Fig. 5. Dose-dependent protective effect of daily CR against primary tumor growth in post-reproductive females.

a Experimental layout. AL-fed retired BALB/cJ breeders were randomized to varying degrees of CR (10–40%) using a ramp-down approach. Mice were then injected with 4T1 cells (10⁶ cells/mL) at day 0 and remained on the specified doses of CR until tissue collection at day 28. Another AL-fed group of retired breeders was also injected with 4T1 cells and 7 days later was randomly divided into three groups; mice were maintained either on AL or subjected to two low caloric cycles of FMD or LCC before tissue collection at day 28. b Average body weight during the course of the study. c Average caloric intake. d Growth rates of the primary tumor. e Average tumor area at day 28. f Tumor mass and g spleen mass per unit of body weight (BW). h Histological quantification of primary tumor viability and i mitotic counts. j Macroscopic quantification of lung metastases [india ink-stained lungs]. k Histological quantification of lung metastatic areas. l Representative images of primary tumors (top) and corresponding lungs (bottom) under different experimental conditions [H&E staining, original magnification ×200]. Scale bar = 100 μm. Most of the data are represented as scatter plots with mean values ±  SEM. One-way ANOVA with Tukey post hoc analysis was used to determine statistical significance with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to AL. b, c AL, n = 7; FMD, n = 7; LCC, n = 8; 10% CR, n = 6; 20% CR, n = 7; 30% CR, n = 7; 40% CR, n = 6. d, e AL, n = 7; FMD, n = 7; LCC, n = 8; 10% CR, n = 8; 20% CR, n = 8; 30% CR, n = 7; 40% CR, n = 6. f–h, j AL, n = 7; FMD, n = 7; LCC, n = 8; 10% CR, n = 8; 20% CR, n = 8; 30% CR, n = 7; 40% CR, n = 5. i AL, n = 6; FMD, n = 6; LCC, n = 7; 10% CR, n = 7; 20% CR, n = 8; 30% CR, n = 7; 40% CR, n = 5. k AL, n = 6; FMD, n = 7; LCC, n = 8; 10% CR, n = 7; 20% CR, n = 8; 30% CR, n = 7; 40% CR, n = 5 mice per treatment group. Source data are provided as a Source Data file.