Adipocyte-derived endotrophin promotes malignant tumor progression

Abstract

Adipocytes represent a major cell type in the mammary tumor microenvironment and are important for tumor growth. Collagen VI (COL6) is highly expressed in adipose tissue, upregulated in the obese state, and enriched in breast cancer lesions and is a stimulator of mammary tumor growth. Here, we have described a cleavage product of the COL6α3 chain, endotrophin (ETP), which serves as the major mediator of the COL6-mediated tumor effects. ETP augmented fibrosis, angiogenesis, and inflammation through recruitment of macrophages and endothelial cells. Moreover, ETP expression was associated with aggressive mammary tumor growth and high metastatic growth. These effects were partially mediated through enhanced TGF-β signaling, which contributes to tissue fibrosis and epithelial-mesenchymal transition (EMT) of tumor cells. Our results highlight the crucial role of ETP as an obesity-associated factor that promotes tumor growth in the context of adipocyte interactions with tumor and stromal cells.

Figure 1. Regression of tumor growth in Col6a1^–/– mice.

Met-1 cells (0.5 × 10⁶ cells/mouse) were implanted into either FVB WT or Col6a1^–/– (COL6KO) mice (mean ± SEM; n = 5 per group). (A) Tumor volume, determined by caliper measurements. ***P < 0.001 vs. WT, 2-way ANOVA. (B) Tumor weight. **P = 0.0022 vs. WT, unpaired t test.

Figure 2. Expression profiles of ETP.

(A) COL6 is composed of COL6α1, -α2, and -α3 chains. The C5 domain of the α3 chain, cleaved off the COL6 microfilament, is highlighted in red. Shown are amino acid sequences compared between the human and mouse COL6α3-C5 domain. Conserved sequences are highlighted in yellow. (B) Western blots showing abundant secretion of ETP from adipocytes. Conditioned media from 3T3-L1 preadipocytes and fully differentiated adipocytes were subjected to Western blotting using α-mETP and α-COL6. Arrows indicate the secreted form of ETP. (C) ETP immunostaining of mammary gland tissues from obese animals, ob/ob and db/db mice, compared with lean controls (n = 5 per group). Arrows indicate crown structure. Scale bars: 50 μm. (D and E) holo-COL6 and ETP immunostaining of tumor tissues from 9-week-old PyMT mice with α-COL6 (D) and α-mETP antibody (E), respectively. Lung tissues from 13-week-old PyMT mice were used for metastasized tumor lesions (F). Scale bars: 50 μm. (G) α-COL6– and α-ETP–positive staining area in tumor or stroma for the primary tumors. Data are mean ± SEM of multiple fields in n = 5 per group. ***P < 0.001 vs. tumor, unpaired t test.

Figure 3. ETP levels in human cancer specimens and its target tissues.

(A and B) Human cancer tissues compared with those of benign tissues were immunostained with human ETP–specific polyclonal antibody. Human samples for breast cancer (A) and colon cancer (B) were analyzed. Scale bars: 25 μm. (C and D) Whole body in vivo imaging of injected ETP. IRD-800 fluorescence–labeled ETP protein (10 μg) was intravenously injected into WT, 8-week-old PyMT, and 10-week-old PyMT mice by tail vein. (C) ETP levels were visualized by the Licor Infrared Scanner 10–90 minutes after injection. Arrows indicate mammary tumors. L, liver; B, bladder; T, tumor. IgG was used as a negative control. (D) Tissues were excised 2 hours after injection, and ETP-positive fluorescence signals were determined with a Licor Infrared Scanner. Quantified values were normalized to total area and represented as percentage of WT. ***P < 0.001.

Figure 4. Transgenic mice expressing ETP under the MMTV promoter.

(A) ETP immunostaining showing strong ETP positive signal in mammary ductal epithelium in the transgenic mice lines (ETP low and high), but not WT and Col6a1^–/– mice. Scale bars: 50 μm. (B and C) Antiapoptotic effects of ETP. (B) Apoptosis for mammary epithelial cells during involution was determined by TUNEL assay on mammary glands of WT, ETP, and Col6a1^–/– mice 2 days after forced weaning. Arrows indicate TUNEL-positive apoptotic cells. Scale bars: 50 μm. (C) Quantification of TUNEL-positive cells, represented as mean ± SEM (multiple images, n = 3 per group). ***P < 0.001, *P < 0.05 vs. WT, unpaired t test. (D and E) Promitotic effects of ETP. High ETP expressers (32 weeks old) spontaneously developed tumors. (D) Whole body image (left; arrows indicate tumors) and H&E staining of mammary gland (middle) and lung (right) tissue. Boxed regions are shown at higher magnification below. Scale bars: 200 μm. (E) Cell proliferation was determined by Ki67 staining with mammary glands of 32-week-old ETP high-expressing and WT mice. Scale bars: 50 μm.

