Effect Of Cell Density On The Malignant Biological Behavior Of Breast C Ancer By Altering The Subcellular Localization Of ANXA2 And Its Clinical Implications

Objective:To investigate the subcellular localization of ANXA2 in breast cancer of different cell densities in humans and its relationship with the clinicopathological features of patients.To investigate the differences of ANXA2 subcellular localization in MDA-MB-231 cells with different cell densities and to compare the proliferation,invasion and migration ability of MDA-MB-231 cells under different ANXA2 subcellular localization.METHODS:Sixty breast cancer tissue specimens from the Department of Thyroid Breast Surgery of the Affiliated Hospital of Chuanbei Medical College were collected from January 2021 to October 2021 after surgical resection,and immunohistochemistry was applied to detect the subcellular localization of ANXA2 in the tissue sections of 60 breast cancer patients,and the association with ANXA2 subcellular localization was verified by combining with cell density.The relationship between cell density and clinicopathological data of breast cancer patients was investigated.To establish high and low density MDA-MB-231 breast cancer cell line models,and to verify the subcellular localization of ANXA2 using immunofluorescence and observation under confocal microscopy.The proliferation,migration and invasion ability of MDA-MB-231 cells under different subcellular localization of ANXA2 were detected and compared using CCK-8 assay and Transwell assay.After changing the subcellular localization of ANXA2 in high-density breast cancer cells with PY-60,the proliferation,migration and invasion ability of MDA-MB-231 cells under different subcellular localization of ANXA2 were compared.Two different groups of 4T1 cell lines with high and low densities were implanted subcutaneously in nude mice to observe the effects of different cell densities on tumor growth in nude mice.SPSS23 was used for statistical analysis,and the test standard was α=0.05,and P<0.05 was considered as statistically significant difference.Results:1.The clinical data showed that breast cancer with high cell density had higher T-stage and higher TNM-stage,and the cell density was positively correlated with the size of breast cancer masses.Immunohistochemical results showed that ANXA2 was mainly localized in the cell membrane when the cell density was high and in the cytoplasm when the cell density was low in humans.2.The immunofluorescence results showed that ANXA2 was mainly localized in the cell membrane at higher density of MDA-MB-231 cells and in the cytoplasm at lower density;the addition of PY-60 to high-density MDA-MB-231 cells could shift the subcellular localization of ANXA2 from the cell membrane to the cytoplasm.Transwell invasion assay showed that the invasive migration ability of high-density cells was significantly higher than that of the control group after the addition of PY-60(P<0.05);Transwell migration assay showed that the migration ability of high-density cells was significantly higher than that of the control group after the addition of PY-60(P<0.05).3.The results of animal experiments showed that high-density breast cancer cells could promote the growth of subcutaneous tumors in nude mice relative to low-density breast cancer cells.Conclusion:1.Breast cancer patients with high cell density had ANXA2 localized on the cell membrane and had later tumor stage;high cell density was related to tumor malignancy.2.Cellular models showed that the subcellular localization of ANXA2 in breast cancer cells with different cell density;changing the membrane localization of ANXA2 to cytoplasmic localization in MDA-MB-231 cells could enhance the proliferation,migration and invasion of breast cancer cells.3.Animal experiments showed that high density 4T1 breast cancer cells promoted tumor growth more significantly.