1. Introduction
The tumor microenvironment, which includes fibroblasts, vascular endothelial cells, and immune cells, is an important component of cancer and affects its progression, metastasis, and sensitivity to therapies. Vitamin D
3—mainly its active form, calcitriol, 1,25-dihydroxycholecalciferol—can influence almost every cell in the body, including the tumor-building ones [
1]. Thus, although cancer cells (e.g., murine cancer cell lines) are not sensitive to the antiproliferative activity of vitamin D
3 in vitro (despite the presence of the vitamin D receptor, VDR), they show significant sensitivity to vitamin D derivatives in vivo. One such example is 4T1 mouse breast cancer cells. Previous studies have shown that calcitriol and its analogs may increase the rate of tumor metastasis [
2,
3] or accelerate the progression of primary tumors in young mice [
4]. However, in aged, ovariectomized animals (as a postmenopausal model), these compounds inhibited 4T1 tumor metastasis [
5]. Besides the age of mice, other factors may also affect the outcome of the in vivo treatment of mammary gland tumors with vitamin D compounds. The abovementioned observations were from experiments in mice in which the therapy was started when tumors were already formed. On the contrary, an antimetastatic effect was observed in young mice bearing 4T1 tumors when calcitriol treatment was started prior to tumor inoculation [
6]. The beneficial effect of vitamin D analogs in young mice was also evident when these substances were used in combination therapy with cyclophosphamide [
7]. In the case of E0771, another mouse mammary gland cancer model that is also not sensitive to the antiproliferative effect of calcitriol [
3], the antitumor effect of cholecalciferol was observed in normal young mice, whereas protumor activity was observed in obese mice. This effect was related to the impact of the treatment on tumor-infiltrating CD8+ T lymphocytes which are increased in number in normal mice and decreased in obese mice [
8].
In recent clinical studies much attention has been paid on the effects of vitamin D in the development of various cancers, including breast cancer. These studies focused mainly on the relationship between tumor progression and plasma 25(OH)D concentrations (the most studied metabolite of vitamin D), vitamin D supplementation or the expression of various proteins related to vitamin D activity or metabolism (recently reviewed in [
9]). However, based on these studies it is difficult to draw firm conclusions concerning beneficial or adverse effects of vitamin D on breast cancer development. For example, a systematic review and meta-analysis by Hossain et al. showed an inverse relationship between vitamin D intake and breast cancer occurrence along with a direct relationship between vitamin D deficiency and breast cancer risk [
10]. Other studies reported the occurrence of vitamin D deficiency in newly diagnosed breast cancer patients [
11]. They also indicate that vitamin D supplementation significantly reduced total cancer mortality but did not affect total cancer incidence [
12]. On the other hand, e.g., O’Connor et al. indicated the lack of the relationship between vitamin D and breast cancer [
13]. Moreover, Kanstrup et al. [
14] found an inverse correlation between 25(OH)D plasma concentrations and breast cancer survival, however, this study also indicated poorer breast cancer survival among patients with high 25(OH)D concentrations [
14]. Likewise, Ganji et al. reported an association of high 25(OH)D plasma concentrations (75–100 nmol/L) with greater risk of breast cancer in postmenopausal women [
14,
15]. Therefore, controlled trials are required to verify the data concerning vitamin D benefits for breast cancer patients.
Cancer-associated fibroblasts (CAFs) resembling myofibroblasts—activated spindle-shaped fibroblasts—are the major cellular component in the tumor microenvironment [
16]. The stimuli produced by tumor cells and stromal cells, such as fibroblasts, in the tumor microenvironment can activate fibroblasts and contain, among others, interleukin-1, interleukin-6 (IL-6), bone morphogenetic protein, sonic hedgehog, reactive oxygen species, transforming growth factor-β (TGF-β), platelet-derived growth factor, and tumor necrosis factor [
17]. The activation of fibroblasts by these stimuli may result in quiescent, tumor-restraining, and tumor-promoting CAFs [
18,
19,
20]. Orimo et al. showed that coimplantation of the tumor xenograft model with CAFs harvested from the breast carcinomas of patients promoted higher growth of breast cancer cells compared with coimplantation with normal mammary fibroblasts derived from the same patients [
21]. CAFs promote tumor growth through the secretion of stromal-cell-derived factor 1 (SDF-1) and angiogenesis by recruiting endothelial progenitor cells into tumor tissue [
21]. Thus, some studies have proposed targeting CAFs as a treatment option for triple-negative breast cancer [
22]. Clinical retrospective studies on breast cancer patients focusing on the expression of caveolin-1, which acts as a tumor-suppressing molecule in CAFs, showed 72 months of cancer-specific survival in caveolin-1-positive patients, whereas only 29.5 months in caveolin-1-negative patients [
23].
