Irradiated Fibroblasts Promote EMT And HDGF Expression Of Esophageal Squamous Cell Carcinoma

Background Radiotherapy is a primary treatment of ESCC and the 5-year survival rate of it has still remained to be less than 20%. Recently, several studies suggested that the presence of activated CAFs was associated with an unfavorable outcome, local recurrence after definitive chemoradiation for ESCC. The classicparadigm of radiation as a cancer initiator is broadened by studies showing that radiation also influences cancer processes through promoting effects on the stromal microenvironment that are mediated by radiation.Recently, HDGF, an acidic polypeptide with mitogenic activity for fibroblasts and a negative prognostic marker for survival incancer progression, has attracted much attention and been proved to be involved in the regulation of a myriad of cancer cell activities during cancer transformation, apoptosis, angiogenesis and metastasis. HDGF played an important role in radiosensitivity and correlated with tumor recurrence of ESCC.CAFs have been linked to a number of prometastatic capabilities, including induction of EMT, which endows cells with migratory and invasive properties and induces stem cell properties. Subsequently, interactions between cancer cells and surrounding stromal fibroblasts have been suggested to play a critical role in tumor invasion and metastasis through EMT. Previous reports have shown that y-ray irradiation to fibroblasts are involved in carcinogenesis and invasive growth of cancer cells that are not exposed to irradiation. However, to our knowledge, it’s absent whether X-ray irradiation to stromal fibroblasts could facilitate ESCC invasion and metastasis through EMT.Objective1. This study investigated the effects of ionizing radiation on stromal fibroblasts and the proactive influence of irradiated CAFs on their ESCC cells counterparts.2. This study also aimed to identify the role of the HDGF and EMT program in this pathological process.Methods1. To characterize stromal fibroblasts in the microenvironment of ESCC, we isolated and cultured CAFs from primary ESCC tissues and normal fibroblasts (NFs).To confirm that CAFs were pure fibroblasts without other cells contamination, the fibroblast marker fibronectin was used to distinguish fibroblasts from ESCC cells. The primary cultured stromal fibroblasts were detected by western blotting and expressed a-SMA highly but did not express E-cadherin, presenting characteristics of CAFs.2. Cells were irradiated with different doses at room temperature using X-rays source (6 MV; Varian 23 EX; Varian, Ltd., USA) with a delivering rate of 400cGy/min and then we prepared conditioned medium of cultured CAFs (CAF-CM) for ESCC cell treatments.3. Using in vitro invasion assay, we examined the invasiveness of ESCC cells (Eca-109 and Eca-9706) cocultured with non-,4Gy-and 8Gy-irradiated fibroblasts (NFs and CAFs).4. Using in vitro scattering assay, we then examined the effect of the supernatant derived from irradiated fibroblasts on ESCC cells scattering.5. ESCC cells (1.3×106) and 0.5×106 4Gy-or non-irradiated fibroblasts were mixed well and implanted subcutaneously into the right flank of 4-5 week-old female nude mice (BALB/c nu/nu; Beijing HFK Bio-Technology.co., LTD, China). Six mice were used in each group. Tumor size was measured every 3 days with the use of a caliper. The tumor volume (V) was calculated according to the formula Volume=(lengthxwidth2)/2.6. To determine whether the effect of irradiated fibroblasts on cell migration and invasion was associated with EMT, expressions of epithelial marker (E-cadherin) and mesenchymal marker (vimentin) were compared between irradiated fibroblasts-CM and control-CM cultured ESCC cells at the 24hr time point by western blotting. Moreover, we examined the expression of β-cateninin tumor cells with irradiated fibroblasts in vivo models by using immunohistochemistry.7. To explore whether the effect of irradiated fibroblasts on cell migration and invasion was associated with HDGF, the expression of HDGF was compared between irradiated fibroblasts-CM and control-CM cultured ESCC cells at the 24hr time point by western blotting. Moreover, we examined the expression of HDGF in tumor cells with irradiated fibroblasts in vivo models by using immunohistochemistry.Results1. Isolation, characterization and purity of fibroblasts These results indicated that the isolated and cultured fibroblasts in vitro from ESCC tissues maintained the features of CAFs. All the fibroblasts used in the experiments were at less than 10 passages and showed spindle like morphology. E-cadherin was not expressed in any fibroblasts after 3 passages, which indicated that the fibroblasts at low passages cultured in vitro retained the features of fibroblasts.2. The supernatant from irradiated fibroblasts promotes scattering of ESCC cells Because the scattering of epithelial colonies possesses characteristics of EMT, such as the loss of epithelial cell-cell junctions and the acquisition of a motile mesenchymal cell phenotype, the scatter assay has been used for studying EMT and for detecting factors able to induce migratory behavior of cells. Exposure to the supernatant from irradiated fibroblasts accelerated the scattering of ESCC cells in a dose-dependent manner.3. Irradiated fibroblasts enhanced invasiveness of ESCC cells Our study demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of ESCC cells and the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts.4. Irradiated fibroblasts promote EMT of ESCC cells in vitro and in vivo The results showed that the expression of all the tested E-cadherin was significantly downregulated, whereas vimentin was slightly upregulated and highly expression in former irradiated groups in a dose-dependent manner. The expression of all the tested β-catenin was upregulated in ESCC cells with irradiated fibroblasts in vitro and in vivo. Remarkably, enhanced β-catenin was demonstrated to localize to the nucleus in tumors with irradiated fibroblasts in vivo.5. Irradiation enhanced HDGF expression in both ESCC cells and fibroblasts in vitro and in vivo. Irradiated fibroblasts promoted tumor growth of ESCC cells, especially in Eca-9706 groups. The expression of HDGF was increased in both fibroblasts and ESCC cells of irradiated group in vitro and in vivo models. Interestingly, the tumor cells adjoining the stromal fibroblasts displayed strong nuclear HDGF immunoreactivity, which suggested the occurrence of a paracrine effect of fibroblasts on HDGF expression.Conclusions1. Irradiated CAFs accelerated invasiveness, EMT and scattering of ESCC cell lines.2. Irradiated fibroblasts induced nuclear β-catenin relocalization in ESCC cells.3. Irradiated fibroblasts promoted growth of ESCC in vivo and increased HDGF expression in vitro and in vivo.