Preliminary Study On The Interaction Between Triple Negative Breast Cancer Cell And Normal Mammary Epithelial Cell

Background: Triple negative breast cancer(TNBC)is a specific subtype of breast cancer which lacks the expression of estrogen/progesterone receptor(ER/PR)and human epidermal growth factor receptor-2(HER-2),accounting for approximately15-20% of all breast cancer types,and currently lacking effective treatment options.TNBC is characterized by high tumor heterogeneity,strong metastatic ability and poor prognosis,which are associated with its unique tumor microenvironment(TME).TME is involved in the induction of tumor cell proliferation,angiogenesis,inhibition of apoptosis,suppression of the immune system and drug resistance development,and may be an effective therapeutic target for TNBC.Studies have shown that a variety of cells in TME are affected or reprogrammed by tumor cells,allowing the promotion of tumor progression and metastasis.As the nearest class of normal host cells to tumor cells,the interactions between normal epithelial cells and tumor cells and their possible mechanisms have not been fully elucidated.In this study,we co-cultured TNBC cells MDA-MB-231 with normal mammary epithelial cells MCF-10 A to explore whether there was any interaction between them;and further investigated whether the interaction between the two cell lines could affect the proliferation and migration of TNBC cells and whether it could have an oncogenic effect on normal mammary epithelial cells.Objective: To investigate the interaction between TNBC cells and normal mammary epithelial cells,as well as the effect of the interaction on the proliferation and migration of cancer cells and the morphological and functional changes of normal mammary epithelial cells,in order to provide new ideas and theoretical support for improving the therapeutic effect of TNBC through the study of the role of normal mammary epithelial cells in the progression of TNBC.Methods:(1)2D and 3D direct cell co-culture were used to observe whether there were morphological and behavioral interactions between triple negative breast cancer cells MDA-MB-231 and normal mammary epithelial cells MCF-10 A.(2)The effect of indirect cell co-culture of MDA-MB-231 cells and MCF-10 A cells on proliferation capacity was determined by the High Content Cell Imaging and Analysis System;the change of cell migration capacity in the indirect cell co-culture system was determined by wound healing assay;and use q RT-PCR and WB to determine m RNA and protein changes of the proliferation pathway PTEN/PI3K/AKT and apoptosis-related regulators Bcl-2、Bax in MDA-MB-231 cells after MCF-10 A cell conditioned medium intervention;and epithelial-mesenchymal transition markers such as E-cadherin and N-cadherin,as well as Bcl-2、Bax,were determined by using WB in MCF-10 A cell after MDA-MB-231 cells conditioned medium intervention.Results:(1)2D direct cell co-culture results showed that MDA-MB-231 cells and MCF-10 A cells could "communicate with each other" by extracellular vesicles;and the results of 3D cell co-culture showed that similar cells aggregated with each other and the aggregated MDA-MB-231 cells gradually formed a spherical structure,while MCF-10 A cells failed to form 3D spheres;after the MCF-10 A cells formed stable spheres,the added MDA-MB-231 cells could gradually adhere to the surface of MCF-10 A cell spheres to form an envelope and affect the stability of the spherical structure of MCF-10 A cells;(2)High Content Cell Imaging and Analysis System showed that the proliferation ability of MDA-MB-231 cells and MCF-10 A cells was reduced in indirect cell co-culture system;the results of wound healing assay showed that the healing rate of MDA-MB-231 cells was low and the healing rate of MCF-10 A cells became higher after indirect co-culture.The results of q RT-PCR and WB showed that after indirect cell co-culture the expression of anti-oncogene PTEN in MDA-MB-231 cells was increased PI3K/AKT expression was decreased,and the ratio of Bcl-2/Bax was decreased;meanwhile,the ratio of Bcl-2/Bax in MCF-10 A cells did not change significantly after indirect co-culture,while E-ca expression was decreased and N-ca expression was increased.Conclusions:(1)TNBC cells and normal mammary epithelial cells can interact with each other by secreting extracellular vesicles;(2)normal mammary epithelial cells may regulate the PTEN/PI3K/AKT pathway and apoptosis in TNBC cells through paracrine secretion to inhibit cancer cell proliferation and migration;TNBC cells may trigger oxymoronic transformation of normal mammary epithelial cells through paracrine secretion.Improving TME by blocking extracellular vesicular delivery of cancer cells may be an effective strategy to improve the therapeutic efficacy of TNBC and inhibit tumor progression.