Studying luminal A and B subtypes of breast cancer under paracrine secretion of fibro- blasts

Abstract

Introduction: Understanding the key role of the tumor microenvironment in specifying molecular markers of breast cancer subtypes is of a high importance in diagnosis and treatment. Therefore, the possibility of interconversion of luminal states and their specific markers alteration under the control of tumor microenvironment (TME), particularly cancer-associated fibroblasts (CAFs) deserves to be further investigated.
Methods: To activate normal human fibroblasts, liquid overlay technique or nemosis was used and α-SMA protein expression, CAFs marker, in fibroblastic spheroids was measured by blotting. The luminal A, MCF-7, and luminal B, MDA-MB 361, cell lines were treated with normal and spheroidal/activated fibroblast conditioned medium for 48 hours. The morphological changes of both luminal A and B cells were evaluated by invert light microscopy and analyzed through the shape factor formula. Moreover, chemo-sensitivity, proliferation, and changes in ER-related and proliferative genes expression levels were assessed respectively via MTT assay, Ki67 expression Immunofluorescence assay, real time PCR and Annexin V-FITC techniques.
Results: Activated (spheroidal) fibroblasts, expressed αSMA marker two folds more than monolayer cultured fibroblasts. Our study indicated a significant increase in IC₅₀ of both luminal A and B cell lines after being treated with conditioned medium particularly in treated group with spheroidal conditioned medium. Studying Morphological changes using shape factor formula demonstrated more aggressiveness with gaining mesenchymal features in both luminal A and B subtypes by increasing exposure time. Changes in the expression of Ki67 were observed following treatment with fibroblastic and spheroidal paracrine secretome. Driven Data from Ki67 assay supports the luminal A and B interconversion by elevated Ki67 expression in luminal A and lowered Ki67 expression in luminal B. Gene expression analysis revealed that anti-apoptotic Bcl2 gene expression in both luminal types treated with condition medium has been increased though there has seen no interchange in expression of ER-related and proliferative genes between luminal A (MCF7) and luminal B (MDA-MB361) subtypes, the results of Annexin V-FITC flow cytometry test indicated a decrease in the population of both early and late apoptotic cells in groups treated with both fibroblastic and spheroidal condition medium compared to of control group.
Conclusion: Under the paracrine influence of fibroblast cells, both luminal A (MCF7) and luminal B (MDA-MB) subtypes of breast cancer gained invasive, anti-apoptotic, and chemoresistance features which are mostly increased by activated(spheroidal) fibroblasts conditioned medium mimicking CAFs. There was no strong proof for interconversion of luminal A and luminal B which share more similarities among breast cancer molecular subtypes.

Keywords: Subtype, Breast cancer, Tumor microenvironment, Cancer-associated fibroblast