Osteopontin (OPN) is an important protein engaged in the crosstalk between cancer cells and stromal fibroblasts. OPN secreted by breast cancer cells induces differentiation of fibroblasts to myofibroblasts. Furthermore, OPN-driven CAFs secrete SDF-1, which in turn triggers epithelial-to-mesenchymal transition in tumor cells [
24]. As reported in previous studies, calcitriol and other agonists of VDR stimulate the transcription of the OPN gene (
Spp1) or secretion of OPN [
25,
26]. In our previous in vitro study, calcitriol and its analogs stimulated OPN secretion in 67NR mouse mammary gland cancer cells, which are sensitive to proliferation inhibition by calcitriol, but not in 4T1 cells [
2]. It was also found that in vitro stimulation by calcitriol and its analogs led to increased OPN secretion by normal mouse fibroblast cells BALB/3T3, but not by the murine macrophage cell line RAW 264.7 [
27]. Although much research has been conducted investigating the effects of vitamin D on fibroblasts in various types of diseases or physiological conditions [
28,
29,
30,
31], its effect on CAFs remains to be elucidated. Studies on fibroblasts derived from colon cancer [
32,
33], pancreatic cancer [
34,
35], and breast cancer [
36,
37] patients have indicated that calcitriol modulates gene expression and inhibits the protumoral properties of CAFs. Therefore, in the present study, we analyzed the effects of calcitriol treatment and vitamin D
3 (cholecalciferol) deprivation or supplementation on tumor and lung fibroblasts in three mouse mammary gland cancer models (4T1, 67NR, and E0771) with different metastatic potential.
2. Materials and Methods
2.1. Cells and Tissues Harvested from Mice Bearing Mammary Gland Tumors
Fibroblasts were isolated from lung and tumor tissues of mice bearing 4T1, 67NR, and E0771 tumors and of healthy BALB/c and C57BL/6 mice. Data regarding tumor growth, metastasis, and vitamin D metabolism in these mice have been previously published [
3]. Origin of the cell lines used: 4T1 cells-the American Type Culture Collection (ATCC, Rockville, MD, USA); 67NR (nonmetastatic counterparts of 4T1) Barbara Ann Karmanos Cancer Institute (Detroit, MI, USA); E0771 cell line [
38], gifted by Dr. Andreas Möller (School of Medicine, University of Queensland; Tumour Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia).
The complete in vivo experimental design has been described earlier [
3]. The animal study protocol was approved by the first Local Committee for Experiments with the Use of Laboratory Animals, Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland (permission number: 66/2018, 18 July 2018). Mice were divided into groups and fed ad libitum with an AIN67 synthetic diet (ZooLab, Sedziszow, Poland) for 6 weeks. The diet fed to groups had a varied content of vitamin D
3 as follows: control amount of vitamin D
3 (1000 IU/kg), supplemented vitamin D
3 (5000 IU/kg), and deficient vitamin D
3 (100 IU/kg). After 6 weeks (day 0), some mice were implanted orthotopically with tumor cells (1 × 104, 2 × 105, and 5 × 104 viable 4T1, 67NR, and E0771 cells, respectively), and the same diet was continued. Seven days after the implantation of tumor cells, calcitriol administration (1 µg/kg per os by gavage; p.o.) was started, which continued thrice a week in the groups receiving food with a normal, control amount of vitamin D
3 (1000 IU) and in the groups receiving a vitamin-D
3-deficient diet (100 IU). On day 23 (C57BL/6) or 28 (BALB/c) after the inculcation of cancer cells, mice were subjected to isoflurane anesthesia and injected with buprenorphine (0.1 mg/kg of body weight) analgesia, subjected to blood collection, and then killed (
Figure 1).
2.2. Blood Flow Assessment
Tumor blood perfusion analysis was performed on day 21 (4T1 and 67NR models) or on day 19 (E0771 model) using the MicroMarker™ contrast agent (VisualSonics, Toronto, ON, Canada) and Vevo 2100 ultrasound imaging system (VisualSonics). Mice were anesthetized by continuous administration of 2–3% (v/v) isoflurane (Baxter, Deerfield, Germany) in synthetic air (600 mL/min) and immobilized. The tumor area was enclosed with air-bubble-free gel, and the central cross-section of the tumor was visualized in the transverse plane using an MS250 scanhead (VisualSonics). Then, 100 μL of the contrast agent was injected intravenously and the first imaging sequence (bolus) was recorded (ca. 15 fps, at least 1000 frames) after the contrast signal in the tumor tissue reached the steady state (ca. 50 s). Next, using the burst mode, contrast microbubbles were destroyed, and the second imaging sequence (replenishment) was recorded. Mice were maintained in a warm environment until fully awakened. The data were analyzed using the Vevo LAB 1.7.1 Software with VevoCQ modality (VisualSonics).
2.3. Tissue Microarrays
Tissue microarrays were prepared using TMA Grand Master (3DHistech, Budapest, Hungary), in accordance with the manufacturer’s instructions. The representative spots for tissue microarrays were selected by a pathologist. Three core punches of 1.5 mm diameter from each tumor block were transferred into the recipient paraffin block.
