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Effect of human breast tumor supernatant on normal CD4+ T cells (Preliminary Experiment)

ABSTRACT: CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. This work aims to characterize Th cells infiltrating invasive primary human breast tumors, analyze the influence by the tumor microenvironment and identify Th cell specific prognostic gene signatures. CD4+ T cells isolated from the tumor (TIL), axillary lymph node (LN) and blood (PB) of 10 patients were analyzed on Affymetrix U133 Plus 2.0 arrays. A confirmation set of 60 patients were studied by flow cytometry, qRT-PCR or immunohistochemistry and analyzed according to the extent of the tumor immune infiltrate. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight or with CD4+ T cells [non-stimulated (NS) or stimulated (S)] from healthy donor PB treated with tumor supernatant (SN). Comparing gene expression profiles of donor blood derived total CD4+ T cells [non-stimulated (NS)] with and without tumor supernatant (SN) treatment Total CD4+ T cells from a healthy donor blood (NS) were treated (and as control: untreated samples in biological triplicate) with SN from fresh breast tumor homogenates of 3 patients and analyzed on Affymetrix U133 Plus 2.0 arrays

ORGANISM(S): Homo sapiens

SUBMITTER: GU Chunyan

PROVIDER: S-ECPF-GEOD-36768 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:BackgroundBreast cancer stem cells (BCSCs) are associated with the invasion of breast cancer. In recent years, studies have demonstrated different phenotypes among BCSCs. Furthermore, BCSCs of diverse phenotypes are present at different tumour sites and different histological stages. Fibroblasts are involved in the phenotypic transformation of BCSCs. Cancer-associated fibroblasts (CAFs) participate in the induction of epithelial-mesenchymal transition, thereby promoting the acquisition of stem cell characteristics, but little is known about the role of normal fibroblasts (NFs) in the phenotypic transformation of BCSCs or about the effect of CAFs and NFs on BCSC phenotypes.MethodsA total of six pairs of primary CAFs and NFs were isolated from surgical samples of breast cancer patients and subjected to morphological, immunohistochemical, cell invasion and proteomics analyses. After establishing a cell culture system with conditioned medium from CAFs and NFs, we used the mammosphere formation assay to explore the effect of CAFs and NFs on the self-renewal ability of BCSCs. The effect of CAFs and NFs on the phenotypic differentiation of BCSCs was further analysed by flow cytometry and immunofluorescence.ResultsThe isolated CAFs and NFs did not show significant differences in cell morphology or alpha-smooth muscle actin (α-SMA) expression, but cell invasion and proteomics analyses demonstrated heterogeneity among these fibroblasts. Both CAFs and NFs could promote the generation of BCSCs, but CAFs displayed a greater ability than NFs in promoting mammosphere formation. Conditioned medium from CAFs increased the proportion of aldehyde dehydrogenase-1 positive (ALDH1+) BCSCs, but conditioned medium from NFs was more likely to promote the generation of CD44+CD24- BCSCs from MCF-7 cells.DiscussionThis study validated the heterogeneity among CAFs and NFs and expanded on the conclusion that fibroblasts promote the generation of cancer stem cells. Our results particularly emphasized the effect of NFs on the phenotypic transformation of BCSCs. In addition, this study further highlighted the roles of CAFs and NFs in the induction of different phenotypes in BCSCs.