The Influence Of Radiotherapy On Tumor Self-seeding And Its Mechanism In Local Recurrence Of Human Breast Cancer

Objective: To observe the tumor self-seeding phenomenon of human triple negative breast cancer cell line MDA-MB-231 after irradiation through establishing orthotopic xenografts on BALB/C-nu or NOD/SCID mice,and evaluate the preliminary mechanisms and interactions underlying tumor self-seeding and radiotherapy through investigating the characteristics of the seeder and the changes of the tumor microenvironment.Methods: BALB/C-nu mice or NOD/SCID mice were used to establish human triple negative breast cancer xenografts on the second mammary glands,human triple negative breast cancer cell line MDA-MB-231 were injected into the left mammary gland as a “recipient tumor”,GFP-expressing MDA-MB-231 were inoculated into the contra-lateral mammary gland to form a “donor” mass of the same tumor.The recipient tumors were treated with irradiation(8Gy).After sacrificing the mice,frozen sections were prepared from the recipient tumors,the proportion of MDA-MB-231-GFP cells versus the total number of cells was calculated as the self-seeding proportion of one animal.Comparing with the control group,we can distinguish the difference between the two groups and evaluate the influence of radiotherapy on self-seeding.ELISA kits were used to quantize the levels of IL-6 and IL-8 in the plasma/recipient tumors/ culture conditioned media before and after irradiation.Western-blot were carried out to detect the level of HIF-1α and VEGF in recipient tumors.Immunohistochemical staining were performed to measure CD68+macrophages and LY6G+neutrophils.Primary culture and screening test using G418 were used to purify the GFP-labeled cells,as termed MDA-MB-231-GFP-S1.MTT assay was performed to quantize the rate of S1 cell and MDA-MB-231 cell proliferation.Transwell assays were carried out to detected the migration activity.Results:(1)The self-seeding in mice model of breast cancer were higher after irradiation.(2)The IL-6 and IL-8 level showed no obvious changes in culture media of MDA-MB-231 after radiotherapy,whereas the level of them were higher in recipient tumors and mice’ plasma compared with the control group.(3)The level of HIF-1α and VEGF in recipient tumors were strengthened after irradiation.(4)CD68+macrophages and LY6G+neutrophils’ recruitment increased after radiotherapy.(5)S1cells have faster proliferation rate than MDA-MB-231 cells.Conclusions:(1)Irradiation can enhance breast cancer self-seeding significantly.(2)Tumor microenvironment changed and facilitated tumor cells secreted more IL-6and IL-8 after radiotherapy that attract the macrophages and neutrophils.(3)Radiotherapy gave rise to the increased level of HIF-1α and VEGF in recipient tumors,which could account for the rise of self-seeding.(4)Self-seeding cells have have faster proliferation rate than primary tumor cells and IL-6 promotes CTCs attraction...