2.4. Immunohistochemical Staining of Tissues
Using the automated immunostainer device Autostainer Link 48 (Dako, Glostrup, Denmark), immunohistochemistry analysis was performed on 4 µm formalin-fixed paraffin-embedded tissue microarray sections. To deparaffinize, rehydrate, and unmask the epitopes, the slides were boiled in EnVision™ (Santa Clara, CA, USA) FLEX Target Retrieval Solution of a high pH for 20 min at 97 °C using PT-Link (both from Dako). The endogenous peroxidase activity was blocked using the EnVision FLEX Peroxidase-Blocking Reagent (Dako). Then, the primary antibody against collagen type I alpha 1 chain (COL1A1) (dilution 1:1600, cat.no. PA5-86949; Invitrogen, Waltham, MA, USA) or against the smooth muscle actin (ready-to-use, cat. no. IR611; Dako, Glostrup, Denmark) were applied for 20 min at room temperature. Afterward, the slides were incubated with EnVision FLEX/HRP (Dako) for (20 min). Subsequently, the sections were incubated for 10 min at room temperature with the substrate for peroxidase, diaminobenzidine. Additionally, using the EnVision FLEX Hematoxylin (Dako), all slides were counterstained for 5 min. After dehydration in graded ethanol concentrations (70%, 96%, absolute) and in xylene, all slides were covered with coverslips in the SUB-X Mounting Medium. Two pathologists evaluated the expression of studied proteins with a routinely used immunoreactive scale of Remmele and Stegner [
39], which is presented in
Table 1.
2.5. Isolation of Lung and Tumor Fibroblasts
Harvested tissues were transferred to a 6 cm tissue culture dish, mechanistically chopped into ~1 mm pieces, and digested for 1 h at 37 °C with gentle shaking. The digestion solution was composed of phosphate-buffered saline containing calcium and magnesium ions (PBS Ca2+ Mg2+; HIIET, Wroclaw, Poland), 1 mg/mL collagenase IV (collagenase from Clostridium histolyticum; Sigma-Aldrich, Saint-Louis, MO, USA), and 1 mg/mL DNase I (Roche, Basel, Switzerland). Digested lung homogenates were centrifuged for 7–10 min (4 °C, 350× g) and washed thrice with a normal fibroblast (NF) medium, Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA), containing 10% (v/v) of fetal bovine serum (FBS), 1% (v/v) nonessential amino acids, 4.0 mM l-glutamine, 100 μg/mL streptomycin (all Sigma-Aldrich, Saint-Louis, MO, USA), and 100 U/mL penicillin (Polfa Tarchomin S.A., Warsaw, Poland). Cell pellets were resuspended in FBS (Sigma-Aldrich, Saint-Louis, MO, USA) with 10% (v/v) DMSO (Sigma-Aldrich, Saint-Louis, MO, USA) and frozen for further analysis. After the digestion process was completed, tumor homogenates were passed through a strainer with a mesh size of 70 μm (Greiner Bio-One, Kremsmünster, Austria) and then washed with fresh PBS containing 2% (v/v) FBS. Cell suspension was centrifuged for 7–10 min (4 °C, 350× g), followed by erythrocytes lysis. In brief, pellets were resuspended with 1 mL of lysis buffer (Sigma-Aldrich, Saint-Louis, MO, USA) and shaken for 1 min; then, PBS with serum was added and the suspension was centrifuged for 7–10 min (4 °C, 350× g). The cell pellets were resuspended in 5 mL of fresh PBS with FBS, and cell quantity was assessed by counting in a Bürker chamber in trypan blue solution (0.4% (w/v); Sigma-Aldrich, Saint-Louis, MO, USA).
2.6. Magnetic Separation
For magnetic separation, whole-cell suspensions (three mice from one group were pooled together for one separation) were used. CAFs were isolated using Anti-Fibroblast MicroBeads (Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer’s protocol. In brief, centrifuged pellets (7–10 min, 4 °C, 350× g) were resuspended in the separation buffer containing PBS of pH 7.2, 0.5% (v/v) bovine serum albumin, 2 mM ethylene diamine tetra-acetic acid (Sigma-Aldrich, Saint-Louis, MO, USA), and TruStain FcX (antimouse CD16/CD32) antibody (BioLegend, San Diego, CA, USA) and then incubated for 10 min at 4 °C in order to block the Fc receptors (0.1 µg/100 µL volume). After blocking, magnetic beads were added (20 µL per 106 of total cells) to the cells and incubated for 30 min in the dark at room temperature. Subsequently, 1 mL of separation buffer was added to the cells and centrifuged (7–10 min, 4 °C, 350× g). The pellets were resuspended in 1 mL of separation buffer and applied onto activated MS columns (Miltenyi Biotec, Auburn, CA, USA) placed in the magnetic field of the MiniMACS Separator (Miltenyi Biotec, Auburn, CA, USA). After three washes with 500 µL of separation buffer, the columns were transferred into new sterile 10 mL tubes, and cells were flushed into collection tubes. The collected cells were counted using a Bürker chamber in trypan blue solution (0.4% (w/v)) and used for further analysis.
