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Obesity-dependent changes in interstitial ECM mechanics promote breast tumorigenesis

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Science Translational Medicine  19 Aug 2015:
Vol. 7, Issue 301, pp. 301ra130
DOI: 10.1126/scitranslmed.3010467
  • 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.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/301/301ra130/DC1

    Materials and Methods

    Fig. S1. Obesity promotes interstitial fibrosis in breast adipose tissue after menopause.

    Fig. S2. Inguinal depots of adipose tissue feature markers of interstitial fibrosis.

    Fig. S3. The profibrotic potential of ob/ob ASCs is not due to leptin deficiency.

    Fig. S4. Obesity-associated ASCs promote fibrotic ECM remodeling in visceral fat.

    Fig. S5. ECMs deposited by obesity-associated ASCs promote myofibroblast differentiation.

    Fig. S6. Physicochemical cues of ob/ob ECMs modulate MDA-MB231 behavior.

    Fig. S7. H&E images of breast tumors with different subtypes.

    Fig. S8. Caloric restriction decreases fibrosis in mammary fat of obese mice.

    Fig. S9. Decellularized ECMs do not contain cellular residuals.

    Table S1. Subtypes, demographics, and desmoplastic grade of lean and obese breast cancer samples.

    Table S2. Gene ontology data analysis.

    Table S3. Correlation of YAP/TAZ-regulated genes with obesity-dependent ECM remodeling/inflammation.

    References (5458)

  • Supplementary Material for:

    Obesity-dependent changes in interstitial ECM mechanics promote breast tumorigenesis

    Bo Ri Seo, Priya Bhardwaj, Siyoung Choi, Jacqueline Gonzalez, Roberto C. Andresen Eguiluz, Karin Wang, Sunish Mohanan, Patrick G. Morris, Baoheng Du, Xi K. Zhou, Linda T. Vahdat, Akanksha Verma, Olivier Elemento, Clifford A. Hudis, Rebecca M. Williams, Delphine Gourdon, Andrew J. Dannenberg, Claudia Fischbach*

    *Corresponding author. E-mail: cf99{at}cornell.edu

    Published 19 August 2015, Sci. Transl. Med. 7, 301ra130 (2015)
    DOI: 10.1126/scitranslmed.3010467

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Obesity promotes interstitial fibrosis in breast adipose tissue after menopause.
    • Fig. S2. Inguinal depots of adipose tissue feature markers of interstitial fibrosis.
    • Fig. S3. The profibrotic potential of ob/ob ASCs is not due to leptin deficiency.
    • Fig. S4. Obesity-associated ASCs promote fibrotic ECM remodeling in visceral fat.
    • Fig. S5. ECMs deposited by obesity-associated ASCs promote myofibroblast differentiation.
    • Fig. S6. Physicochemical cues of ob/ob ECMs modulate MDA-MB231 behavior.
    • Fig. S7. H&E images of breast tumors with different subtypes.
    • Fig. S8. Caloric restriction decreases fibrosis in mammary fat of obese mice.
    • Fig. S9. Decellularized ECMs do not contain cellular residuals.
    • Table S1. Subtypes, demographics, and desmoplastic grade of lean and obese breast cancer samples.
    • Table S2. Gene ontology data analysis.
    • Table S3. Correlation of YAP/TAZ-regulated genes with obesity-dependent ECM remodeling/inflammation.
    • References (5458)

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