ERα36-High Cancer-Associated Fibroblasts as an Unfavorable Factor in Triple-Negative Breast Cancer

Abstract

Background: Cancer-associated fibroblasts (CAFs) are the most abundant cell type in the tumor microenvironment (TME). Estrogen receptor alpha 36 (ERα36), the alternatively spliced variant of ERα, is described as an unfavorable factor when expressed in cancer cells. ERα can be expressed also in CAFs; however, the role of ERα36 in CAFs is unknown. Methods: Four CAF cultures were isolated from chemotherapy-naïve BC patients and characterized for ERα36 expression and the NanoString gene expression panel using isolated RNA. Conditioned media from CAF cultures were used to assess the influence of CAFs on triple-negative breast cancer (TNBC) cells using a matrigel 3D culture assay. Results: We found that ERα36high CAFs significantly induced the branching of TNBC cells in vitro (p < 0.001). They also produced a set of pro-tumorigenic cytokines compared to ERα36low CAFs, among which hepatocyte growth factor (HGF) was the main inducer of TNBC cell invasive phenotype in vitro (p < 0.001). Tumor stroma rich in ERα36high CAFs was correlated with high Ki67 expression (p = 0.041) and tumor-associated macrophages markers (CD68 and CD163, p = 0.041 for both). HGF was found to be an unfavorable prognostic factor in TCGA database analysis (p = 0.03 for DFS and p = 0.04 for OS). Conclusions: Breast cancer-associated fibroblasts represent distinct subtypes based on ERα36 expression. We propose that ERα36high CAFs could account for an unfavorable prognosis for TNBC patients.

Keywords: breast cancer; cancer-associated fibroblasts; estrogen receptor alpha 36; triple-negative breast cancer.

Figure 1

Characterization of CAF cultures from BC patients, (A). ERα36 staining (green) by IF and nuclear DAPI staining (blue) of the obtained CAF cell cultures, scale bar: 50 µm. (B). nCounter PanCancer Immune Profiling panel analysis of gene expression in ERα36^hihg CAFs vs ERα36^low CAFs, genes with logFC > 1 (FC—fold change) were considered upregulated, genes with logFC < −1 were considered downregulated, (C). Human Cytokine XL Profiler analysis of conditioned media obtained from CAF cultures. The left panel shows secretomes of ERα36^high and ERα36^low CAFs with marked spots that were secreted differently between the two groups, the right panel quantifies the changes in the six selected cytokines levels, (D). 3D matrigel cultures of the TNBC MDA-MB-231 cells treated with the condition media from ERα36^high or ERα3 ^low CAFs. ERα36^high CAF-conditioned media significantly induced branching in MDA-MB-231 cells, ** p < 0.0001 calculated vs control; colony photographs were taken using 10× magnification.

Figure 1

Characterization of CAF cultures from BC patients, (A). ERα36 staining (green) by IF and nuclear DAPI staining (blue) of the obtained CAF cell cultures, scale bar: 50 µm. (B). nCounter PanCancer Immune Profiling panel analysis of gene expression in ERα36^hihg CAFs vs ERα36^low CAFs, genes with logFC > 1 (FC—fold change) were considered upregulated, genes with logFC < −1 were considered downregulated, (C). Human Cytokine XL Profiler analysis of conditioned media obtained from CAF cultures. The left panel shows secretomes of ERα36^high and ERα36^low CAFs with marked spots that were secreted differently between the two groups, the right panel quantifies the changes in the six selected cytokines levels, (D). 3D matrigel cultures of the TNBC MDA-MB-231 cells treated with the condition media from ERα36^high or ERα3 ^low CAFs. ERα36^high CAF-conditioned media significantly induced branching in MDA-MB-231 cells, ** p < 0.0001 calculated vs control; colony photographs were taken using 10× magnification.

Figure 2

3D matrigel cultures of MBA-MB-231 cells treated with selected cytokines in two concentrations (10 ng/mL and 50 ng/mL), only HGF induced colony branching. ** p < 0.001 calculated vs control; culture photographs were taken using 10× objective. The graph represents the number of branching colonies per visible field.

Figure 3

Analysis of c-Met pathway involvement in HGF-mediated MDA-MB-231 colony branching (A). Representative colony photographs of 3D matrigel cultures, HGF and HGF with capmatinib treatment on the left panel, conditioned media (CM) from ERα36^high (CAF4) culture and capmatinib treatment on the right panel, 10× magnification, capmatinib significantly inhibited colony branching induced by HGF as well as by CAF4-CM (B). Graphs representing data from 3D matrigel cultures, ** p < 0.005 between HGF or CAF4-conditioned media treatment and addition of capmatinib (C). Western blot analysis of c-Met-signaling pathway after HGF and CAF4-CM treatment at different times (15–60 min), with or without capmatinib treatment. Activation of c-Met receptor and Akt was inhibited after capmatinib treatment. (D) Densitometry measurements of protein phosphorylation as a ratio between phosphorylated to total protein signal.

Figure 4

Levels of protein expression in CAFs subgroups, (A) Ki67 expression in ERα36 negative and positive CAFs groups, (B) CXCR4 expression in ERα36 negative and positive CAFs groups, (C) CD68 expression in ERα36 negative and positive CAFs groups, (D) CD163 expression in ERα36 negative and positive CAFs groups. Mann-Whitney U test was used in the analysis.

Figure 5

Kaplan–Meier survival curves of (A) disease-free survival (DFS) and (B) overall survival (OS) according to HGF expression in TCGA database.

Figure 6

Significantly affected biological processes in BC patients with ERα36high tumor stroma fibroblasts depicted as the number of genes involved in a particular process.