Adipocyte mesenchymal transition contributes to mammary tumor progression

Abstract

Obesity is associated with increased cancer incidence and progression. However, the relationship between adiposity and cancer remains poorly understood at the mechanistic level. Here, we report that adipocytes from tumor-invasive mammary fat undergo de-differentiation to fibroblast-like precursor cells during tumor progression and integrate into the tumor microenvironment. Single-cell sequencing reveals that these de-differentiated adipocytes lose their original identities and transform into multiple cell types, including myofibroblast- and macrophage-like cells, with their characteristic features involved in immune response, inflammation, and extracellular matrix remodeling. The de-differentiated cells are metabolically distinct from tumor-associated fibroblasts but exhibit comparable effects on tumor cell proliferation. Inducing de-differentiation by Xbp1s overexpression promotes tumor progression despite lower adiposity. In contrast, promoting lipid-storage capacity in adipocytes through MitoNEET overexpression curbs tumor growth despite greater adiposity. Collectively, the metabolic interplay between tumor cells and adipocytes induces adipocyte mesenchymal transition and contributes to reconfigure the stroma into a more tumor-friendly microenvironment.

Keywords: CP: Cancer; CP: Metabolism; adipocyte; breast cancer; de-differentiation; obesity.

Figure 1.. Adipocytes undergo de-differentiation during mammary tumor infiltration into the stroma

(A) H&E staining reveals smaller adipocytes upon mammary tumor invasion in patients and PyMT mice. Scale bar, 300 μm. (B) Immunofluorescent staining of adipocyte marker Perilipin (red) and endothelial cell marker CD31 (green) for patient and mouse tumor samples (n = 3). Scale bar, 50 μm. (C and D) Frequency and average adipocyte size in regions distant (>500 μm) and adjacent (<500 μm) to the tumor lesion in patient samples (n = 9). (E) Down-regulation of adipocyte marker genes upon tumor invasion in mammary adipose tissues from PyMT-mice (n = 12). “Normal fat” indicates corresponsive fat from mice without tumor, “tumor fat” is distal fat from PyMT-tumor, “tumor edge” is fat connected with tumor, and “tumor interior” is tissues from tumor core. (F) Scheme of PyMTChaser and AlloChaser mice. (G) Both PyMTChaser (a-c) and AlloChaser (d-f) reveal mammary adipocyte de-differentiation upon tumor infiltration (n = 3). (a, d) No dox control; (b, e) distal fat from the tumor lesion; (c, f) tumor adjacent region. Arrows indicate de-differentiated EGFP+/Perilipin− cells with fibroblast-like morphology. Scale bar, 300 μm. Each dot represents an individual sample. Data are presented as mean ± SEM. Unpaired Student’s t test was used in (D), and one-way ANOVA with Holm-Sidak’s multiple comparisons test was used in (E). ***p < 0.001.

Figure 2.. Single cell-RNA sequencing reveals adipocyte mesenchymal transition during mammary tumor progression

EGFP-labeled de-differentiated adipocytes from PyMTChaser and AlloChaser mice were isolated by FACS and subjected to scRNA-seq. (A and B) T-distributed stochastic neighbor embedding (t-SNE) plots. Clustering of PyMT-EGFP + or allograft (Allo)-EGFP + cells, with mean number of genes per cell equals 1, 163 or 2, 173, respectively. (C) Representative adipocyte early differentiation marker genes. (D) Representative mesenchymal stem cell/adipocyte precursor marker genes. The y axis is the log-scale normalized read count.

Figure 3.. Mammary tumor-induced de-differentiated adipocytes contribute to tumor microenvironment concerning inflammation and ECM remodeling

