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    2.4. Statistical analysis
    Data shown are from single representative experiments of two or more repeats and are presented as the mean ± standard error of six independent cultures per treatment group. Data were analyzed by ANOVA with Tukey's post-test to determine significance between groups. Experiments with two independent variables (DNA synthesis, total DNA content, and total p53 time courses) were analyzed with two-way ANOVA with Bonferroni's modification of Student's t-test post-tests to determine the variance between row and column factors. The wound closure assay was analyzed with a repeated measures ANOVA test with comparisons between time points. P-values < .05 were considered significant.
    3. Results
    3.1. 17β-Estradiol supplementation stimulated MCF7 breast cancer tumor growth in mice
    MCF7 (estrogen receptor positive, ER+, progesterone receptor po-sitive, PR+) breast cancer RVX-208 were injected into the duct of the fourth mammary gland. Mice receiving 17β-estradiol injections grew tumors
    A. Verma, et al. BBA - General Subjects xxx (xxxx) xxx–xxx
    Fig. 1. 17β-estradiol stimulates MCF7 tumor growth in a mammary fat pad xenograft model in NSG mice. (A) Non-ovariectomized mice injected with 17β-estradiol showed a significant increase in tumor growth as compared to the vehicle after 3 weeks of injection until the end of the study at week 10. (B) Similar trends were observed in a separate study with ovariectomized animals, with animals treated with 17β-estradiol showing a significant increase in tumor growth as compared to vehicle at weeks 4 and 5 and weeks 7–10 of the study. (C) At harvest, tumors grown in ovariectomized mice supplemented with 17β-estradiol reached an average volume of 120 mm3, nearly 6 times greater than tumors grown in vehicle-treated mice. (D) Histology of the fourth mammary fat pad revealed neoplastic tissue characteristic of MCF7 tumors. (E) Final tumor volume was largest in control mice supplemented with 17β-estradiol. Ovariectomized mice supplemented with 17β-estradiol had tumor volumes that were smaller than those from normal mice, but still larger than tumors grown in ovariectomized mice given a vehicle control. Groups that share a letter are not statistically significant at α = 0.05 with two-way ANOVA.
    3–5 times their original size. In contrast, tumors grown in animals given a vehicle control injection showed minimal growth over 10 weeks [Fig. 1]. Tumors in mice given 17β-estradiol increased from approxi-mately 40 mm3 to an average of 200 mm3, approximately 500% of their original volumes, with a linear growth increase over 8 weeks (week 2 to week 10). However, tumors grown in mice given a vehicle injection reached an average size of 55 mm3, an increase of approximately 10% of their original volume [Fig. 1A]. In ovariectomized animals, the dif-ference between vehicle versus 17β-estradiol injected mice was even more pronounced, with tumors grown in 17β-estradiol injected mice reaching an average size of 100 mm3 versus only 25mm3 in vehicle-injected ovariectomized mice [Fig. 1B & C]. Tumor morphology was similar in both 17β-estradiol and vehicle-injected mice, and there was no observed difference in tumor morphology in samples taken from normal vs. ovariectomized mice [representative image, Fig. 1D]. At harvest, the final tumor volume in 17β-estradiol animals was approxi-mately 4 times greater than tumor volume in vehicle-treated animals in both the normal and ovariectomized mouse models [Fig. 1E]. None of the animals in either experiment showed signs of estrogen toxicity.
    3.2. Treatment with 24R,25(OH)2D3 reduced tumor aggression and increased animal survival
    RFP-overexpressing MCF7 mammary fat pad tumors were grown in female NSG mice for 10 weeks. Estrogen supplementation was given for 2 weeks before tumor initiation, then continued for the first 4 weeks of tumor growth. Dose-dependent 24R,25(OH)2D3 was initiated at the