Breast-Associated Adipocytes Secretome Induce Fatty Acid Uptake and Invasiveness in Breast Cancer Cells via CD36 Independently of Body Mass Index, Menopausal Status and Mammary Density

Abstract

Breast adiposity is correlated with body mass index, menopausal status and mammary density. We here wish to establish how these factors influence the cross-talk between breast adipocytes and normal or malignant breast cells. Adipocyte-derived stem cells (ASCs) were obtained from healthy women and classified into six distinct groups based on body mass index, menopausal status and mammary density. The ASCs were induced to differentiate, and the influence of their conditioned media (ACM) was determined. Unexpectedly, there were no detectable differences in adipogenic differentiation and secretion between the six ASC groups, while their corresponding ACMs had no detectable influence on normal breast cells. In clear contrast, all ACMs profoundly influenced the proliferation, migration and invasiveness of malignant breast cells and increased the number of lipid droplets in their cytoplasm via increased expression of the fatty acid receptor CD36, thereby increasing fatty acid uptake. Importantly, inhibition of CD36 reduced lipid droplet accumulation and attenuated the migration and invasion of the breast cancer cells. These findings suggest that breast-associated adipocytes potentiate the invasiveness of breast cancer cells which, at least in part, is mediated by metabolic reprogramming via CD36-mediated fatty acid uptake.

Keywords: CD36; adiposity; body mass index; breast cancer; fatty acid; mammary density; menopausal status.

Figure 1

Adipocyte differentiation of adipocyte-derived stem cells (ASCs) populations. Breast ASCs were differentiated into adipocytes for 14 days. (A) The differentiation process was monitored by microscopic imaging after Oil Red O-staining (upper panel). Subsequently, Oil Red O-stained areas were quantified by ImageJ analysis. Scale bars, 70 µm. Results are presented as means ± standard error of the mean (SEM) (lower panel). (B) Whole cell lysates were extracted at day 0 and 14 of ASCs differentiation and were subjected to immunoblotting with anti-PPARγ and anti-FABP4 antibodies (left panel). Immunoblot signals were quantified by densitometry, and normalized with tubulin. Data are representative of three individual samples from three independent experiments. Results are presented as means ± SEM (right panel). (C) Dosage of Free Fatty Acids (FFAs). C16.0, palmitic acid; C18.0, stearic acid; PUFA, polyunsaturated fatty acids. Results are presented as means ± SEM. The uncropped blots and molecular weight markers are shown in Supplementary Materials.

Figure 2

Adipokine expression by different ASCs populations. (A) Serum-free conditioned medium from the indicated adipocytes was applied onto human adipokine array membranes. The boxes indicate adipokines further tested by ELISA. (B) Scatter plots of leptin, adiponectin, MCP1 and IL6 concentrations in conditioned medium are shown from 16 breast ASCs populations after 14 days of differentiation, as measured by ELISA, * p < 0.05.

Figure 3

Adipocyte conditioned medium stimulates proliferation of tumor cells independently of BMI, menopausal status and mammary density. (A) Graphs representing the proliferation of HMEC, MCF7 and SUM159 cells treated with control medium (CTRL) or conditioned medium from ASCs with the indicated characteristics differentiated into adipocytes. The proliferation rate was assessed by the xCELLIgence system RTCA system. Data are representative of three individual samples from three independent experiments. (B) Cell extracts were prepared from HMEC, MCF7 and SUM159 cells treated for 1 h with either control medium or the indicated adipocyte conditioned media (ACM) and characterized by Western blot analysis. The uncropped blots and molecular weight markers are shown in Supplementary Materials.

Figure 4

The influence of adipocyte-derived conditioned medium on the migration and invasion of breast cells. Dynamic real-time monitoring of the migration (A) or invasion (B) of HMEC, MCF7 and SUM159 cells towards either control media or the indicated ACM from ASCs differentiated into adipocytes. Results are expressed as mean ± SEM for three individuals from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

Adipocyte-conditioned medium increases lipids droplets in breast tumor cells. (A) Representative images of HMEC, MCF7 and SUM159 cells cultured with control medium or ACM from ASCs differentiated into adipocytes for the indicated times, followed by Bodipy staining. Scale bars, 10 µm (upper panels). The total lipid droplet area was calculated with ImageJ (lower panels). (B) Representative side scatter of HMEC, MCF7 and SUM159 cells cultured with control media or with ACM for 24 h. n.s. p ≥ 0.05; * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 6

CD36 inhibition reduces adipocyte-induced fatty acid uptake. (A) The expression of FA-binding proteins and transporters was determined by qRT-PCR. Fold induction is calculated as the expression of the indicated marker in treated cells compared to untreated controls. Results are expressed as mean ± SEM for three individuals from three independent experiments. ** p < 0.01; *** p < 0.001. (B) Western blot analysis of CD36 were performed for HMEC, MCF7 and SUM159 cells cultured with ACM for 24 h, (C) The expression of cell surface CD36 was determined by flow cytometry for HMEC, MCF7 and SUM159 cells cultured with ACM for 24 h. The graph shows the mean ± SEM from three independent experiments. * p < 0.05; **** p < 0.0001. (D) Fatty acid uptake was measured with BODIPY-FA in presence or absence of 150 µM sulfo-N-succinymidyl (SSO). Scale bars, 10 µm (upper panels). Changes in intracellular fatty acid levels are quantified with CellProfiler. The graph shows the mean ± SEM from three independent experiments. ** p < 0.01; *** p < 0.001; **** p < 0.0001. The uncropped blots and molecular weight markers are shown in Supplementary Materials.

Figure 6

CD36 inhibition reduces adipocyte-induced fatty acid uptake. (A) The expression of FA-binding proteins and transporters was determined by qRT-PCR. Fold induction is calculated as the expression of the indicated marker in treated cells compared to untreated controls. Results are expressed as mean ± SEM for three individuals from three independent experiments. ** p < 0.01; *** p < 0.001. (B) Western blot analysis of CD36 were performed for HMEC, MCF7 and SUM159 cells cultured with ACM for 24 h, (C) The expression of cell surface CD36 was determined by flow cytometry for HMEC, MCF7 and SUM159 cells cultured with ACM for 24 h. The graph shows the mean ± SEM from three independent experiments. * p < 0.05; **** p < 0.0001. (D) Fatty acid uptake was measured with BODIPY-FA in presence or absence of 150 µM sulfo-N-succinymidyl (SSO). Scale bars, 10 µm (upper panels). Changes in intracellular fatty acid levels are quantified with CellProfiler. The graph shows the mean ± SEM from three independent experiments. ** p < 0.01; *** p < 0.001; **** p < 0.0001. The uncropped blots and molecular weight markers are shown in Supplementary Materials.

Figure 7

Inhibition of CD36 reduces adipocyte-induced migration and invasion. (A) The expression of cell surface CD44 and CD36 were determined by flow cytometry for SUM159 cells cultured with ACM for 24 h in the presence or absence of SSO. (B,C) Influence of the CD36-specific inhibitor SSO on breast cancer cell migration and invasion. Cancer cells were incubated with ACM in the presence or absence of SSO for 24 h. Dynamic real time monitoring of the migration (B) and invasion (C) of SUM159 cells towards the indicated ACM. Results are expressed as mean ± SEM for three individual samples from three independent. n.s. p ≥ 0.05; ** p < 0.01; *** p < 0.001.