The Effect Of TGFβ1 In Cancer-Associated Fibroblasts On The Proliferation And Radiotherapy Response Of SCLC And The Underlying Mechanisms

Background: Lung cancer is one of the leading causes of cancer deaths in the world,there are almost two million patients diagnosed with lung cancer each year the two major histologic subtypes of lung cancer including non-small cell lung cancer and small cell lung cancer(SCLC),which account for 13%-15% of lung cancer.Due to the rapid progression and early metastasis of SCLC,patients are often in the progressive stage of the disease at the time of diagnosis.Radiotherapy and chemotherapy are one of the main treatment methods for SCLC.However,although patients with SCLC in the initial stages of radiotherapy and chemotherapy has good curative effect,but most patients relapse rate is still high.Tumor microenvironment(TME)plays an important role in tumor development.TME is composed of extracellular matrix,immune cells such as lymphocytes and macrophages,fibroblasts,mesenchymal stem cells,adipocytes,and vascular cells.Tumor-associated fibroblasts(CAFs),as an important part of them,can secrete a variety of cell growth factors such as transforming growth factor(TGFβ1),epidermal growth factor(EGF),hepatocyte growth factor(HGF),IL-6,etc.,which play a promoting role in the process of promoting tumor development.However,the effect of TGFβ1 on tumor is complex,and its mechanism in tumor growth remains unclear.The effect of TGFβ1 expression level in CAFs on tumor cell growth and tumor immune microenvironment,as well as the effect on tumor radiosensitivity still need to be further studied.Objective: The purpose of this study is to explored the effect of CAF cell on the growth and proliferation of SCLC,and to further investigated the effect of TGFβ1 expression in CAFs on the progression,immune microenvironment and radiotherapy sensitivity of SCLC and its potential mechanism.Methods: To investigate the effect of TGFβ1 expression in CAF cells on the development of SCLC tumor cells,the expression of TGFβ1 and other immune-related factors and immune check point in tumors and stroma of 90 patients with SCLC were analyzed by immunohistochemistry and multicolor immunofluorescence.TGFβ1 knockdown stable cell lines were constructed using TGFβ1 sh RNA of lentiviral vector,TGFβ1 overexpression stable cell lines were also constructed.CCK 8 experiment,clonogenic survival assays,transwell invasion test,flow cytometry and Western blotting were used to investigated the effect of CAFs and the effect of TGFβ1 expression in CAFs on the growth,invasion ability and radiotherapy sensitivity of lung cancer cells.In order to explored the effect of TGFβ1 expression in CAFs on the immune microenvironment of lung cancer,we mixed the CAF cells of knockdown or overexpression of TGFβ1 with tumor cells to establish a mouse xenograft tumor model,and observed the growth of the xenograft tumor and the changes of the tumor immune microenvironment in mice.Results:1.The expression of TGFβ1 in CAF cells is positively correlated with the prognosis of patients with SCLCBy immunohistochemical analysis we found that the patients with TGFβ1 high expression in CAFs had better survival benefit than the patients with TGFβ1 low expression in CAFs.To further verify the effect of TGFβ1 expression level of CAFs on tumor growth,multiplex immunohistochemistry analysis showed that the median survival time of patients with low TGFβ1 expression in CAF cells was 26.9 months(95% CI: 20.95-32.94),the median survival time of patients with high TGFβ1 expression was 53.9 months(95%CI: 43.43-54.87,P = 0.037).Univariate and multivariate analyses showed that high expression of TGFβ1 in CAF cells was an independent predictor of good prognosis.It was also found that TGFβ1 expression level in CAF cells was positively correlated with prognosis in patients receiving postoperative radiotherapy,which enhanced the radiotherapy sensitivity of patients.The correlation between the expression of immune factors in tumors and the expression level of TGFβ1 in CAF cells showed that TGFβ1 in CAF cells played an inhibitory role in the anti-tumor immunity.2.CAF cells promote the proliferation of lung cancer cells and inhibit the radiosensitivity of tumor cellsCAF cells were co-cultured with murine lung cancer cells LLC and KLN205,CAF cells could promote the growth and proliferation of tumor cells,as well as the invasion ability of tumor cells in vitro and in vivo experiments.After different doses of radiotherapy were applied to tumor cells,it was found that the tumor cells treated with CAF conditional medium were more resistant to radiotherapy than the control group.3.TGFβ1 expression in CAF cells affects the proliferation ability and radiotherapy sensitivity of lung cancer cellsTGFβ1 knockdown and overexpression of CAF cells were co-cultured with tumor cells,respectively.In vitro and in vivo experiments showed that compared with CAF cells with high TGFβ1 expression,tumor cells co-cultured with CAF cells with low TGFβ1 expression showed enhanced clonal formation ability,cell proliferation activity and invasion ability of tumor cells.Further,through the Annexin Ⅴ-FITC flow cytometry,we found after improve CAF cell expression of TGFβ1 enhanced the radiation sensitivity of tumor cells and activate cell apoptosis pathway.4.TGFβ1 expression in CAF cells affects the immune microenvironment of tumor cellsCAF cells expressing different levels of TGFβ1 were mixed with LLC cells to establish a mouse tumor-bearing model.The expression level of TGFβ1 in CAF cells was negatively correlated with the expression of CD8,CD56,CD86,and positively correlated with the expression of Foxp3,PDL1,and CD206.There was no significant difference between TGFβ1 expression and CD11 b expression in tumor tissues.Conclusions: This study is the first to demonstrate that TGFβ1 expression in CAFs is an independent prognostic factor for good prognosis in SCLC patients.We found that TGFβ1 expression in CAFs has different effects on tumor cell development and anti-tumor immunity.Therefore,the effect of TGFβ1 on tumor cells and tumor immunity should be treated differently.Our results provide a basis for TGFβ1 expression in CAFs as a new predictor of prognosis in SCLC patients.In addition,targeting TGFβ1 expression in CAFs may be a new therapeutic strategy.