Fig. 2

Effect of SAM, 25(OH)D, and their combination on mammary tumor emergence, growth, and lung metastasis in MMTV-PyMT transgenic female mice. a Schematic representation of treatment protocol for the transgenic MMTV-PyMT mice. Briefly, female MMTV-PyMT mice were treated with vehicle alone as control, SAM (160 mg·kg⁻¹ per day) by daily oral gavage, 25(OH)D (40 ng·kg⁻¹ per day) by daily intraperitoneal (i.p) injection, and SAM + 25(OH)D in combination from day 28 (week 4) after birth until the experimental endpoint at day 77 (week 11) when the animals were all sacrificed (n = 8 animals per group) and different tissues were collected for downstream experiments. b Kaplan–Meier curve showing the percentage of mice no tumor in control and different treatment groups, separately (n = 8 animals per group). c Tumor volumes were measured at weekly intervals using calipers and are shown as a bar graph. Results are shown as the mean ± SEM of eight animals per group. Significant differences were determined using ANOVA followed by post hoc Tukey’s test and are represented by asterisks. d Tumor growth inhibition (TGI) index at sacrifice was calculated using the formula described in ‘Materials and Methods’ and represented in a tabular format. e The formalin-fixed histologic sections of the mammary tumors from control and different treatment groups were probed with the antibody for ki67 proliferation marker, and representative images of the ki67 positive cells (brown color staining) is shown for different groups [scale bar size = 60 μm]. The percentage of ki67 positive cells was determined and plotted as bar graphs (n = 4 animals per group). f For the evaluation of lung metastasis, formalin-fixed histologic sections of the whole lung tissue sections from control and treated animals were stained with Haemotoxylin and Eosin (H&E) (left panel), and the relative area of metastases was quantified using the Fiji plugin (ImageJ) (right panel). The metastatic sites on the lung show darker staining patterns, as indicated by red arrowheads. Results are shown as mean ± SEM (n = 4 per group). The metastatic area in the control group was set to 1 for the statistical analysis for the bar graph. Here, C = control, S = SAM, V = 25(OH)D, S + V = SAM + 25(OH)D treated group