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Decellularized extracellular matrix scaffolds identify full-length collagen VI as a driver of breast cancer cell invasion in obesity and metastasis

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Science Advances  21 Oct 2020:
Vol. 6, no. 43, eabc3175
DOI: 10.1126/sciadv.abc3175
  • Fig. 1 Reseeding tumor cells onto dECM scaffolds reveals whole-tissue ECM effects on tumor cell proliferation and invasion.

    (A) MDA-MB-231 xenografts (231-tumor) and tumors from PyMT-MMTV (PyMT-tumor) mice were used. (B) Schematic of experimental pipeline. (C) H&E of mammary gland, 231-tumors, and PyMT-tumors before and after decellularization (scale bar, 200 μm). (D) Representative Western blot of 231-tumor before and after decellularization for cellular compartments. (E) Photo of PyMT-tumor before and after decellularization Photo credit: Jackson Fatherree, Tufts University. (F) Representative tracks of 231-GFP or PyMT-GFP cells seeded on dECM from mammary gland (MG), 231-tumors, or PyMT-tumor. Each line represents the track of a cell over 16 hours. (G) Percentage of 231-GFP cells proliferating on healthy mammary gland or 231-tumor. (H) Cell migration speed of 231-GFP seeded on dECM from mammary gland and 231-tumors. (I) Percentage of PyMT-GFP cells proliferating on mammary gland or PyMT-tumor scaffolds. (J) Cell migration speed of PyMT-GFP seeded on dECM from mammary gland and PyMT-tumors. For proliferation, data point is a field of view. For cell migration, each point is the average speed of a cell over 16 hours. Data obtained from at least three dECM scaffolds from three mice show mean ± SEM. Significance was determined by unpaired two-tailed Mann Whitney t test. **P < 0.01 and ***P < 0.005. ns, not significant.

  • Fig. 2 ECM from the mammary gland of obese mice increases cell invasion and overlaps with breast tumor ECM composition.

    (A) Diet-induced obesity model in female C57BL/6 mice. (B) dECM of lean and obese mammary glands by decellularization was used for reseeding and proteomics. Representative tracks (C) and quantification (D) of cell migration speed of 231-GFP cells seeded on dECM from lean and obese mammary gland. Each point represents the average speed of a cell over 16 hours. (E) Percentage of proliferating 231-GFP cells on ECM scaffolds from lean and obese mammary gland. Data show mean ± SEM, obtained from at least three dECM scaffolds from three mice. Significance was determined by unpaired two-tailed Mann-Whitney t test. ***P ≤ 0.005. (F) Average log2 fold change (FC) differences of 68 ECM proteins identified by proteomics of the ECM fraction and principal components analysis (G). (H) ECM proteins found enriched in obese mammary gland were overlapped with proteins enriched in 4T1 orthotopic syngeneic mammary (9) tumor and in MDA-MB-231-LM2 highly metastatic (10) xenografts in a Venn diagram. (I) List of nine matrisome proteins present in obese mammary gland ECM, 4T1 tumor ECM, and metastatic LM2 tumors.

  • Fig. 3 Collagen VI, ECM protein up-regulated in obese and tumor ECM, drives TNBC cell adhesion, migration, and invasion.

    (A) Representative images from an adhesion assay of MDA-MB-231 cells seeded onto wells coated with FN (20 μg/ml), collagen VI, laminin, or elastin (scale bar, 50 μm). (B) Representative rose plots of cell migration tracks of MDA-MB-231 cells on ECM substrates, with each colored line representing a single cell track over 16-hour time course. (C) 2D migration speed of MDA-MB-231 cells seeded on glass, FN (20 μg/ml), collagen VI, laminin, or elastin. (D) 2D migration speed of MDA-MB-468 cells seeded on wells coated with PBS (n = 111), FN (20 μg/ml) (n = 133), collagen VI, laminin, or elastin. Each point represents the average speed of a cell over 16 hours. (E) Representative images of spheroid 231-GFP cells after 5 days within medium without ECM, in collagen I (1 mg/ml) and in a collagen I + collagen VI (50 μg/ml) matrix (scale bar, 300 μm). (F) Quantification of fold change in 231-GFP spheroid area after 5 days. Each dot represents an individual spheroid. For all graphs in this figure, data were pooled from at least three independent experiments and show mean ± SEM. Significance was determined by a nonparametric Kruskal-Wallis test with Dunn’s multiple testing correction. *P < 0.05, **P < 0.01, and ***P < 0.005.

  • Fig. 4 Collagen VI is enriched in obese mammary gland ECM, and TNBC patients with high tumor collagen VI have lower overall survival.