Figure 5. ETP augments primary tumor growth and pulmonary metastasis in the background of PyMT mice.

(A) ETP immunostaining, with a high ETP-positive signal in tumor tissues from 12-week-old PyMT and PyMT/ETP mice. Scale bars: 50 μm. Intensity of ETP staining was quantified and represented as mean ± SEM (n = 5 per group). *P = 0.02 vs. PyMT, unpaired t test. (B) Whole-mount staining of mammary gland tissues from 8-week-old PyMT and PyMT/ETP mice, with early neoplastic lesion areas increased by ETP. (C) Tumor volume was determined by weekly caliper measurements from PyMT (n = 35) and PyMT/ETP (n = 38) mice. Results are represented as mean ± SEM. P = NS, 2-way ANOVA. (D and E) ETP augmented pulmonary metastasis. (D) Pulmonary metastatic growth was determined by measuring the tumor incidence in lung tissues (8- to 17-week-old, n = 22–25 per group). H&E-stained preparations for lung tissues were used for analysis. Shown is percent metastasis-free mice over time. *P = 0.025, log-rank test. (E) Representative H&E stain for lung tissues showing the degree of pulmonary metastasis in 15- and 17-week-old PyMT and PyMT/ETP mice. (F) Representative whole-body images for tumor burden. Tumor volume for 13-week-old FP635/PyMT and FP635/PyMT/ETP mice was monitored by IVIS fluorescence scanner. Metastatic burden was determined by fluorescence signals in lung tissues. Quantified results are represented as mean ± SEM (n = 8–9 per group). *P = 0.0117, **P = 0.0011 vs. PyMT, unpaired t test.

Figure 6. Histological analysis for tumor tissues of PyMT/ETP versus PyMT mice.

(A) Proliferation indices were determined by immunostaining with Ki67. Quantified results represent mean ± SEM (n = 5 per group). P = NS vs. PyMT, unpaired t test. (B) Fibrosis indices were determined by Masson’s Trichrome C stain. Percent fibrotic area over the tumor lesions was quantified. Data represent mean ± SEM (n = 5 per group). **P = 0.01 vs. PyMT, unpaired t test. Arrows indicate collagen fibrils. (C) Functional blood vessel areas were determined by lectin perfusion. Podoplanin (lymphangiogenesis marker) and DAPI (nucleus) were costained. Quantified results represent mean ± SEM (n = 5 per group). **P = 0.003 vs. PyMT, unpaired t test. (D) Hypoxia was determined by pimonidazole-HCl injection. Hypoxic lesions were stained in dark brown. Quantified results represent mean ± SEM (n = 5 per group). ***P = 0.0007 vs. PyMT, unpaired t test. (E–H) Total RNA was prepared from the tumor tissues from PyMT/ETP and PyMT mice. mRNA levels for the genes responsible for fibrosis and EMT (E and F), angiogenesis and lymphangiogenesis (G), and inflammation (H) were determined by qRT-PCR. mRNA levels were normalized with β-actin and represented as mean ± SEM (n = 8 per group). Relative values of each gene are represented as fold change relative to PyMT. *P < 0.05, ***P < 0.001 vs. PyMT, 2-way ANOVA. Scale bars: 50 μm (A–C); 100 μm (D). Insets in A are enlarged ×5.

Figure 7. ETP augments metastasis through enforcing TGF-β–dependent EMT.

(A) E-cadherin immunostaining for tumor tissues from PyMT and PyMT/ETP. (B and C) SBE-luciferase reporter assay. See Supplemental Methods for details. Data represent fold increase (3 independent experiments). **P < 0.01, *P < 0.05, 2-way ANOVA. pRA-ctrl, empty; pRA-sETP, secretion form; pRA-ETP, intracellular form. (D–G) Allografts of Met-1 cells in the presence of either ETP (20 ng/plug) or PBS mixed with 1D11 or IgG (10 μg/plug) within a Matrigel plug. 10 days after implantation, additional 1D11 or IgG (100 μg) was i.p. injected once a week during tumor progression. (D) Tumor volumes represent means ± SEM (n = 5 per group). *P < 0.05, 2-way ANOVA. (E) H&E staining. The ratio of stromal area in tumor tissues was quantified. Data represent mean ± SEM (n = 5 per group). *P < 0.05, unpaired t test. T; tumor and S; stroma. (F) Fibrosis was determined by Masson’s Trichrome C stain. Data represent mean ± SEM (n = 5 per group). **P < 0.01, ***P < 0.001, unpaired t test. (G) Western blotting for EMT markers E-cadherin, vimentin, and α-SMA. β-actin, loading control. Data represent fold increase (n = 5 per group). *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t test. (H) Control and ETP⁺-cancer cells were isolated from FP635/PyMT and FP635/PyMT/ETP mice and conveyed into WT mice by tail vein injection (0.5 × 10⁶ cells/mouse). Either IgG or 1D11 (100 μg) was i.p. injected every 5 days. 20 days post injection, metastasized cancer cells in the lung tissues were determined by fluorescence intensity. Data represent fold increase (n = 3–4 per group). **P < 0.01, *P < 0.05, unpaired t test. Scale bars: 20 μm (A); 50 μm (E); 100 μm (F).