(A) Heat map of top 20 genes across different cell groups from allograft (Allo)-EGFP + cells. (B) Gene ontology (GO) pathway enrichment analysis. (C) Representative macrophage and fibroblast markers genes. The y-axis is the log-scale normalized read count. (D) Co-staining of α-SMA (green), EGFP (red), and Perilipin (magenta) of PyMTChaser-tumor. Scale bar, 50 μm. (E) Co-staining of EGFP (green), Mac2 (red), and Perilipin (magenta) of PyMTChaser-tumor. ‘*’ indicates macrophage-like cells (EGFP+/Perilipin−/Mac2+); ‘^a’ indicates adipocytes (EGFP+/Perilipin+/Mac2−); ‘^m’ indicates macrophages (EGFP−/Perilipin−/Mac2+). Scale bar, 50 μm. (F) mRNA levels of genes related to ECM remodeling in cultures of de-differentiated adipocytes (EGFP+, tdTomato−) and tumor-associated fibroblasts (EGFP−, tdTomato+) isolated from AlloChaser-tumor (n = 6). (G) mRNA levels of cytokines/chemokines in cultures of EGFP+ and EGFP− cells treated with vehicle (PBS) or LPS (100 ng/mL) for 3 h (n = 6). (H) Trichrome, collagen (Col), and Mac2 staining indicates enhanced fibrosis, collagen production and inflammation in the mammary fat at a distance from the tumor lesion in patients (n = 9). Scale bar, 100 μm for trichrome staining and 50 μm for Col and Mac2 staining. (I) Average fibrous area and thickness in distant and adjacent regions from the tumor lesion in patients (n = 9). Each dot represents an individual sample. Data are presented as mean ± SEM. Unpaired Student’s t test was used in (F), (G), and (I). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4.. Distinct metabolic characteristics of tumor-induced de-differentiated adipocytes

(A and B) Interactive PCA analysis and heatmap of metabolite profile in the culture medium of de-differentiated adipocytes (EGFP+, tdTomato−) and tumor-associated fibroblasts (EGFP−, tdTomato+) isolated from AlloChaser-tumor (n = 3). At fasted condition, medium was analyzed in the absence of serum for 6 hr. (C and D) The metabolome map of matched metabolic pathways in fed and fasted condition according to the p values from the enrichment analysis and impact values from the pathway topology analysis. Colors varying from yellow to red indicate the metabolites are in enrichment analysis with different levels of significance. (E and F) Enhance glycolysis in EGFP + cells (n = 6–7). (G–I) EGFP + cells exhibit similar basal and maximal oxygen consumption rate (OCR) but have higher coupling efficiency (n = 6–7). Conditioned medium (CM) from EGFP + cells and EGFP− cells shows similar effects on the proliferation of various human (J) and mouse (K) breast cancer cells (n = 4). Data are presented as mean ± SEM. Unpaired Student’s t test was used in (E)-(I); two-way ANOVA with Holm-Sidak’s multiple comparisons test was used in (J) and (K). *p < 0.05; **p < 0.01.

Figure 5.. Adipocyte de-differentiation promotes mammary tumor growth

(A and B) Ad-Xbp1s mice display lower adiposity and smaller size of different fat pads (n = 5). (C) Up-regulation of total Xbp1 (Xbp1t) and spliced Xbp1 (Xbp1s) upon tumor invasion (n = 6). (D) Downregulation of adipocyte marker genes in mammary fat from Ad-Xbp1s mice (n = 8–9). (E) Scheme of the Xbp1sChaser mice. (F) Two weeks of Xbp1s induction causes adipocyte de-differentiation. Scale bar, 50 μm. (G and H) Increased total tumor mass in Ad-Xbp1s-PyMT mice in C57B/L6 (n = 9–12) or FVB background (n = 15–16) at 23 or 14 weeks old, respectively. (I) H&E staining indicates mammary adipose tissue is largely engulfed by tumor in Ad-Xbp1-PyMT mice (n = 3). Scale bar, 200 μm. (J) Ad-MitoNEET-PyMT mice promotes triglycerides tolerance (n = 5–7). (K) Increased fat mass percentage in Ad-MitoNEET-PyMT mice (n = 5–7). (L and M) Decrease in total tumor mass at 23-week-old Ad-MitoNEET-PyMT mice fed with chow (n = 18–19) or HFD (n = 11–12), respectively. (N) H&E staining indicates mammary adipose tissue is largely preserved in Ad-Xbp1-PyMT mice (n = 3). Scale bar, 200 μm. Each dot represents an individual mouse. Data are presented as mean ± SEM. Unpaired Student’s t test was used in (A), (D), (G), (H), and (K)–(M); one-way ANOVA or two-way ANOVA with Holm-Sidak’s multiple comparisons test was used in (C) or (J), respectively. *, ^#, or ^&p < 0.05; **p < 0.01; ***or ^###p < 0.001.