2.7. Flow Cytometry Analysis of Isolated Fibroblasts
CAFs isolated from tumor tissues were analyzed immediately after the separation. Lung fibroblasts from tumor-bearing mice were processed directly after thawing, and those from healthy mice were subjected to cytometry analysis after stimulation with TGF-β1 or cancer-cell-conditioned medium. To assess cell viability, cell suspensions of 3–5 × 104 cells per sample were resuspended in pure PBS and incubated with eBioscience™ Fixable Viability Dye eFluor™ 780 (Invitrogen, Waltham, MA, USA) for 30 min at 4 °C. Cell surface markers were stained extracellularly in the FACS buffer (2% (v/v) FBS in FBS) for 30 min at 4 °C. To carry out intracellular staining, cells were fixed in a fixation buffer (BioLegend, San Diego, CA, USA) for 20 min at room temperature, subsequently washed, and permeabilized using the Intracellular Staining Perm Wash Buffer (BioLegend, San Diego, CA, USA) thrice (7 min, 20 °C, 350× g). The samples resuspended in the FACS buffer were read using a BD LSR Fortessa cytometer with FACSDiva V8.0.1 software (BD Biosciences, Franklin Lakes, NJ, USA). Cytometric analysis was performed for four replicates (pooled from three mice each). For each of the markers, the median fluorescence intensity (MFI) of stained cells in relation to the isotype control and the percentage of positive cells were determined. Following antibodies were used for CAF staining: α-smooth muscle actin (α-SMA)-PE (Abcam, Cambridge, UK), CD31-BV-421, CD90.2-PerCP/Cy5.5, EpCam-FITC, platelet-derived growth factor receptor β (PDGFRβ)-APC, Podoplanin-PE-Cy7 (all Biolegend, San Diego, CA, USA), and Tenascin C (TNC)-Alexa Fluor 700 (Novus Biologicals, Centennial, CO, USA). For NF staining CD90.2 was replaced with CD45 with the same dye (BD Biosciences, San Jose, CA, USA).
2.8. Preparation of Conditioned Media (CM) from 4T1, 67NR, and E0771 Cell Cultures
Cancer cell lines (4T1, 67NR, and E0771) were grown on appropriate media on 10 cm tissue culture dishes (4T1 in RPMI 1640 medium with GlutaMAX, supplemented with 10% (v/v) FBS HyClone (both Thermo Fisher Scientific, Waltham, MA, USA), 1 mM sodium pyruvate, 3.5 g/L glucose, 100 μg/mL streptomycin (all Sigma-Aldrich, Saint-Louis, MO, USA), and 100 U/mL penicillin (Polfa Tarchomin S.A., Warsaw, Poland); 67NR in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) calf bovine serum (ATCC, Manassas, VA, USA), 1× nonessential amino acids, 2.0 mM l-glutamine, 100 μg/mL streptomycin (all Sigma-Aldrich, Saint-Louis, MO, USA), and 100 U/mL penicillin (Polfa Tarchomin S.A., Warsaw, Poland); E0771 in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) FBS HyClone, 2.0 mM l-glutamine, 100 μg/mL streptomycin (all Sigma-Aldrich, Saint-Louis, MO, USA), and 100 U/mL penicillin (Polfa Tarchomin S.A., Warsaw, Poland)). The dishes were washed with PBS after the cells reached confluence, and cells were incubated for 24 h with an appropriate medium without serum. Then, CM were collected, centrifuged (7 min, 4 °C, 400× g), and applied in a 1:1 ratio (CM:NFs medium) for 72 h on NF cultures for stimulation.
2.9. Stimulation of Lung Fibroblasts from Healthy Mice with TGF-β and Mammary Gland Cancer CM
NFs from healthy mice were thawed in NF medium onto a 10 cm tissue culture dish and cultured at 37 °C in a humidified atmosphere with 5% (v/v) CO2. After reaching >60% confluence, cells were transferred onto a 15 cm tissue culture dish (passage 1). Cells from the second passage were plated on a 6-well plate (3 × 105 per well) for stimulation. The medium was refreshed after 24 h with stimulation media for 72 h: (1) control medium—NF medium with 2% of FBS instead of 10%; (2) control medium with 1 ng/mL TGF-β1 (763102; BioLegend, San Diego, CA, USA); (3) conditioned medium from 4T1; (4) conditioned medium from 67NR; and (5) conditioned medium from E0771. Media (1), (2), (3), and (4) were applied on BALB/s NFs, whereas media (1), (2), and (5) were applied on C57BL/6 NFs.
