High-Fat, High-Calorie Diet Enhances Mammary Carcinogenesis and Local Inflammation in MMTV-PyMT Mouse Model of Breast Cancer

Abstract

Epidemiological studies provide strong evidence that obesity and the associated adipose tissue inflammation are risk factors for breast cancer; however, the molecular mechanisms are poorly understood. We evaluated the effect of a high-fat/high-calorie diet on mammary carcinogenesis in the immunocompetent MMTV-PyMT murine model. Four-week old female mice (20/group) were randomized to receive either a high-fat (HF; 60% kcal as fat) or a low-fat (LF; 16% kcal) diet for eight weeks. Body weights were determined, and tumor volumes measured by ultrasound, each week. At necropsy, the tumors and abdominal visceral fat were weighed and plasma collected. The primary mammary tumors, adjacent mammary fat, and lungs were preserved for histological and immunohistochemical examination and quantification of infiltrating macrophages, crown-like structure (CLS) formation, and microvessel density. The body weight gains, visceral fat weights, the primary mammary tumor growth rates and terminal weights, were all significantly greater in the HF-fed mice. Adipose tissue inflammation in the HF group was indicated by hepatic steatosis, pronounced macrophage infiltration and CLS formation, and elevations in plasma monocyte chemoattractant protein-1 (MCP-1), leptin and proinflammatory cytokine concentrations. HF intake was also associated with higher tumor-associated microvascular density and the proangiogenic factor MCP-1. This study provides preclinical evidence in a spontaneous model of breast cancer that mammary adipose tissue inflammation induced by diet, enhances the recruitment of macrophages and increases tumor vascular density suggesting a role for obesity in creating a microenvironment favorable for angiogenesis in the progression of breast cancer.

Keywords: angiogenesis; high-fat diet; inflammation; obesity; tumor progression.

Figure 1

Body composition and weight gain in MMTV-PyMT mice following short-term (8 weeks) dietary intervention with high-fat (HF) and low-fat (LF) diets, n = 20/group. (A) Visceral adipose tissue as % body weight for the HF and LF-fed groups (p = 0.002); (B) Body weight gain over the 8 week observation period for HF [■] and LF [▲] fed mice. The greater weight gain in the HF group was statistically significant (p = 0.005). (C) Body weight gain adjusted for tumor weight over the 8 week diet period for HF [■] and LF [▲] fed mice was significant (p < 0.05).

Figure 2

Period of tumor latency and primary tumor volume as measured by ultrasound. (A) Lack of effect of high-fat HF [■] and low-fat LF [▲] diets on latency for mammary tumor development, n = 20/group; (B) Primary tumor as measured in the 4th and 5th mammary for each mouse over the 8 week observation period for HF [■] and LF [▲] fed mice, n = 20 HF and n = 19 LF mice. The overall greater growth rate for the HF group was statistically significant (p = 0.0495). (C) Lack of effect on the total number of lung metastases over the 8 week observation period for LF and HF diet, n = 18/group.

Figure 3

Macrophage infiltration into peritumoral adipose tissue and adjacent tumor mass. The macrophages were identified by their immunoreactivity with an anti-CD68 antibody and counted as described in the text for HF and LF fed mice. (A/B) Infiltration was significantly greater in the tumors from the HF-fed mice, n = 20/group compared to LF, n = 19/group (p = 0.0012); (C) Quantitation of tumor-associated macrophages at 4 and 8 weeks after diet consumption; (D/E) Representative peritumoral adipose tissues from HF and LF-fed mice showing crown-like structures (CLS) formed by the localization of macrophages around a single adipocyte. Their presence was increased in tissues from mice fed a HF diet; (F) Quantitation of crown-like structures at 4 and 8 weeks after diet consumption, n = 9–10 mice/diet/timepoint; (G/H) Tumor-associated angiogenesis and inflammatory changes in mice fed HF or LF diets. Microvessel density is indicated by endothelial cell immune-reactivity with anti-CD31 antibody and was significantly greater in tissues from HF-fed mice (p = 0.0002); (I), quantitation of microvessel density at 4 and 8 weeks after diet consumption, n = 9–10 mice/diet/timepoint.

Figure 4

Tissue and plasma levels of MCP-1 and the effects of feeding a high-fat (HF) diet in tumor-bearing MMTV-PyMT mice. (A) Visceral adipose MCP-1 concentrations were higher at 4 weeks in the mice on HF diet (* p < 0.05); mammary adipose MCP-1 concentrations were higher at 4 and 8 weeks in mice fed HF diet (* p < 0.05); tumor tissue MCP-1 levels were significantly increased in both dietary groups (*** p < 0.001); plasma MCP-1 concentrations were significantly higher in the LF/HF groups (** p < 0.01). Data are depicted as mean ± S.E.M.; (B) MCP-1 expression in tissues (adipose, tumor) significantly correlates with plasma levels in mice fed HF diet (r_s = 0.3591, p = 0.002).