Obesity-dependent changes in interstitial ECM mechanics promote breast tumorigenesis

Abstract

Obesity and extracellular matrix (ECM) density are considered independent risk and prognostic factors for breast cancer. Whether they are functionally linked is uncertain. We investigated the hypothesis that obesity enhances local myofibroblast content in mammary adipose tissue and that these stromal changes increase malignant potential by enhancing interstitial ECM stiffness. Indeed, mammary fat of both diet- and genetically induced mouse models of obesity were enriched for myofibroblasts and stiffness-promoting ECM components. These differences were related to varied adipose stromal cell (ASC) characteristics because ASCs isolated from obese mice contained more myofibroblasts and deposited denser and stiffer ECMs relative to ASCs from lean control mice. Accordingly, decellularized matrices from obese ASCs stimulated mechanosignaling and thereby the malignant potential of breast cancer cells. Finally, the clinical relevance and translational potential of our findings were supported by analysis of patient specimens and the observation that caloric restriction in a mouse model reduces myofibroblast content in mammary fat. Collectively, these findings suggest that obesity-induced interstitial fibrosis promotes breast tumorigenesis by altering mammary ECM mechanics with important potential implications for anticancer therapies.

Fig. 1. Obesity increases interstitial fibrosis in mouse mammary fat pads

(A) Schematic showing the dietary [high-fat diet (HFD) and low-fat diet (LFD), 15 weeks] and genetic [ob/ob and wild-type (WT), 11 weeks] mouse models of obesity that were used to compare fibrotic remodeling between lean and obese mammary fat. (B) H&E-stained sections of mammary fat from lean and obese mice. Scale bars, 200 μm. (C and D) Immunofluorescence and Western blot analysis of α-SMA (C) and fibronectin (Fn) (D) in mammary fat. Scale bars, 200 μm. Western blot quantification is relative to the corresponding HSP90 levels. (E) SHG imaging of collagen fiber linearity in mammary fat. Box plots show medians with whiskers from minimum to maximum values. Scale bars, 100 μm. (C to E) Data are means ± SD (n = 6 to 11 per group). *P < 0.05, **P < 0.01, unless otherwise noted, by unpaired Student's two-tailed t tests for two conditions and one-way analysis of variance (ANOVA) for multiple comparisons. DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 2. Obesity enhances the profibrotic phenotype of ASCs

(A) Schematic showing ASC isolation from inguinal fat of age-matched lean and obese mice. (B) Immunofluorescence analysis of α-SMA⁺ ASCs isolated from ob/ob relative to WT fat. Western blot analysis of α-SMA relative to β-actin. (C) Percentage of α-SMA⁺ and BrdU⁺ ASCs as analyzed by immunofluorescence. (D) SDF-1 secretion of WT and ob/ob ASCs as determined by enzyme-linked immunosorbent assay (ELISA) and normalized to DNA content. (E) Confocal analysis of collagen type I or fibronectin deposition by WT and ob/ob ASCs. (F) Western blot quantification of fibronectin deposition relative to β-actin. (G) Comparison of ASCs isolated from low-fat diet– and high-fat diet–fed mice for α-SMA levels and BrdU incorporation by immunofluorescence; SDF-1 secretion by ELISA; and fibronectin matrix deposition by confocal microscopy. Scale bars, 200 μm. (B to G) Data are means ± SD (n = 3 to 6 per condition). P values by unpaired two-tailed t tests.

Fig. 3. Obesity-associated ASCs deposit partially unfolded and stiffer ECMs

(A) FRET analysis of fibronectin conformation in ECMs deposited by WT or ob/ob ASCs. Schematic shows separation of donor (Do) and acceptor (Ac) fluorophores due to partial fibronectin unfolding. Pseudocolored immunofluorescence micrographs depict FRET intensities of fibronectin fibers in the different ECMs. Scale bars, 50 μm. (B) Representative histogram of FRET intensity distribution as analyzed from the fields of view shown in (A). Box and whisker plots of FRET intensities as analyzed from six to eight representative fields of view per condition. Data are medians with whiskers from minimum to maximum values. (C) SFA indentation analysis of decellularized ECMs between two silvered mica surfaces mounted on a cantilever spring of constant k. F is normal force; R is the radius of curvature of the cylindrical discs; D₀ and D are the undistorted and force-applied thicknesses of the matrix, respectively. (D) Compressive elastic moduli of decellularized ECMs with corresponding fibronectin immunofluorescence micrographs. Box plots show medians with whiskers from minimum to maximum values from four random fields per condition. Scale bars, 100 μm. (E) AFM analysis to assess the elastic moduli of interstitial mammary fat. (Left) Picrosirius Red–stained histological cross sections showing representative regions of analysis. (Middle) Representative force versus indentation curve. (Right) Data are average compressive elastic moduli ± SD, analyzed from four samples per condition (20 areas per sample). Scale bars, 100 μm. P values in (B), (D), and (E) were determined by unpaired two-tailed t tests. PBS, phosphate-buffered saline.