2.10. Fluorescence Microscopy Analyses
A total of 2.5 × 10
3 cells/well were cultured for imaging on a Falcon
® 96-well Black/Clear Flat Bottom TC-treated Imaging Microplate (Corning, New York, NY, USA). Staining was carried out after stimulation with TGF-β1 or cancer-cell-conditioned medium. Before staining, cells were washed twice with PBS solution, fixed in freshly prepared 4% (
v/
v) paraformaldehyde (Avantor Performance Materials Poland, Gliwice, Poland) for 10–15 min, washed thrice with PBS, and permeabilized in 0.25% (
v/
v) Triton X-100 (Sigma-Aldrich, Saint-Louis, MO, USA) for 15 min at room temperature (only wells for VDR staining). After washing thrice in the PBS solution, cells were blocked for 30 min in 1% (
w/
v) bovine serum albumin (Sigma-Aldrich, Saint-Louis, MO, USA) solution in 0.1% (
v/
v) PBS/Tween 20 (Sigma-Aldrich, Saint-Louis, MO, USA) at room temperature. Then, the fixed cells were rinsed thrice with PBS (5 min each) and incubated with primary antibodies against VDR (bs-2987R, dilution 1:100; Bioss Antibodies, Woburn, MA, USA) and fibroblast activation protein (FAP) (ab28244, dilution 1:500; Abcam, Cambridge, UK) in blocking solution at 4 °C overnight. Next, after washing thrice with PBS, a secondary antibody (Anti-rabbit Antibody Alexa Fluor 488, ab150077; Abcam, Cambridge, UK) in the blocking solution was used for 1 h at room temperature. Then, the samples were rinsed thrice with PBS and stained with DAPI (dilution 1:1500; Cell-Signaling, Danvers, TX, USA) and DyLight™ 554 Phalloidin (dilution 1:100; Cell-Signaling, Danvers, MA, USA) in PBS solution for 15 min at room temperature. Cells were photographed using an Olympus IX81 fluorescence microscope (Olympus, Warsaw, Poland) with CellSense software (Olympus, Warsaw, Poland). Fluorescence was determined from the images using ImageJ according to the protocol of Luke Hammond (QBI, The University of Queensland, Australia; accessed on 18 October 2021;
www.theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html). The areas of interest (whole cell for FAP staining and cell nucleus for VDR staining) and areas of background were measured, and corrected total cell fluorescence (CTCF) was calculated using the following formula:
2.11. Real-Time qPCR Analysis of Cultured NFs Isolated from Lungs of Healthy BALB/c or C57BL/6 Mice Fed Control Diet and Vitamin-D3-Deficient Diet and/or Treated with Calcitriol
From the stimulated ex vivo NFs, total RNA was extracted using 1 mL Tri-reagent (Sigma Aldrich, Saint-Louis, MO, USA) followed by RNA purification with Direct-zol™ RNA Miniprep (ZYMO RESEARCH, Tustin, CA, USA) according to the manufacturer’s protocol. Using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA), 1 µg of purified RNA was reverse transcribed into complementary DNA (cDNA). Then, the expression of the following genes was determined in cDNA with ready-to-use primers and probes (TaqMan® Gene Expression Assays; Thermo Fisher Scientific, Waltham, MA, USA): Acta2 (Mm01546133_m1), Mmp9 (Mm00442991_m1), Spp1 (Mm00436767_m1), and Vdr (Mm00437297_m1). Real-time qPCR was carried out in a 10 µL reaction volume composed of 20× presented probes, 50 ng cDNA, and 2× TaqMan™ Gene Expression Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) in a ViiA™ 7 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using the following program: 10 min at 95 °C for initial denaturation and 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Expression was calculated according to the comparative ΔΔCt method in which Gadph (Mm99999915_g1) and Rps27a (Mm01180369_g1) were used as endogenous controls and normalized to each untreated control using QuantStudio™ Real-Time PCR Software and ExpressionSuite Software (Thermo Fisher Scientific, Waltham, MA, USA).
2.12. Tumor Tissue Preparation for Enzyme-Linked Immunosorbent Assays (ELISA)
Tumor specimens were frozen in liquid nitrogen, suspended in RIPA buffer (Sigma-Aldrich, Saint-Louis, MO, USA) that contained protease and phosphatase inhibitors (both Sigma-Aldrich, Saint-Louis, MO, USA), and then mechanically homogenized (MP Biomedicals, Santa Ana, CA, USA). The samples were centrifuged at 10,000× g for 10 min at 4 °C after homogenization, and supernatants were transferred to fresh Eppendorf tubes. Protein concentration in the homogenates was measured using the Quick Start™ Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Tumor protein samples were used for ELISA.
2.13. Quantitative Protein Evaluation by ELISA
ELISA kits were used to assess the expression of selected proteins in mouse plasma and tumor homogenates. Assays were performed according to the manufacturer’s protocols, and absorbance values were measured using a plate reader (BioTek Instruments, Winooski, VT, USA). The expression of the following proteins was measured in plasma: C-C motif chemokine ligand 2 (CCL2), TGF-β (eBioscience, Amsterdam, The Netherlands), IL-6 (Wuhan EIAab Science Co, Ltd., Wuhan, China), OPN (Thermo Fisher Scientific, Waltham, MA, USA), and vascular endothelial growth factor (VEGF; Invitrogen, Carlsbad, CA, USA). The expression of the following proteins was measured in tumor samples: CCL2 (eBioscience, Amsterdam, The Netherlands), fibroblast growth factor 23 (FGF-23; Abcam, Cambridge, UK), IL-6 (Wuhan EIAab Science Co, Ltd., Wuhan, China), OPN (Thermo Fisher Scientific, Waltham, MA, USA), SDF-1, TGF-β, and VEGF (Invitrogen, Carlsbad, CA, USA). The results obtained were analyzed using the CurveExpert ver. 1.4 software.
