Effect And Mechanism Of Radiation On Invasive And Metastatic Potential Of Breast Cancer Cells—an Experimental Study

Background and purposeRadiation therapy can reduce local-regional recurrence risk and improve survival in both the breast conserving and post-mastectomy setting, and paly a role in definitive treatment for both early-stage and locally advanced breast cancer,but it has been reported that radiation may also promote the invasive and metastatic potential of cancer cells by causing changes in the tumor microenvironment. In this study, we determined whether an irradiation could alter the invasive phenotype of breast cancer cells and its molecular machenism.Methods and Materials1.Human breast cancer cell line MDA-MB-435 was irradiated with different doses(0~8 Gy). Matrix Metalloproteases-2(MMP-2) protein activity was detected by gelatin zymogram and MMP-2 protein expression were tested by Western bloting-at 24 h and 48 h after irradiation. Invasion of cancer cells were also measured by matrigel-coated transwell inserts at the same time.2.MDA-MB-435 cells were transfected with Lentivirul vector with expression of green fluorescent protein(GFP) and luciferase(Luc) MDA-MB-435 cells which can express GFP and luciferase stablely were inoculated in nude mice for establishment of animal model of orthatopic breast cancer. In second week after inoculation, 8 Gy single dose irradiation of orthotopic tumor, circulating tumor cells(CTCs) in nude mice ear artery were detected by in vivo flow cytometry(IVFC) weekly, and tumor growth and metastasis were detected synchronously by in vivo image system(IVIS). At sixth week after inoculation, D-luciferin was injected into the mice immediately prior to necropsy, the lung tissue was excised and imaged. Lung tissue were subsequently fixed in formalin and prepared for standard HE staining. The expression of MMP-2 was measured through immunohistochemical staining of primary tumor.Results1. The invasiveness of MDA-MB-435 cells,the activity and expression of MMP-2 protein were significantly increased after radiation in a dose- and time-dependent manner. After radiation with 8Gy for 48 h, they reached the peak.successfully established :significant expression of green fluorescence,GFP transfection efficiency reached 99.58%; Luminescence in vitro was positively correlated with cell number(R2= 0.998). CTCs signal peak in vivo could be detected by IVFC in the second week postimplantation of tumor cells, and IVIS imaging could detect pulmonary micrometastasis.There was no significant change in morphological characteristics, the growth of cancer cells, invasion and migration characteristics after transfection.3.The tumor volume, the number of CTCs, fluorescence intensity in tumor location of irradiation group decreased firstly and then increased after irradiation. In sixth week after inoculation, tumor volumes of control group and irradiation group were519.66±32.89 mm3, 497.28±45.86 mm3, respectively(p > 0.05). The numbers of CTCs for each hour were 7.8±1.9,11.8±2.3, respectively(p < 0.05). Lung metastases and MMP-2 protein expression in the primary tumor tissue of irradiated group was significantly increased compared with the non-irradiated group.Conclusion1.The combination of IVFC and IVIS can detect early, real-time and dynamic monitoring of the orthotopic breast cancer model CTCs of MDA-MB-435-GFP-Luc cell line, tumor growth and micrometastasis.2. Radiation can enhance invasive and metastatic potential of breast cancer cells through activating MMP-2.