Postmenopausal obesity promotes tumor angiogenesis and breast cancer progression in mice

Abstract

Obese postmenopausal women have a 50% higher risk of breast cancer than non-obese women. There is not an animal model that mimics postmenopausal obesity related to breast cancer progression. Using age-relevant C57BL/6 mice, this study determined whether postmenopausal obesity increases VEGF expression, tumor angiogenesis, and breast tumor growth. Ovariectomy (OVX) was performed in 12 sixty week-old female mice, then followed by a low-fat (5%, LF, n=6) or a high-fat (60%, HF, n=6) diet for 12 weeks. In the eighth week of the dietary program, 10(6) E0771 (mouse breast cancer) cells were injected in the left fourth mammary gland. Tumor size was monitored for 4 weeks. Body weights were monitored weekly. At the end of the experiment, blood samples, visceral fat and tumors were collected for measuring VEGF expression using ELISA and intratumoral microvessel density (IMD) using CD31 immunochemistry. Body weight was significantly increased in OVX/HF mice, compared to OVX/LF group (55.3±1.7 vs. 41.5±1.5 g; p < 0.01). There was a two-fold increase in the ratio of visceral fat/BW in OVX/HF mice, compared to those in OVX/LF group (0.062±0.005 vs. 0.032±0.003; p < 0.01). Postmenopausal obesity significantly increased breast tumor weight over the control (4.62±0.63 vs. 1.98±0.27 g; p < 0.01) and IMD (173±3.7 vs. 139±4.3 IM#/mm^2; p < 0.01). Tumor VEGF levels were higher in OVX/HF mice, compared to OVX/LF group (73.3±3.8 vs. 49.5±4.3 pg/mg protein; p < 0.01). Plasma VEGF levels (69±7.1 vs. 48±3.5 pg/ml) and visceral fat VEGF levels (424.4±39.5 vs. 208.5±22.4 pg/mg protein) were significantly increased in OVX/HF mice, compared to OVX/LF group, respectively (n=6; p < 0.01). Interestingly, adipose tissue primary culture showed that subcutaneous fat released more VEGF, compared to visceral fat (6.77±1.14 vs. 0.94±0.16 pg/mg tissue; n=6; p < 0.01). These findings support the hypothesis that postmenopausal obesity promotes tumor angiogenesis and breast cancer progression, possibly through increased adipose tissue mass and adipokines such as VEGF that could systemically and locally affect breast cancer progression.

Figure 1

(A) Shows the images of both ovaries (OVX) removed from a mouse and the body weight profiles for >60 wk old OVX female C57BL/6 mice fed HF and LF for 12 weeks. High-fat (60%) diet significantly increased the body weight in >60 wk old ovariectomized (OVX) mice, compared to the OVX mice with low-fat (5%) diet, after 4 weeks of dietary program (p < 0.05). After 12 weeks of dietary program, body weight was significantly increased in OVX/HF mice, compared to OVX/LF group (55.3 ± 1.7 vs. 41.5 ± 1.5 g; p < 0.01). (B) Shows the images of visceral fat isolated from OVX female C57BL/6 mice fed with LF and HF. (C) The visceral fat weight was significantly increased in OVX/HF mice, compared to OVX/LF group (3.43 ± 0.47 vs. 1.31 ± 0.14 g; p < 0.01). (D) There was a two-fold increase in the ratio of visceral fat/BW in OVX/HF mice, compared to those in OVX/LF group (0.062 ± 0.005 vs. 0.032 ± 0.003; p < 0.01).

Figure 2

Twelve weeks of high-fat diet significantly increased breast tumor weight in >60 wk old ovariectomized (OVX) mice, compared to those mice with low-fat diet (4.62 ± 0.63 vs. 1.98 ± 0.27 g; N = 6; p < 0.01).

Figure 3

Linear regression analysis showed there was a strong positive linear correlation between increased breast tumor weight and visceral fat weight in OVX mice (R² = 0.7379; N = 12; p < 0.01).

Figure 4

The digital images of CD31 immunohistochemistry staining in OCT-embedded cryosections of mouse breast cancer tumors obtained from >60 wk old OVX female C57BL/6 mice fed LF (A) and HF (B) for 12 weeks. Sections were incubated with rat anti-mouse CD31 antibody followed by mouse anti-rat IgG (Vector laboratories, Burlingame, CA), Extravadin Peroxidase (Sigma, St. Louis, MO) and peroxidase substrate (Vector laboratories, Burlingame, CA). Sections were counterstained with hemotoxylin. The brown staining indicated microvascular vessels. Morphometric analysis (C) indicated that postmenopausal obesity (OVX/HF) caused a significant increase in average microvessel density (AMVD, the number of microvessels per mm² area) of breast cancer tumors when compared to the control (OVX/LF) breast cancer tumors (173 ± 3.7 vs. 139 ± 4.3 microvessels number per mm²; n = 6; p < 0.01).

Figure 5

Effects of a high-fat diet on VEGF protein levels in plasma and breast tumors of OVX mice. ELISA assay showed that plasma VEGF levels (69 ± 7.1 vs. 48 ± 3.5 pg/ml) and tumor VEGF levels (73.3 ± 3.8 vs. 49.5 ± 4.3 pg/mg protein; p < 0.01) were significantly increased in OVX/HF mice, compared to OVX/LF group, respectively (n = 6; p < 0.01).

Figure 6

Differential expression of VEGF protein in visceral fat between OVX/LF and OVX/HF mice and those in visceral fat compared to subcutaneous fat in postmenopausal obese (OVX/HF) mice. (A) ELISA assay showed that VEGF levels in visceral fat (424.4 ± 39.5 vs. 208.5 ± 22.4 pg/mg protein) were significantly increased in OVX/HF mice, compared to those in OVX/LF group (n = 6; p < 0.01). (B) The abdominal subcutaneous fat expressed more VEGF proteins than visceral fat in OVX/HF mice (692 ± 72 vs. 431 ± 44 pg/mg protein; n = 6; p < 0.01). (C) The adipose tissue primary culture showed that subcutaneous fat released more VEGF, compared to visceral fat (6.77 ± 1.14 vs. 0.94 ± 0.16 pg/mg tissue; n = 6; p < 0.01).