2.14. Statistical Analysis
Statistical analysis was performed using the GraphPad Prism 7.1 software. Using the Shapiro–Wilk data normality test, the normality of the data distribution was analyzed (assumption of significance of the test for p < 0.05). The datasets that did not meet normality requirements (did not pass the Shapiro–Wilk test with α = 0.05) were further tested using the Kruskal–Wallis test using Dunn’s post-test for multiple comparisons (if not otherwise stated in the dataset description). Datasets that met the normality requirement were further analyzed using a one-way ANOVA followed by Sidak’s post-hoc test for multiple comparisons (since only selected, biologically/experimentally meaningful comparisons were challenged Sidak’s post-test provides better correction for multiple comparisons, thus, provides a better test power). The statistical analysis of individual data, depending on their distribution, is presented in table and figure legends. Differences between groups for which p < 0.05 were considered statistically significant.
4. Discussion
To further understand the effect of varying vitamin D
3 status on the growth and metastasis of breast cancer, we implanted two metastatic mouse breast cancer cells (4T1 and E0771) and nonmetastatic cells (67NR) into mice fed with vitamin-D
3-normal, vitamin-D
3-deficient, and vitamin-D
3-supplemented diets. Implantation was performed when differences in the plasma 25(OH)D
3 concentrations statistically differed between normal and experimental diets, which was achieved after 6 weeks of feeding [
3]. On day 7 after tumor transplantation, calcitriol gavage (1 µg/kg/day) was started in mice fed with normal and deficient diets and the same diets were continued [
3]. As discussed earlier, the effect of the above treatments on tumor tissue blood flow was the highest in deficiency groups in both 4T1 and E0771 tumors: vitamin D
3 deficiency significantly increased selected blood-flow parameters, whereas a deficient diet followed by calcitriol gavage led to a significant decrease in most of them. However, in 4T1 tumors (not in E0771), some parameters describing the blood flow were also decreased, but other parameters were increased in mice fed with a normal diet and administered with calcitriol. The modulation of tumor vascularization is not only important in the context of metastasis but may also have an impact on the response to treatment. Decreased blood flow in the tumor may cause a decrease in the response to chemotherapy [
40]. A previous study showed that calcitriol treatment led to decreased vascularization of MCF-7 human breast cancer tumors with overexpression of VEGF [
41]. However, other studies have shown that calcitriol stimulated in vitro expression of VEGF and downregulated the expression of thrombospondin 1. In T47D human breast cancer, calcitriol in vivo inhibited tumor growth, but had no effect on vascular density. Moreover, calcitriol showed a stimulating effect on the plasma VEGF concentration in the tested mice [
42]. In the present study, no effect of the used treatment on VEGF expression was observed in 4T1 or E0771 tumor tissue or in mice plasma; however, in 67NR tumor-bearing mice, in which no effect of treatments on blood flow was noted, the VEGF plasma concentration was decreased irrespective of the treatment as compared to control mice. Therefore, it can be concluded that the effect of vitamin D
3 on tumor vascularization or angiogenic factors may depend on the type of tumor. Our studies have indicated that the basal expression of molecules, which can be implicated in the angiogenesis process, differ significantly between mouse mammary gland tumors and the plasma of tumor-bearing mice. For instance, in this study, the concentration of CCL2 was significantly higher in the tumor-bearing mice and in the plasma of E0771-bearing mice; however, OPN was high in concentration in 4T1 plasma and tumor tissue. Both cytokines can modify the tumor microenvironment, either by directly influencing angiogenesis [
43,
44] or via recruiting macrophages (CCL2) [
45] and activating fibroblasts (OPN) [
24]. This effect may also depend on the dose and schedule of treatment with calcitriol or vitamin D
3 (as can be seen from different effects of calcitriol gavage on, for example, blood flow in tumor tissue in mice fed with vitamin-D
3-normal or vitamin-D
3-deficient, or vitamin-D
3-supplemented diet) as well as the observation time. In our previous studies, a decrease in the 4T1 tumor tissue VEGF concentration was found on day 14 of tumor progression in mice injected subcutaneously (0.5 µg/kg/day) with calcitriol or its analogs, with increased PE and TTP observed in young mice on day 24 [
2]. However, the effect on 4T1 cancer metastasis was the same, namely in mice from which tissue samples were collected in the present study [
3], as well as in our previous work [
2]: in all groups of mice in which calcitriol was administered or vitamin D
3 content in the diet was increased, an enhanced metastatic process was observed [
2,
3].