Fig. 4. ECMs deposited by obesity-associated ASCs stimulate MDA-MB231 mechanosensitive growth

(A) Experimental setup to analyze human breast cancer cell line MDA-MB231 behavior in response to decellularized WT and ob/ob ECMs. (B) Number of MDA-MB231 cells after culture on decellularized ob/ob or WT ECMs as determined by image analysis. Scale bar, 100 μm. (C) Immunofluorescence analysis of pFAK[397]⁺ MDA-MB231 (red arrows) after culture on the different ECMs and corresponding Western blot analysis of pFAK[397] relative β-actin. Scale bars, 50 μm. (D) Immunofluorescence analysis of the nuclear/cytoplasmic YAP/TAZ ratio of MDA-MB231 after culture on ob/ob or WT ECMs. Scale bars, 50 μm (top); 20 μm (bottom). P values in (B) to (D) determined by unpaired two-tailed t tests. (E) Effect of Y27632 on MDA-MB231 growth on ob/ob or WT ECMs as assessed by image analysis of cell number. *P < 0.05 versus all other groups, determined by two-way ANOVA. (B to E) Data are means ± SD (n = 3 per condition).

Fig. 5. Obesity-associated ECMs promote the tumorigenic potential of premalignant human breast epithelial cells

(A) Number of MCF10AT cells after culture on WT or ob/ob ECMs in the presence and absence of Y27632. Data are means ± SD (n = 3). *P < 0.05 versus all other groups, determined by two-way ANOVA. Scale bars, 50 μm. (B) Methodology used to assess the effect of WT and ob/ob ECMs on the disorganization of MCF10AT acini. Spreading was analyzed by measuring acini surface area and height. (C) Confocal image analysis of surface area and height of acini and representative confocal micrographs of intact versus spread MCF10A acini (n = 100 acini per condition). Horizontal lines indicate means. Scale bar, 50 μm. (D) Time-lapse imaging of MCF10AT migration on ob/ob relative to WT ECMs. (i) Representative image sequence visualizing MCF10AT migration along fibronectin fibers. Scale bar, 20 μm. (ii) x-y coordinate maps tracking individual cells over 300 min. (iii) Computed MCF10AT motility from (ii) (n = 41 cells per condition). Horizontal lines indicate means. P values in (C) and (D) determined by unpaired two-tailed t tests. FOV, field of view.

Fig. 6. Obesity-associated, tumor-free human breast tissue displays profibrotic features

(A and B) Immunofluorescence analysis of α-SMA levels (A) and SHG image analysis of collagen fibers (B) in tumor-free breast tissue from normal, overweight, and obese women. Scale bar, 100 μm (A); 50 μm (B). Data are medians with minimum and maximum values from 9 to 11 samples per group. P values by one-way ANOVA. (C) Correlation of α-SMA levels with fibronectin levels (determined by immunofluorescence), collagen fiber thickness and length (analyzed by SHG), TGF-β expression (measured by quantitative reverse transcriptase polymerase chain reaction), and obesity-associated chronic inflammation (as defined by CLS-B index). Spearman's rank correlation was used to determine P values, and its coefficient was denoted as rho (ρ). Data points represent individual samples (n = 28).

Fig. 7. Obesity-associated ECM remodeling in patient samples

(A) Histopathological scoring of clinical tumor specimens for degree (1 to 3) of desmoplasia (n = 17 to 18 patients per group). Scale bars, 200 μm. Statistical significance was assessed by Fisher's exact test. (B and C) Immunofluorescence analysis of α-SMA (B) and fibronectin (C) content in breast tumor specimens from obese and lean patients (n = 10 patients per group). (D) SHG image analysis of collagen fiber thickness and length. Data are medians with minimum and maximum values (n = 10 patients per group). P values in (B to D) were determined by Mann-Whitney U tests. (E) Immunofluorescence image analysis of epithelial total and nuclear YAP/TAZ in tumors. Data points are individual images (n =10 images per patient, 10 patients per group). P values by mixed-model design ANOVA. Scale bars, 100 μm. (F) Bioinformatics analysis of published data (39) to correlate obesity-induced transcriptomic changes with ECM- and inflammation-related gene signatures.