Studying Luminal, A and B subtypes of the breast cancer under paracrine secretion of fibroblasts

Abstract

Introduction Understanding the role of the tumor microenvironment in specifying molecule markers of breast cancer subtypes could involve tumor microenvironment properties in subtyping and clinical assessment of breast cancer. Therefore, interconversion of luminal states and or their specific markers alteration under the control of TME particularly CAFs deserve to be more investigated. Methods To activate normal human fibroblasts, liquid overlay technique or nemesis was used and α-SMA expression, CAFs marker, in fibroblastic spheroids was measured by blotting. The luminal A, MCF-7, and luminal B, MDA-MB 361, cell lines are treated with normal and spheroidal/activated fibroblast conditioned medium for 48 hours. The morphological changes of luminal A and B cells were evaluated by invert microscopy and analyzed by the shape factor formula. Moreover, sensitivity changes to chemotherapeutic agents, Ki67 expression, and gene expression level including ER-related and proliferative genes were assessed respectively by MTT assay, Immunofluorescence, and real time qRT-PCR methods. Results Activated fibroblast, spheroid form, by LOT/nemosis expressed α-SMA marker two folds more than monolayer cultured fibroblasts. Our study underlined a significant increase in IC50 of both luminal A and B cell lines after being co-cultured with conditioned medium markedly in spheroidal conditioned medium. Morphological changes study by shape factor formula demonstrated more aggressiveness with gaining mesenchymal features both in luminal A and B subtypes by increasing exposure time. Changes in Ki67 expression level were observed after fibroblasts and spheroidal paracrine conditioning. Only this data supports the luminal interconversion by elevated Ki67 expression in luminal A and lowered Ki67 expression in luminal B. Gene expression results revealed that the MCF7 and MDA-MB cells as representatives luminal A and B subtypes in this study did not interchange their own specific gene pattern, however both luminal types in co-culture with condition medium increased the expression of anti-apoptotic Bcl2 gene. Conclusion Under the paracrine influence of fibroblast cells, both luminal A(MCF7) and B (MDA-MB) subtypes of breast cancer gained invasive, anti-apoptotic, and chemoresistance features which are mostly increased by activated fibroblast conditioned medium mimicking CAFs. There was no strong proof for interconversion of luminal A and B, with more similarities, subtypes of breast cancer.