Insulin Resistance Increases TNBC Aggressiveness and Brain Metastasis via Adipocyte-Derived Exosomes

Abstract

Patients with triple-negative breast cancer (TNBC) and comorbid type 2 diabetes (T2D), characterized by insulin resistance of adipose tissue, have a higher risk of metastasis and shorter survival. Adipocytes are the main nonmalignant cells of the breast tumor microenvironment (TME). However, adipocyte metabolism is usually ignored in oncology, and the mechanisms that couple T2D to TNBC outcomes are poorly understood. In this study, we hypothesized that exosomes, small vesicles secreted by TME breast adipocytes, drive epithelial-to-mesenchymal transition (EMT) and metastasis in TNBC via miRNAs. Exosomes were purified from conditioned media of 3T3-L1 mature adipocytes, either insulin-sensitive (IS) or insulin-resistant (IR). Murine 4T1 cells, a TNBC model, were treated with exosomes in vitro (72 hours). EMT, proliferation, and angiogenesis were elevated in IR versus control and IS. Brain metastases showed more mesenchymal morphology and EMT enrichment in the IR group. MiR-145a-3p is highly differentially expressed between IS and IR and potentially regulates metastasis.

Implications: IR adipocyte exosomes modify the TME, enhance EMT, and promote brain metastasis-likely via miRNA pathways-suggesting that metabolic diseases such as T2D foster a prometastatic TME, reducing survival and warranting close monitoring and potential metabolic interventions in patients with TNBC and T2D.

Figure 1.. Exosomes derived from IR adipocytes increase transcription of EMT genes and migration ability of 4T1 cells in vitro.

A. 4T1 cells were treated with IS and IR adipocyte-derived exosomes compared to control (no treatment); cellular mRNA was analyzed by EMT array. B. Ingenuity pathway analysis of differentially expressed EMT array genes. C. Representative images of IHC staining with osteopontin and E-cadherin antibodies in Control, IS and IR groups, scale bar represents D. Representative images of migration assay of exosome-treated 4T1 cells; TGFB, TGF-β positive control at 5ng/ml for EMT induction. D. Quantification of C (n=4, *, p<0.05, **, P<0.01). E. Pathway enrichment analysis of differentially expressed genes; pathways are compared by exosome status. Bubble size indicates number of genes. Color bar indicates false discovery rate (FDR); red represents lower value, blue represents higher value. F. Survival analysis of breast cancer patients in Metabric Basal data using genes differentially expressed between IS and IR states.

Figure 2.. Expression of EMT, proliferation and angiogenesis marker proteins in primary tumor in vivo.

A. Timeline of experimental procedure. B. Representative images of IHC staining with Vimentin, Ki67 and Cd31 antibodies in Control, IS and IR groups, scale bar represents 100 μm in Ki67 staining and 50 μm in Vimentin and Cd31 staining. C-E. Quantification of vimentin (C), Ki67 (D), Cd31 (E) antibody staining. (n=4, *, p<0.05, **, P<0.01)

Figure 3.. Quantification and morphology analysis of clonogenic assay from brain metastases.

A. Representative images of stained colonies (top) and morphology of cells in colonies (bottom) B. Quantification of clonogenic assay from brain metastases (n=5). C-I. Quantification of area per cell (C), equivalent diameter (D), solidity (E), compactness (F), eccentricity (G), extent (H) and form factor (I) from A (n>=4 per group, *, p<0.05, **, P<0.01, ***, p<0.001, ****p<0.000)

Figure 4.. Transcriptome analysis of brain metastatic cells.

A. Differentially expressed genes across Control, IS and IR group. B. Pathway enrichment analysis using genes upregulated in IR comparing to IS. C. Pathway enrichment analysis using genes upregulated in Control comparing to IS. D. Correlation matrix across all differentially expressed gene sets. The row and column labels indicate the differentially expressed gene sets. The Spearman correlation was calculated for each pair of results. The absolute value of these correlations is plotted in the heatmap. E. Survival analysis of breast cancer patients in METABRIC Basal data using differentially expressed genes between IS and IR. F. Survival analysis of breast cancer patients in TCGA data using differentially expressed genes between IS and IR.

Figure 5.. Exosomal miR-145a-3p is functional in migration assays.

A. Representative images of migration assay with 4T1 cells treated with exosomal miRNA. B. Quantification of migration assay. C. miR-145a-3p is differentially expressed between IS and IR adipocyte exosomes. D. Top table, Trp53bp2 and Ocln has complementary sequence of miR-145a-3p in the 3’-UTR regions, based on TargetScan prediction. Bottom table, average counts of Trp53bp2 and Ocln, fold change, and adjusted p-value when comparing miR-145a-3p treated group with scrRNA control from bulk RNA sequencing. E. Representative images of migration assay treated with scrambled miRNA (scrRNA) control and miR-145a-3p for 3 days and 1 day without treatment. F. Quantification and statistical analysis of E (n=4, *, p<0.05, **, P<0.01, ***, p<0.001). G. Differentially expressed gene between scrRNA and miR-145a-3p treated group. H. Pathway enrichment analysis using genes downregulated in miR-145a-3p treated group VS scrRNA group. I. Quantification of the number of colonies in brain clonogenic assay (n>=3, *, p<0.05)