Comparison of the paracrine effect of fibroblast on molecular changes and phenotypes of cancer stem cells in luminal and basal subgroups of breast cancer

Abstract

Fibroblast, as the most abundant component of the microenvironment, probably plays a major role in obtaining the characteristics of stem cells in breast cancer, and cancer cells, by changing their phenotype towards stem characteristics, play a major role in resistance to treatment and recurrence. Therefore, in this study, the effect of secretions or medium conditioning from normal human fibroblasts on the acquisition of fundamental characteristics in basal and luminal cell lines with OCT4/SOX2/NANOG, CD44+, CD24-, and ALDH1 markers has been investigated. Materials and methods: For this study, 2 MCF-7/MDA-MB231 cell lines were prepared, which are luminal and basal type respectively. After preparation of fibroblast medium condition from HFF-2 cells, luminal and basal cell lines were cultured with normal human fibroblast medium condition for 7 days. Then, the expression changes of CD44+, CD24- and ALDH1 markers were investigated by flow cytometry and western blot. Also, the degree of sphere formation strength among the luminal and basal subgroups of breast cancer after co-cultivation with fibroblast medium condition was compared with an inverted microscope, and finally, the gene expression level of cancer stem cells in both luminal and basal subgroups was compared by real time RT method. - PCR was checked. Findings: SOX-2 expression level in MCF-7 cell line showed a significant decrease in gene expression from 0.1 to 0.2 in comparison with the cell line treated with conditioned medium obtained from fibroblast and also in MDA-MB 231 line. The significance of gene expression showed 1 to 0.1. Also, the Nanog gene expression level was measured in MCF-7 and MDA-MB 231 cells treated with the conditioned medium obtained from normal human fibroblast and compared with the control group. This level of Nanog expression in the MCF-7 cell line shows a meaningless decrease after co-culture, while in the MDA-MB 231 cell line, it showed a significant decrease in gene expression compared to the control line. Oct-4 gene expression was measured in MCF-7 and MDA-MB 231 cells after treatment with conditioned medium obtained from normal human fibroblast and compared with the control group. In the MCF-7 cell line, it showed a meaningless decrease in expression from 1 to 0.8 after the same vessel, and also in the MDA-MB 231 cell line, it showed a significant decrease in Oct-4 gene expression. In the examination of ALDH1 enzyme expression in both MCF-7 and MDA MB 231 strains, a significant decrease in ALDH1 enzyme expression was observed. In examining the expression level of CD44+ and CD24- markers, it was able to create the basic characteristics of cancer cells by increasing the CD44+/CD24- cell population. No significant difference was observed in terms of the number and size of MCF-7 cells. Also, in the basal category, both the number and the size of mammospheres increased. Statistically, the increase in the size of mammospheres was not significant, but the increase in the number of mammospheres was significant.