Caloric restriction reduces growth of mammary tumors and metastases

Abstract

We investigated the effects of caloric restriction (CR) on growth of tumors and metastases in the 4T1 mammary tumor model and found that CR, compared with normal diet, reduced the growth of mammary tumors and metastases and the total number of metastases that originated both spontaneously from the primary tumor and also experimentally from i.v. injection of the tumor cells. CR also decreased proliferation and angiogenesis and increased apoptosis in tumors. CR reduced levels of insulin, leptin, insulin-like growth factor 1, insulin-like growth factor binding protein 3 and increased adiponectin in tumors. We also demonstrated that tumors from CR mice possessed lower levels of transforming growth factor-β, lower intratumor deposition of collagen IV and reduced invasiveness due to a decrease in tumor secretion of active matrix metalloproteinase 9. Our results suggest that CR-induced metabolic and signaling changes affect the stroma and the tumor cells resulting in a microenvironment that prevents proliferation of breast tumors and their metastases.

Fig. 1.

CR causes a significant inhibition of 4T1 murine mammary tumor formation in syngeneic mice. (A) Tumor volume was significantly increased over 30 days after inoculation with 4T1 cells and this growth was significantly reduced in CR mice. (B) CR mice weighed significantly less than ND mice. (C) The average of tumor wet weight at 30 days was significantly lower in CR mice. (D) Tumor wet weights normalized by body weight were also significantly different. *P < 0.05, **P < 0.01 versus ND; n = 7 per group.

Fig. 2.

CR causes a decrease in the number of spontaneous and experimental lung metastases. (A) Representative reduced spontaneous metastases in lung from a ND and a CR mouse. (B) The average ± SE of spontaneous lung metastases showed that the differences in reduction between CR with respect to ND mice were greater for larger metastases. In addition, the total number of lung nodules was significantly reduced in the CR mice. (C) Representative reduced experimental metastases in lung from a ND and a CR mouse. (D) The average ± SE of experimental lung metastases showed that the differences in reduction between CR with respect to ND mice were greater for larger metastases. In addition, the total number of lung nodules was significantly reduced in the CR mice. **P ≤ 0.01 versus ND; *P < 0.05 versus ND; n = 7 per group (spontaneous); n = 5 per group (experimental).

Fig. 3.

CR reduces intratumor microvessel density and in vivo angiogenesis induced by 4T1 tumors. (A) CD31 antigen staining to assess vascular density was reduced in CR mice. (B) Quantification of microvessel density shows a reduction in CR mice versus ND mice. (C) Representative photographs showing reduced vessels in an example of CR mouse. Black arrows indicate the vessels in the vicinity of tumor implant. (D) Reduced total length of vessels around tumor implants in CR mice. (E) Serum VEGF is also reduced in CR mice. *P < 0.05 versus ND; n = 4 per group (microvessel density); **P < 0.01 versus ND; n = 5 per group (in vivo angiogenesis); **P < 0.01 versus ND; n = 7 per group (VEGF).

Fig. 4.

Levels of TGF-β are reduced in CR mice. (A) Reduced serum TGF-β in CR (white bars) with respect to the ND (black bars) mice. (B) Intratumor levels of TGF-β are also reduced in CR mice. (C) TGF-β staining is reduced in tumors from CR mice. *P < 0.05 and versus ND; n = 7 per group.

Fig. 5.

CR mice possess reduced intratumor collagen IV expression. (A) Quantification of the percentage of total collagen volume fraction shows a significant reduction in CR (white bar) versus ND (black bar) mice. (B) Tumors from CR mice express lower levels of collagen IV. (C) Quantification of the mean pixels shows that collagen IV levels were significantly lower in tumors from CR mice. *P < 0.05 and **P < 0.01 versus ND; n = 4 per group.