    (A) Immunostaining of mammary gland sections from lean and obese C57BL/6 mice stained for collagen VI (green) and nuclei (blue) (scale bar, 100 μm). (B) Collagen VI intensity relative to DAPI signal shows increased abundance in obese ECM. Data are from more than 75 fields of view from at least three mammary gland tissues from three lean and obese mice. Unpaired two-tailed Mann-Whitney test was performed. Data represent mean ± SEM. ***P ≤ 0.005. (C) Kaplan-Meier curve of human TNBC breast cancer patients with low versus high mRNA of the most abundant form of collagen VI (COL6A1/COL6A2/COL6A3); data from (38). (D) Collagen VI (green), perilipin (red), and DAPI (blue) staining of a human TNBC biopsy samples from an average weight TNBC patient (BMI = 23.66), an overweight TNBC patient (BMI = 26.26), and an obese TNBC patient (BMI = 34.04) (scale bar, 100 μm). Correlation between BMI and collagen VI intensity relative to region area for the tumor area (as identified by DAPI staining) (E), adipose tissue (as identified by perilipin) (F), and peritumoral region (G) for nine patients of ranging BMI. a.u., arbitrary units.

  • Fig. 5 Collagen VI–driven TNBC cell migration is mediated by NG2/EGFR cross-talk and MAPK signaling.

    (A) Schematic of collagen VI signaling. (B) Migration speed of 231-GFP cells on collagen VI (20 μg/ml) treated with IgG2a, NG2 targeting antibody (NG2ab), or β1 integrin inhibitory antibody (β1ab). (C) Migration speed of 231-GFP cells on collagen VI (20 μg/ml) treated with DMSO, lapatinib (EGFRi, 10 μM), and lapatinib/NG2ab (NG2ab + EGFRi) combination. Significance was determined by a Kruskal-Wallis test with Dunn’s multiple testing correction, with *P < 0.05, **P < 0.01, and ***P < 0.005. (D) Immunostaining for phalloidin and phospho-p44/42ERK (Thr202/Tyr204) in MDA-MB-231 cells seeded on collagen VI (20 μg/ml) and treated with IgG and DMSO or NG2ab + EGFRi. Quantification of MDA-MB-231 cell adhesion (E) and phospho-p44/42 MAPK (F) in cells treated with NG2ab, EGFRi, or combination, relative to vehicle control. (G) Representative images of spheroid 231-GFP cells after 5 days in collagen I + collagen VI (50 μg/ml) matrix treated with DMSO, NG2ab/EGFRi, or trametinib (MAPKi) (scale bar, 300 μm). (H) Quantification of fold change in 231-GFP spheroid area after 5 days. Each dot represents an individual spheroid. For all graphs, data were pooled from at least three independent experiments and show mean ± SEM. Significance was determined by Kruskal-Wallis test with Dunn’s multiple testing correction. *P < 0.05, **P < 0.01, and ***P < 0.005.

  • Fig. 6 Inhibition of collagen VI–driven signaling reduces TNBC cell migration on dECM derived from the mammary gland of obese and tumor-bearing mice.

    (A) Representative rose plots of 231-GFP cell migration seeded on dECM from obese mammary gland and treated with IgG2a or NG2ab, where each colored line represents the tracked movement of a cell over the time course (16 hours). (B) Cell migration speed of 231-GFP cells seeded on mammary gland ECM derived from lean mice, obese mice, and tumor-bearing mice treated with IgG2a or NG2ab. Graphs show mean ± SEM, with dot representing data from a single cell. Unpaired two-tailed Mann-Whitney tests were performed between respective IgG2a control and NG2ab conditions. Data were obtained from at least three different ECM scaffolds from three different mice, with an average of 108 total cells per condition. Significance by t test, with ***P < 0.005. (C) Representative rose plots of PyMT-GFP cell migration seeded on dECM from PyMT-tumors and treated with vehicle, NG2ab+ EGFRi or MAPKi, where each colored line represents the tracked movement of a cell over the time course (16 hrs) (D) Cell migration speed of PyMT-GFP cells seeded on mammary gland ECM derived from PyMT-mice treated with vehicle, NG2ab+ EGFRi or MAPKi. Data obtained from at least 3 different ECM scaffolds from 3 different mice, statistic by one-way Anova, with ***P < 0.005.

Supplementary Materials

  • Supplementary Materials

    Decellularized extracellular matrix scaffolds identify full-length collagen VI as a driver of breast cancer cell invasion in obesity and metastasis

    Andrew L. Wishart, Sydney J. Conner, Justinne R. Guarin, Jackson P. Fatherree, Yifan Peng, Rachel A. McGinn, Rebecca Crews, Stephen P. Naber, Martin Hunter, Andrew S. Greenberg, Madeleine J. Oudin

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