As evidenced from previous studies, CAFs play an important role in invasion and metastasis as well as in angiogenesis of tumors [
20,
46]. Tumor cells can stimulate stromal fibroblasts to differentiate into CAFs, and the crosstalk between cancer cells and stromal fibroblasts leads to tumor progression [
24]. In the present study, the phenotype of CAFs as well as lung NFs from tumor-bearing and healthy mice was analyzed. Upon various vitamin D
3 or calcitriol treatments, significant changes in the phenotype of CAFs were observed only in E0771 tumors. Interestingly, in lung NFs from healthy C57BL/6 mice, calcitriol did not influence the expression of studied molecules, but in NFs from BALB/c mice, α-SMA expression was decreased in all treatment groups. However, in tumor-bearing mice, the effect of various treatments on NFs was dependent on the tumors implanted. No significant effects were observed on NFs from 67NR nonmetastatic and E0771 metastatic tumors. Whereas in highly metastatic 4T1 tumor-bearing mice, signs of fibroblast activation were observed in both groups of mice administered with calcitriol but were more pronounced in mice on a deficient diet (100 IU+cal). NFs from this group of mice showed increased expression of α-SMA and podoplanin, as well as TNC, which suggests increased activation of NFs [
47]. Interestingly, the expression of these proteins was significantly higher in the 100 IU+cal group also as compared to the supplemented group. In this tumor model, metastases were located in the lungs and were significantly increased in both groups treated with calcitriol as well as in the 5000 IU group but by a lower number (count for macrometastases presented earlier [
3] as mean number ± standard deviation is as follows—control 40 ± 4; 1000 IU+cal 67 ± 27; 5000 IU 52 ± 11; 100 IU 44.5 ± 15; 100 IU+cal 77 ± 27) [
3]. This proves the impact of the mammary gland tumor type on the biological effects of vitamin D
3. For example, the expression of OPN and SDF-1, which are some of the important molecules secreted from cancer cells stimulating the differentiation of fibroblasts to myofibroblasts [
24,
48], was the highest in plasma and/or tumor tissue of 4T1 as compared to 67NR and E0771 tumor-bearing mice. In the present study, no significant effects of vitamin D
3 on plasma OPN were observed, but previously, in later stages of 4T1 tumor progression (days 28 and 33), calcitriol injections led to an increased plasma concentration of OPN [
2]. Moreover, increased features of NF activation noted in 4T1 tumor-bearing mice, which were the most visible in the 100 IU+cal group, may also be an effect of the increased tumor tissue concentration of IL-6, a cytokine that may activate fibroblasts through the induction of STAT3 phosphorylation [
49]. As a consequence of myofibroblast activation, the increased expression of α-SMA on NFs may result in macrophage recruitment and M2 polarization as well as myeloid-derived suppressor cells (MDSC) and regulatory T lymphocytes (Tregs) recruitment and their differentiation with Th2 polarization [
50,
51]. Similarly, the expression of podoplanin on fibroblasts may be responsible for M2 polarization and immunosuppression of the tumor microenvironment [
52]. In addition, TNC-expressing fibroblasts modulate the recruitment of monocytes/macrophages [
53]. Our previous study has shown that treatment of 4T1 tumor-bearing young mice with calcitriol or its analogs (mice on a diet containing a normal concentration of vitamin D
3) led to an increase in the percentage of monocytes in the blood [
54] and an increase in the ratio of inflammatory (Ly6C
highCXCR1
lowCCR2
+) spleen monocytes to monocytes with repair and anti-inflammatory properties (Ly6C
lowCXCR1
high) [
27]. Moreover, previous ex vivo studies confirmed that cultured media from 4T1 cells enhanced the M2 polarization stimulated by calcitriol; moreover, M2 macrophages differentiated in the presence of calcitriol stimulated the migration of 4T1 and 67NR cells in vitro [
55]. Treg prevalence with Th2 polarization was also observed in calcitriol-treated mice bearing 4T1 tumors [
4,
54]. Together, these factors lead to the modulation of blood flow and/or increased metastasis as an effect of calcitriol treatment of mice bearing 4T1 tumors observed in our studies [
3].
