Normalizing glucose levels reconfigures the mammary tumor immune and metabolic microenvironment and decreases metastatic seeding

Abstract

Obesity and diabetes cumulatively create a distinct systemic metabolic pathophysiological syndrome that predisposes patients to several diseases including breast cancer. Moreover, diabetic and obese women with breast cancer show a significant increase in mortality compared to non-obese and/or non-diabetic women. We hypothesized that these metabolic conditions incite an aggressive tumor phenotype by way of impacting tumor cell-autonomous and tumor cell non-autonomous events. In this study, we established a type 2 diabetic mouse model of triple-negative mammary carcinoma and investigated the effect of a glucose lowering therapy, metformin, on the overall tumor characteristics and immune/metabolic microenvironment. Diabetic mice exhibited larger mammary tumors that had increased adiposity with high levels of O-GlcNAc protein post-translational modification. These tumors also presented with a distinct stromal profile characterized by altered collagen architecture, increased infiltration by tumor-permissive M2 macrophages, and early metastatic seeding compared to non-diabetic/lean mice. Metformin treatment of the diabetic/obese mice effectively normalized glucose levels, reconfigured the mammary tumor milieu, and decreased metastatic seeding. Our results highlight the impact of two metabolic complications of obesity and diabetes on tumor cell attributes and showcase metformin's ability to revert tumor cell and stromal changes induced by an obese and diabetic host environment.

Keywords: Breast cancer; Diabetes; Macrophages; Metastasis; O-GlcNAc; Obesity.

Figure 1:

A. Kaplan Meier overall survival curve for diabetic vs non-diabetic breast cancer patients from i2b2 database. Differences in survival probabilities were statistically significant in both groups; Log rank test p < 0.001 B. Nelson–Aalen cumulative hazard estimates for diabetic vs non-diabetic breast cancer patients from i2b2 database. Differences in survival probabilities were statistically significant in both groups p < 0.001

Figure 2:. High-fat diet/low-dose streptozotocin-induces a state of type 2 diabetes in mice that is mitigated by metformin

A. Schematic for in vivo T2DM induction and metformin treatment. B. Average body weight (g) weekly measurements through the course of experiment. The difference is statistically significant between groups (2-way ANOVA, p = 0.0001). C. Blood glucose (mg/dl) weekly measurements through the course of experiment; red arrow represents the start of metformin at week 6. The difference was statistically significant (2-way ANOVA, p = 0.0002). D. Blood glucose measurements (mg/dl) during the intraperitoneal glucose tolerance test (1 g/kg) following fasting for 0, 15, 30, 45, 60, 90 & 120 min. Difference was statistically significant (2-way ANOVA, =<p = 0.0001). E. Area under the curve for glucose was calculated using the Receiver Operator Characteristic curve (ROC). Results are represented as mean ± SD (n = 5–7) (one-way ANOVA, p = 0.04).

Figure 3:. Blood glucose levels correlate with mammary tumor growth

A. Representative bio-luminescence images of mice with mammary tumors at D19 of E0771-FLUC tumors (n = 7 in each group). The color scale represents radiance (Photons/sec/cm³). B. Luminescence total flux (photons/sec) for the mammary fat pad tumors measured at D7, D14 D19 after injection of E0771 cells. Difference is statistically significant (2-way ANOVA, p= 0.03) C. Total Flux (p/s) between the groups at D19 after injection (n=7 per group). Results shown are mean ± SD (One-way ANOVA p=0.04) (unpaired t-test lean vs Ob/D p=0.04, Ob/D vs Ob/D+Met p=0.07). D. Mean tumor volume (mm³) = (length × width²) / 2 at Days 12, 15, 18 & 20 from injection; results are depicted as mean ± SD (n=7 each group) (2-way ANOVA p=ns). E. Graph depicting correlation between luminescence signal (p/s) at D19 and blood glucose measurement (mg/dl) at D14 (R square=0.49) (p=0.02).

Figure 4:. Host obesity and diabetes alter tumor O-GlcNAcylation and adiposity

A. Ob/D tumors showed significantly higher O-GlcNAcylation than the lean tumors (p=<0.0001) Ob/D vs Ob/D+Met (p <0.0001) (one-way ANOVA p <0.0001) O-GlcNAc was evaluated by immunohistochemical staining with RL2 antibody. B. H&E stainingof tumor tissues demonstrates significantly greater amounts of adipose tissue in Ob/D compared to lean and metformin treated mice tumors (p=0.0007, 0.016 respectively). The graph represents quantified percentage of adipose tissues per tumor. C. Tumor sections were assessed for tumor collagen orientation using Picrosirius red staining. Upper panel shows polarized light images, lower panel shows bright field images. Tumors from Ob/D group shows more aligned collagen orientation while lean and Ob/D+Met tumors shows more disoriented networked collagen alignment.

Figure 5:. Metformin blunts the malignant features of mammary tumors from diabetic and obese mice

Tumor sections were assessed for A. Proliferation by PCNA staining, showing significantly lower proliferating cell levels in lean vs Ob/D mice tumors (p=0.005) and in Ob/D+Met vs Ob/D mice tumors (p=0.03). B. Apoptosis by TUNEL staining. Tumors from lean mice had significantly higher levels of apoptotic cells compared to Ob/D group (p=0.027). C. Endothelial cell staining (Griffonia Simplicifolia Lectin I) which shows a significant decrease in vascular density in Ob/D+Met tumors compared to Ob/D tumors (p=0.002). The vascular density was not statistically different between lean and Ob/D tumors. The graph represents quantitated percentage of vascular area per field. D. H&Estaining of mouse lungs showing early metastatic foci in Ob/D groups unlike Lean and Ob/D+Met groups.

Figure 6:. Obesity and diabetes reprogram the tumor immune-microenvironment

A. Tumor sections from lean, Ob/D, and Ob/D+Met mice were assessed for M2 macrophages by immunofluorescence staining for CD206 (M2 polarized macrophages). Tumors from Ob/D mice show a significant increase (p=0.02) in the abundance of CD206+ cells. Metformin treatment decreases this significantly (p=0.02). The graph represents quantification of CD206+ cells/field. B. Tumor sections from lean, Ob/D, and Ob/D+Met mice were assessed for M1 macrophages by dual immunohistochemistry for CD80/68 staining. CD68 marks all macrophages, while CD80 depicts M1-polarized macrophages. Tumors from lean mice show significantly greater numbers of infiltrating CD80+/CD68+ cells relative to Ob/D tumors (p=0.003). Metformin-treated tumors show a significant increase in M1 macrophages relative to Ob/D mice tumors (p=0.008). The graph represents quantification of double positive cells/field. C. The graph represents a ratio of quantified M2/M1 polarized macrophages per field in tumor sections. The results are statistically significant (One-way ANOVA, p=0.01)