GSE137528
ECM mediated FGF-induced estrogen receptor activity and therapy response
We wanted to investigate the effects of stromal signaling on ER+ breast cancer cells as recent work implicates the tumor microenvironment in the acquisition of resistance to endocrine therapy. We desired to examine the effects of both coculture with fibroblasts, and 2.5D monoculture of breast cancer cells on decellularized, fibroblast-derived extracellular matrix scaffolds. We cultured MCF7 cells alone (Unc_#_Mono) or in coculture (Co_#) with BJ fibroblasts to examine stromal signalling on ER+ breast cancer. Cocultured MCF7 were isolated by magnetically activated cell sorting. We also cultured MCF7 cells in decellularized ECM scaffolds generated by BJ fibroblasts (ECM1_#) or IMR90 fibroblasts (ECM2_#) or on an uncoated, plastic cell culture dish (Unc_#). Total RNA was extracted from cells using TRIzol (Invitrogen) and purified using Direct-zol RNA mini kit (Zymo Research) with DNase I treatment. After RNA purification, samples were confirmed to have a RIN value > 9.0 when measured on an Agilent Bioanalyzer. Libraries for RNA-Seq were prepared with KAPA Stranded RNA-Seq Kit. The workflow consisted of mRNA enrichment, cDNA generation, end repair to generate blunt ends, A-tailing, adaptor ligation and 14 cycles of PCR amplification. Unique adaptors were used for each sample in order to multiplex samples into several lanes. Sequencing was performed on Illumina Hiseq 4000 with a 150bp pair-end run. A data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq2 v 2.17 program. Overall design: Replicate coculture sets were generated by culturing MCF7 cells alone or in coculture with BJ fibroblasts prior to magnetic separation. Replicate ECM sets were generated by culturing MCF7 cells on ECM generated by IMR90 or BJ fibroblasts or on uncoated surfaces.