Further analysis of the effects of calcitriol on fibroblast activation was performed on lung NFs from healthy BALB/c and C57Bl/6 mice treated with various diets and/or calcitriol. It is worth emphasizing that NFs from these two strains of mice react differently to various vitamin D
3 or calcitriol treatments. The differences between these two strains of mice, for example, in the concentration of vitamin D metabolites were already reported. For example, higher plasma concentrations of 25(OH)D
3, 24,25(OH)
2D
3, and 3-epi-25(OH)D
3 were observed in C57BL/6 than in BALB/c mice in the control (1000 IU), 1000 IU+cal, and 5000 IU groups [
3]. In the present study, decreased α-SMA expression on NFs from BALB/c mice was observed in all treatment groups, but no effect was observed in C57BL/6 mice. In both strains of healthy mice, vitamin D
3 deficiency combined with calcitriol gavage (100 IU+cal) led to an increased plasma concentration of OPN, but CCL2 and IL-6 were increased in these groups only in BALB/c mice; in C57BL/6 mice, the opposite tendency was observed. NFs growing in such microenvironments modified by varying vitamin D
3 treatments were stimulated ex vivo with TGF-β or cancer cell CM. Because the most significant changes in fibroblasts were observed in deficiency groups, the gene expression in NFs of these mice was also analyzed. However, these ex vivo studies did not fully confirm the observations of lung NF activation in 4T1 tumor-bearing mice. In the ex vivo experimental conditions of the present study, there were limited components of the full microenvironment in which CAFs interact with tumor cells and with TAMs, and their interactions are important for their final phenotype [
56]. In NFs from deficiency groups, 4T1 CM stimulated
Mmp9 expression as well. Circulating MMP9 was previously described as inversely correlated with the vitamin D plasma concentration, with the highest concentrations in vitamin-D-deficient healthy adults [
57]. Furthermore, E0771 CM stimulated the expression of
Spp1. Our previous study demonstrated that calcitriol stimulated OPN secretion in vitro in a mouse BALB/3T3 NF cell line [
27], and others reported a similar effect of calcitriol on other mouse and human fibroblast cell lines [
32]. This effect is dependent on the interaction of VDR with the
Spp1 gene [
58]. Moreover, the authors of these studies reported that CAFs and NFs expressed VDR, and the expression of selected genes such as CD82 and S100A4 correlated with stromal VDR expression and clinical outcome in patients with colorectal patients [
32]. VDR expression was also analyzed in stimulated lung NFs in our study. In E0771-stimulated NFs, the expression of
Vdr mRNA was the highest in the 100 IU+cal group, in which the expression of
Spp1 was also the highest, but on the protein level, the expression of VDR was lower in this group of mice as compared to the control. Thus, in this study, VDR expression did not correspond to the observed effects of vitamin D deficiency/supplementation, also when measured in whole tumor tissue, as we previously described [
3].
The differences between two metastatic mouse mammary gland cancer models observed in response to vitamin D
3 can be clearly visualized. Of particular note are the differences in pulmonary NFs responses to different modes of vitamin D
3 delivery in the 4T1 tumor model. Mice fed with a vitamin-D
3-supplemented diet (5000 IU) showed decreased expression of proteins responsible for protumoral activity of myofibroblasts (α-SMA, PDGFRβ, and TNC). This effect was accompanied by high plasma concentrations of vitamin D metabolites 25(OH)D
3 and 24,25(OH)
2D
3 at the beginning of tumor progression, but significantly decreased during continued supplementation in the late stages of tumor growth. High concentrations of 3-epi-25(OH)D
3 were observed during the whole period of observation [
3]. In the E0771 tumor model, no change in lung NF activity was observed, but CAFs in mice fed with a vitamin-D-supplemented diet (5000 IU) were activated as podoplanin, TNC, and PDGFRβ increased. In these mice, however, all vitamin D metabolites measured were high during the whole experiment [
3]. Thus, it can be noticed that vitamin D
3 supplementation may lead to decreased or increased fibroblast activation, which is dependent on the tumor model/microenvironment and may also be dependent on the mice strain studied [
3]. Interestingly, vitamin D deficiency (which in general does not lead to increased metastatic process, like vitamin D
3 supplementation or calcitriol administration in the 4T1 model, but shows a tendency for an increased number of micrometastases [
3]) also led to decreased NFs activation in the 4T1 model (decreased podoplanin and TNC) and to some signs of increased activation of CAFs in the E0771 model (increased podoplanin). In these deficient mice, a low concentration of vitamin D metabolites was observed during the whole experiment [
3]. The most interesting observation is the comparison of calcitriol administration in mice fed with a normal vs. deficient diet, especially in the 4T1 model. Only α-SMA induction on NFs was observed in the former, but in the latter, podoplanin and TNC also increased significantly. Mice fed with a deficient diet and treated with calcitriol showed the lowest concentrations of all vitamin D metabolites, also as compared to mice on a deficient diet [
3].
In summary, the impact of vitamin D
3 supplementation or calcitriol administration on blood flow or CAFs and lung NFs is not visible in less invasive tumor models, such as 67NR, despite induced changes in plasma and/or tumor tissue concentrations of OPN, CCL2, TGF-β, VEGF, and FGF23. Moreover, differences between invasive tumor models were observed in response to the above treatments. In E0771-tumor-bearing mice, the influence on the metastatic process was not visible, but a tendency of tumor growth inhibition was observed in the 5000 IU diet as well as in 100 IU+cal groups [
3]. In the latter, a decrease in the blood flow was also observed, which was accompanied by a decrease in FGF23 and CCL2 in tumor tissue, but an increase in TNC on CAFs. Interestingly, similar effects on blood flow (decrease) were also observed in 4T1 tumors from the 100 IU+cal group, but it did not affect the CAF phenotype. At the same time, increasing α-SMA, podoplanin, and TNC on lung NFs and increasing metastasis.