In our previous study, calcitriol and its analogs PRI-2191 and PRI-2205 stimulated 4T1 mouse mammary gland cancer metastasis. Therefore, we aimed to analyze the inflammatory response in 4T1-bearing mice treated with these compounds. Gene expression analysis of the splenocytes and regional lymph nodes demonstrated prevalence of the T helper lymphocytes (Th2) response with an increased activity of regulatory T (Treg) lymphocytes in mice treated with these compounds. We also observed an increased number of mature granulocytes and B lymphocytes and a decreased number of TCD4⁺, TCD4⁺CD25⁺, and TCD8⁺, as well as natural killer (NK) CD335⁺, cells in the blood of mice treated with calcitriol and its analogs. Among the splenocytes, we observed a significant decrease in NK CD335⁺ cells and an increase in TCD8⁺ cells. Calcitriol and its analogs decreased the levels of interleukin (IL)-1β and IL-10 and increased the level of interferon gamma (IFN-γ) in the plasma. In the tumor tissue, they caused an increase in the level of IL-10. Gene expression analysis of lung tissue demonstrated an increased level of osteopontin (Spp1) and transforming growth factor β (TGF-β) mRNA. The expression of Spp1 was also elevated in lymph nodes. Calcitriol and its analogs caused prevalence of tumor-conducive changes in the immune system of 4T1 tumor-bearing mice, despite the induction of some tumor-disadvantageous effects.

Low vitamin D status is considered as a risk factor for breast cancer and has prognostic significance. Furthermore, vitamin D deficiency increases after adjuvant cancer therapy, which alters bone metabolism increasing the risk of osteoporosis. It is now postulated that vitamin D supplementation in breast cancer treatment delays the recurrence of cancer thereby extending survival. We evaluated the impact of calcitriol and its low-calcemic analogs, PRI‑2191 and PRI‑2205, on the tumor growth, angiogenesis, and metastasis of 4T1 mouse mammary gland cancer. Gene expression analysis related to cancer invasion/metastasis, real‑time PCR, ELISA, western blotting, and histochemical studies were performed. In vitro studies were conducted to compare the effects of calcitriol and its analogs on 4T1 and 67NR cell proliferation and expression of selected proteins. Calcitriol and its analogs increased lung metastasis without influencing the growth of primary tumor. The levels of plasma 17β-estradiol and transforming growth factor β (TGFβ) were found to be elevated after treatment. Moreover, the results showed that tumor blood perfusion improved and osteopontin (OPN) levels increased, whereas vascular endothelial growth factor (VEGF) and TGFβ levels decreased in tumors from treated mice. All the studied treatments resulted in increased collagen content in the tumor tissue in the early step of tumor progression, and calcitriol caused an increase in collagen content in lung tissue. In addition, in vitro proliferation of 4T1 tumor cells was not found to be affected by calcitriol or its analogs in contrast to non-metastatic 67NR cells. Calcitriol and its analogs enhanced the metastatic potential of 4T1 mouse mammary gland cancer by inducing the secretion of OPN probably via host cells. In addition, OPN tumor overexpression prevailed over the decreasing tumor TGFβ level and blood vessel normalization via tumor VEGF deprivation induced by calcitriol and its analogs. Moreover, the increased plasma TGFβ and 17β-estradiol levels contributed to the facilitation of metastatic process.

