroblasts On Chemo- And Radio-sensitivity Of Esophageal Squamous Cell Carcinoma And The Involved Mechanisms

Objective Esophageal carcinoma (EC) is the eighth in incidence and the forth in mortality with approximately 90% of esophageal squamous cell carcinoma (ESCC) in China. Since the early symptoms of ESCC were not obvious, approximately 70 %-80% of patients were diagnosed at moderate and advanced stages. Chemoradiotherapy was used as the major treatment modality for these patients. However, five years survival rates of ESCC patients were lower than 15% because of significant resistance to chemotherapeutic drugs such as cisplatin and taxol and to radiotherapy, which led to tumor recurrence and metastasis. Previous studies on the machanisms of ESCC chemoradioresistance only focused on tumor cells themselves while tumor microenvironment has been completely ignored. In fact, cancer-associated fibroblasts (CAFs), as the major component of tumor microenvironment, were significantly associated with tumor initiation and progression via multiple mechanisms. Thus, the study on the effect of CAFs in ESCC on chemoradiosensitivity is being urgently needed.Methods 1. CAFs and matched NFs (normal fibroblasts) had been isolated by primary culture from cancer tissues and matched normal esophageal epithelial tissues of ESCC patients, respectively, and further characterized by Immuohistochemical analysis (IHC) and Western Blotting (WB) analysis.2. The effect of isolated CAFs and NFs on ESCC chemoradiosensitivity and involved mechanisms were studied in vitro and in vivo by MTT assay, qRT-PCR analysis, WB analysis, IHC analysis, immunofluorescence analysis, Annexin V/PI staining, Elisa assay, human cytokines antibody array, colony formation assay, establishment of xenograft tumor models and TUNEL assay.Results 1. The staining of mesenchymal marker vimentin was positive in CAFs and matched NFs, while the staining of epithelial marker E-cadherin was negative; furthermore, CAFs-related markers including a-SMA, FSP-1 and FAP were highly expressed in CAFs, compared with in NFs, suggesting that CAFs and matched NFs had been successfully isolated and cultured.2. CAFs conferred significant resistance to cisplatin, taxol,5-Fu, CPT-11, vincristine, pharmorubicin, docetaxel and carboplatin compared with matched NFs. Mechanism studies revealed that CAFs in comparison with NFs secreted great amount of TGFβ1 (Transforming Growth Factor β1) that mediated ESCC chemoresistance by activation of anti-apoptotic NF-κB signaling pathway and induction of epithelial-mesenchymal transition (EMT). The crosstalk of CAFs and ESCC cells stimulated autocrine/paracrine signaling loop of TGFβ1, which further enhanced the chemoresistance. Survival analysis showed that the expression of TGFβ1 in CAFs was significantly associated with overall survival of ESCC patients treated with chemotherapy after surgery.3. CAFs, in comparison with NFs, secreted great amount of CXCL-1, which mediated the radioresistance of ESCC cells by regulating DNA damage repair.Conclusion 1. CAFs mediated significant ESCC chemoresistance via secretion of TGFβ1.2. CAFs-secreted TGFβ1 was able to activate anti-apoptotic NF-κB signaling pathway and induce epithelial-mesenchymal transition in ESCC cells.3. TGFβ1 expressed in CAFs could serve as an independent prognostic factor of ESCC patients treated with chemotherapy after surgery.4. CAFs-secreted CXCL-1 mediated significant ESCC radioresistance by regulation of DNA damage response. Together, these finding in our study first clarified the mechanisms of ESCC chemoradioresistance from the viewpoint of tumor microenvironment, and may provide some novel insights into how to improve chemoradiotherapy efficacy of ESCC patients in clinics.