Effect And Mechanism Of Cancer-associated Fibroblasts On The Malignant Biological Behavior Of Lung Cancer Cells After Ionizing Radiation

Background and Objective: Lung cancer is a disease with high incidence and high fatality rate in the field of cancer,which poses a great threat to the long-term survival of human beings.Cancer-associated fibroblasts(CAFs),as the main interstitial cell component of the cancer surrounding,can directly or indirectly affect the development of lung cancer in a variety of ways.In this study,We have investigated the effect of CAFs after ionizing radiation on malignant biological behavior of lung cancer cells.In addition,the effect of exosomes secreted by CAFs on lung cancer cells was further explored.Methods: Paracancerous tissues from patients with lung adenocarcinoma in the Department of Thoracic Surgery of People’s Hospital of Wuhan University were collected and quickly transferred to our laboratory at 4℃ and asepsis.Human lung cancer CAFs were extracted from surgical specimens by adherent living cell extraction technology.The cells were cultured to 4-8 generations and no other hybrid cells were found under microscope before use in the experiment.The proteins specifically expressed in the extracted primary cells were detected by immunofluorescence and western blot assay to identify CAFs.Part of CAFs were given 8 Gy ionizing radiation and continued subculture.Collect CAFs and 8 Gy irradiated CAFs supernatant under the same conditions,namely conditioned medium(CAFs supernatant was referred to as CAFs-0 Gy-CM for short,and CAFs-8 Gy-CM for short after 8 Gy irradiated).Then,lung cancer cell line A549 and A549 irradiated by 6 Gy were divided into groups and treated with ordinary DMEM medium(blank),CAFs-0 Gy-CM,CAFs-8 Gy-CM,etc.After a period of culture,cell apoptosis was detected by flow cytometry,cell migration was detected by scratch test,and cell proliferation was detected by CCK-8 reagent.Finally,an animal subcutaneous transplantation tumor model was established.A549 cells were inoculated subcutaneously alone or A549 cells and CAFs were inoculated subcutaneously or A549 cells and 8 Gy irradiated CAFs were inoculated subcutaneously,and the tumor formation and growth status of each group of mice were observed.In addition,for further study,the exosome inhibitor GW4869 was used to pretreat CAFs and 8 Gy irradiated CAFs,and their supernatants were collected to culture A549 cells,and the apoptosis of A549 cells was detected by flow cytometry.Results: The extracted primary cells highly expressed α-smooth muscle actin(α-SMA),fibroblast activation protein(FAP)and vimentin,which are common markers of CAFs.After receiving a single dose of 8 Gy radiation,the appearance(size,morphology)of CAFs did not change significantly,but the growth rate slowed down significantly.In the A549 unirradiated group,the apoptosis rates of A549 cells treated with blank medium,CAFs-0 Gy-CM and CAFs-8 Gy-CM were 9.24±3.5%,4.40±0.9% and 2.04±0.49%,respectively(P < 0.05).Apoptosis rates in the A549 group were 33.83±1.15%,23.20±1.55% and 16.30±2.00%(P < 0.05).Moreover,the CAFs supernatant can promote the migration and proliferation of lung cancer cells before and after irradiation.The tumor volume growth curve showed that the tumor volume of mice in the A549 plus 8 Gy irradiated group(abbreviated as A549+CAF8)and A549 plus CAFs group(abbreviated as A549+CAF0)increased faster,A549 plus8 Gy irradiated group showed the fastest increase in tumor volume.The tumor mass after dissection was 208.3±42.9 mg in the A549 group,300.0±40.8 mg in the A549 plus CAFs group,and 433.3±86.9 mg in the A549 plus 8 Gy irradiated CAFs group,respectively,with statistically significant differences.There was no statistically significant difference in the weight change of mice during the experiment.GW4869 pretreated CAFs and 8 Gy irradiated CAFs.After the supernatant was used to culture A549 cells,the apoptosis rates of the cells in each group were 13.56±0.23%,14.96±0.67%,13.96±0.31%,22.87±0.61%,17.89±0.67%,18.49±1.01%,respectively.Conclusion: Ionizing radiation can activate CAFs and promote the survival of tumor cells.The activated CAFs not only have significant apoptosis protection,proliferation,and migration promotion effects on lung cancer cells that have not been irradiated,but also have a significant promotion effect on lung cancer cells after ionizing radiation.In addition,the exosomes secreted by CAFs may have a protective effect on lung cancer cells.In ionized and irradiated lung cancer cells,except for exosomes,there may be other factors that affect cell apoptosis.This study provides evidence from cell and animal experiments to support that ionizing radiation can activate CAFs and promote radiotherapy resistance of lung cancer cells.Further studies on the molecular mechanisms of both of them need to be further explored.