Vitamin D3 and its analogs are known to modulate the activity of fibroblasts under various disease conditions. However, their impact on cancer-associated fibroblasts (CAFs) is yet to be fully investigated. The aim of this study was to characterize CAFs and normal fibroblasts (NFs) from the lung of mice bearing 4T1, 67NR, and E0771 cancers and healthy mice fed vitamin-D3-normal (1000 IU), -deficient (100 IU), and -supplemented (5000 IU) diets. The groups receiving control (1000 IU) and deficient diets (100 IU) were gavaged with calcitriol (+cal). In the 4T1-bearing mice from the 100 IU+cal group, increased NFs activation (increased α-smooth muscle actin, podoplanin, and tenascin C (TNC)) with a decreased blood flow in the tumor was observed, whereas the opposite effect was observed in the 5000 IU and 100 IU groups. CAFs from the 5000 IU group of E0771-bearing mice were activated with increased expression of podoplanin, platelet-derived growth factor receptor β, and TNC. In the 100 IU+cal group of E0771-bearing mice, a decreased blood flow was recorded with decreased expression of fibroblast growth factor 23 (FGF23) and C-C motif chemokine ligand 2 (CCL2) in tumors and increased expression of TNC on CAFs. In the 67NR model, the impact of vitamin D3 on blood flow or CAFs and lung NFs was not observed despite changes in plasma and/or tumor tissue concentrations of osteopontin (OPN), CCL2, transforming growth factor-β, vascular endothelial growth factor, and FGF23. In healthy mice, divergent effects of vitamin D3 supplementation/deficiency were observed, which lead to the creation of various body microenvironments depending on the mouse strain. Tumors developing in such microenvironments themselves modified the microenvironments by producing, for example, higher concentrations of OPN and stromal-cell-derived factor 1 (4T1), which influences the response to vitamin D3 supplementation/deficiency and calcitriol administration.Keywords: CAFs; angiogenesis; breast cancer; calcitriol; cancer-associated fibroblasts; vitamin D.

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