Calcitriol and its analogues are considered drugs supporting the anticancer treatment of breast cancer and preventing the osteoporosis that results from the development of cancer or from chemotherapy or hormone therapy. Following the orthotopic implantation of 4T1 mammary carcinoma cells into aged ovariectomized (OVX) mice, we evaluated the effects of calcitriol and its two analogues, PRI-2191 and PRI-2205, on metastatic spread and bone homeostasis. Calcitriol and its analogues temporarily inhibited the formation of metastases in the lungs. Unexpectedly, only mice treated with calcitriol analogues showed a deterioration of bone-related parameters, such as bone column density, marrow column density and the CaPO4 coefficient. These findings correlated with an increased number of active osteoclasts differentiated from bone marrow-derived macrophages in mice treated with the analogues. Interestingly, in the tumours from mice treated with PRI-2191 and PRI-2205, the expression of Tnfsf11 (RANKL) was increased. On the other hand, osteopontin (OPN) levels in plasma and tumour tissue, as well as TRAC5b levels in tumours, were diminished by calcitriol and its analogues. Despite a similar action of both analogues towards bone metabolism, their impact on vitamin D metabolism differed. In particular, PRI-2191 and calcitriol, not PRI-2205 treatment significantly diminished the levels of both 25(OH)D3 and 24,25(OH)2D3. In conclusion, though there is evident antimetastatic activity in old OVX mice, signs of increased bone metabolism and deterioration of bone mineralization during therapy with calcitriol analogues were observed.

In this study, we differentiated murine bone marrow-derived macrophages (BMDMs) into M0, M1, and M2 in the presence or absence of calcitriol. Real-time PCR analysis of gene expression, FACS analysis of surface markers, and chemokine/cytokine production assays were performed. In addition, the effect of the conditioned media (CM) from murine breast cancer 4T1 (metastatic) and 67NR (non-metastatic) and Eph4-Ev (normal) cells with and without calcitriol on the polarization of M1/M2 cells was determined. We found that calcitriol enhanced the differentiation of M2 macrophages, which was manifested by increased expression of Cd206 and Spp1 mRNA and CD36, Arg, and CCL2 in M2 BMDMs and by decreased expression of Cd80 and Spp1 mRNA and IL-1, IL-6, OPN, and iNOS in M1 BMDMs. 4T1 CM showed a higher effect on the gene and protein expression in macrophages than 67NR and Eph4-Ev, with the greatest effect observed on M2 macrophages which increased their differentiation and properties characteristic of alternative macrophages. Moreover, M2 macrophages differentiated with calcitriol-stimulated migration of 4T1 and 67NR cells through fibronectin and collagen type IV, respectively. Overall, our results indicated that vitamin D supplementation may not always be beneficial, especially in relation to cancers causing excessive, pathological activation of the immune system.

To analyze if the prometastatic activity of calcitriol (active vitamin D₃ metabolite), which was previously observed in a 4T1 breast cancer model, is also found in other breast cancers, and to assess the impact of various schemes of vitamin D supply, we used 4T1 and E0771 mouse metastatic and 67NR nonmetastatic cells in this study. BALB/c and C57BL/6 healthy and tumor-bearing mice were exposed to a control (1000 IU), low- (100 IU), and high- (5000 IU) vitamin D₃ diets. Additionally, from day 7 of tumor transplantation, the 1000 and 100 IU groups were gavaged with calcitriol (+cal). After 8 weeks of feeding, plasma levels of 25(OH)D₃, 24,25(OH)₂D₃, and 3-epi-25(OH)D₃ were significantly lower in calcitriol-treated and vitamin D-deficient groups than in the control, whereas the levels of all metabolites were increased in the 5000 IU group. The ratio of 25(OH)D₃:24,25(OH)₂D₃ was increased in both calcitriol-treated groups, whereas the ratio of 25(OH)D₃:3-epi-25(OH)D₃ was increased only in the 100 IU group but decreased in the 5000 IU group. In contrast to E0771, 4T1 lung metastasis was accelerated in all vitamin D-supplemented mice, as well as in the deficient group with an increased inflammatory response. 67NR tumor growth was transiently inhibited in the 1000 IU+cal group, but single metastases were observed in the 5000 and 100 IU groups. Based on the results, we conclude that various schemes of vitamin D supply and vitamin D deficiency led to similar metabolite profiles irrespective of the mice strain and tumor burden. However, depending on the type of breast cancer, different effects on tumor growth and metastasis were noticed.