Unravelling heterogeneous effects of cancer‑associated fibroblasts on poor prognosis markers in breast cancer EM‑G3 cell line: In vitro‑targeted treatment (anti‑IL-6, anti‑VEGF-A, anti‑MFGE8) based on transcriptomic profiling

Abstract

Breast cancer is the most frequently diagnosed cancer in women worldwide. Although dramatically increased survival rates of early diagnosed cases have been observed, late diagnosed patients and metastatic cancer may still be considered fatal. The present study's main focus was on cancer‑associated fibroblasts (CAFs) which is an active component of the tumor microenvironment (TME) regulating the breast cancer ecosystem. Transcriptomic profiling and analysis of CAFs isolated from breast cancer skin metastasis, cutaneous basal cell carcinoma, and squamous cell carcinoma unravelled major gene candidates such as IL6, VEGFA and MFGE8 that induced co‑expression of keratins‑8/‑14 in the EM‑G3 cell line derived from infiltrating ductal breast carcinoma. Western blot analysis of selected keratins (keratin‑8, ‑14, ‑18, ‑19) and epithelial‑mesenchymal transition‑associated markers (SLUG, SNAIL, ZEB1, E‑/N‑cadherin, vimentin) revealed specific responses pointing to certain heterogeneity of the studied CAF populations. Experimental in vitro treatment using neutralizing antibodies against IL-6, VEGF‑A and MFGE8 attenuated the modulatory effect of CAFs on EM‑G3 cells. The present study provided novel data in characterizing and understanding the interactions between CAFs and EM‑G3 cells in vitro. CAFs of different origins support the pro‑inflammatory microenvironment and influence the biology of breast cancer cells. This observation potentially holds significant interest for the development of novel, clinically relevant approaches targeting the TME in breast cancer. Furthermore, its implications extend beyond breast cancer and have the potential to impact a wide range of other cancer types.

Keywords: breast cancer; cell differentiation; epithelial‑mesenchymal interaction; neutralizing antibody; tumor microenvironment.

Figure 1.

Immunohistochemical analysis of HDFs, BCCFs, SCCFs and BCMFs. Presence of fibronectin (Fibr) and α-SMA. Cell nuclei were counterstained with DAPI. Scale bar, 100 µm; magnification, ×200. HDFs, human dermal fibroblasts; BCCFs, basal cell carcinoma fibroblasts; SCCFs, squamous cell carcinoma fibroblasts; BCMFs, breast cancer metastasis fibroblasts; SMA, smooth muscle actin.

Figure 2.

Transcriptomic analysis of HDFs, BCCFs, SCCFs and BCMFs. (A) The heatmap displays expression profiles of all differentially expressed genes with concordant changes between CAFs and HDFs. (B) The expression profiles of the selected candidate genes IL6, VEGFA, and MFGE8 are shown in boxplots. HDFs, human dermal fibroblasts; BCCFs, basal cell carcinoma fibroblasts; SCCFs, squamous cell carcinoma fibroblasts; BCMFs, breast cancer metastasis fibroblasts.

Figure 3.

Western blot analysis of EM-G3 cells. Protein expression analysis in indirect (CM) co-culture of EM-G3 cells with HDFs, BCCFs, SCCFs and BCMFs. The studied conditions for each studied fibroblast type included: Control culture of EM-G3 cells, EM-G3 cells cultured in CM derived from respective fibroblasts, EM-G3 cells cultured in CM enriched with neutralizing antibodies against either IL-6 or VEGF-A or MFGE8 or combination of all three tested antibodies (anti-IL-6 + anti-VEGF-A + anti-MFGE8). The expression profile of the following markers related to cell differentiation and epithelial-to-mesenchymal transition was evaluated: keratin-8, keratin-14, keratin-18, keratin-19, vimentin, SLUG, SNAIL, TWIST1, ZEB1, E-cadherin, N-cadherin, VE-cadherin. β-Actin was used as sample loading control (representative beta-actin controls are shown). HDFs, human dermal fibroblasts; BCCFs, basal cell carcinoma fibroblasts; SCCFs, squamous cell carcinoma fibroblasts; BCMFs, breast cancer metastasis fibroblasts; CM, conditioned media.

Figure 4.

Immunofluorescence analysis EM-G3 cells. Expression of keratin-8 and keratin-14 in EM-G3 cells directly and indirectly (conditioned media) co-cultured with HDFs, SCCFs, and BCMFs. The co-expression of keratins-8/-14 was increased in EM-G3 cells co-cultured with cancer-associated fibroblasts. Addition of neutralizing antibodies against IL-6, VEGF-A, and MFGE8 attenuated the co-expression of keratins-8/-14. Positive control included supplementation of culture media with human recombinant IL-6, VEGF-A, and MFGE8 and resulted in marked co-expression of keratins-8/-14. HDFs, human dermal fibroblasts; SCCFs, squamous cell carcinoma fibroblasts; BCMFs, breast cancer metastasis fibroblasts.