Figure 8. ETP reconstitution into Col6a1^–/–-cancer cells rescues tumor growth.

Cancer cells isolated from PyMT, PyMT/Col6a1^–/–, and PyMT/Col6a1^–/–/ETP mice were mixed with the same volume of Matrigel (50 μl) and implanted into WT mice (0.5 × 10⁶ cells/mouse). (A) Tumor growth (mean ± SEM; n = 5–8 per group), determined by caliper measurements. *P = 0.024 vs. Col6a1^–/–, unpaired t test. (B) Representative image 35 days after implantation. (C) Tumor weight (mean ± SEM; n = 5–8 per group). *P = 0.023 vs. Col6a1^–/–, unpaired t test. (D–H) Tissue fibrosis was determined by Masson’s Trichrome C stain (D), and EMT was determined by immunostaining for E-cadherin (E), vimentin (F), α-SMA (G), and FSP-1 (H). Cytokeratin (epithelial cells) and DAPI (nucleus) were costained in G and H. Positive staining areas were quantified and represented as fold increase over Ctrl-tumors (multiple images, n = 5 per group). *P < 0.05, ***P < 0.001 vs. Col6a1^–/–, unpaired t test. Scale bars: 50 μm (E and F); 100 μm (D, G, and H).

Figure 9. ETP acts as a chemokine augmenting tumor growth.

(A) Tumor growth significantly increased in ETP⁺-tumor tissue allograft into WT mice. Tumor volume was determined 1 month after implantation (representative images). Data represent mean ± SEM (n = 5 per group). ***P < 0.001, unpaired t test. (B) In vivo Matrigel bioassay. Matrigel (50 μl) was mixed with ETP (100 ng/plug) or PBS and implanted into WT mice in the presence of IgG, 1D11, or 10B6 (20 μg/plug). 2 days after implantation, plugs were excised and stained for H&E. Scale bars: 100 μm. (C–L) Cancer cells were plated in the bottom chamber 1 day prior to seeding MS-1 cells (5 × 10⁵ cells/well) and macrophages (1 × 10⁵ cells/well) atop the membrane chamber in Transwell and incubated for 18–24 hours (C). (D–K) Representative images of multiple independent experiments. Scale bars: 100 μm. (L) Quantitation (mean ± SEM; n = 3 per group). ***P < 0.001, **P < 0.05, *P < 0.01, unpaired t test. (M) MS-1 migration assay. MS-1 cells (5 × 10⁵ cells/well) were plated atop the chamber in Transwell and incubated for 24 hours. Chemotaxis was set up by following cell migration from DMEM/serum-free to DMEM/2% FBS/PBS or DMEM/2% FBS/ETP protein (1 μg/well). Images are representative of multiple independent experiments. Data represent mean ± SEM (n = 3 per group). ***P = 0.008, unpaired t test. Scale bars: 100 μm.

Figure 10. 10B6 blocks ETP-mediated stromal expansion in vivo.

(A) 10B6 (200 μg/mouse) was i.p. injected twice weekly into PyMT mice from 9 to 13 weeks of age. Tumor growth (mean ± SEM; n = 4–6 per group) was determined by weekly caliper measurements. *P < 0.05, **P < 0.01, ***P < 0.001 vs. IgG control, 2-way ANOVA. (B) Primary mammary epithelial cancer cells were isolated from 12-week-old FP635/PyMT and FP635/PyMT/ETP mice and implanted into WT recipients with the same volume of Matrigel. For the 10B6 group, 10B6 was added in a Matrigel plug (10 μg/plug) admixed with ETP⁺-cancer cells (i.e., ETP⁺/10B6). A representative whole-body image was acquired 25 days after implantation using IVIS fluorescence scanner. Artificial color indicates fluorescence signal intensity accounts for tumor volume (AU). Quantitative results are represented as mean ± SEM (n = 3 per group). *P < 0.05, unpaired t test. (C–I) 6 weeks after implantation, tumor tissues were excised from Ctrl-, ETP⁺-, and ETP⁺/10B6-cancer cells allografted mice and stained for H&E (C), Masson’s Trichrome C (D), α-SMA (E), FSP-1 (F), CD31 (G), F4/80 (H), and Ki67 (I). Quantified results in D–I are mean ± SEM (multiple images, n = 3 per group). *P < 0.05, **P < 0.01, unpaired t test. Scale bars: 50 μm. (J) Working model for ETP in mammary tumor progression.