Calcitriol and Its Analogs Establish the Immunosuppressive Microenvironment That Drives Metastasis in 4T1 Mouse Mammary Gland Cancer

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.


Supplementary
. Calcitriol, PRI-2191, and PRI-2205 increased lymphocyte and monocyte percentage with a parallel decrease in granulocytes percentage in mice bearing 4T1 mammary gland tumors.
(A) General white blood cell count (WBC); the number (B) and the percentage (C) of: lymphocytes, (D) and (E) monocytes, (F) and (G) granulocytes. From day 7, vitamin D analogs were administered subcutaneously (s.c.) thrice a week. The single dose of compounds was as follows: calcitriol, 0.5 µg/kg; PRI-2191, 1.0 µg/kg; and PRI-2205, 10.0 µg/kg. Number of mice was 9-12 per group. The blood morphology was evaluated in each blood sample using the Mythic 18 automatic analyzer. Data are presented as mean ± SD. Statistical analysis: Kruskal-Wallis multiple comparison test. *p<0.05. Figure S2. Selected blood morphological parameters of mice bearing 4T1 mammary gland tumors and treated with calcitriol and its analogs: PRI-2191 and PRI-2205.

Supplementary Figure S3. Selected cytokine levels in plasma and supernatants from lipopolisaccharide (LPS)-or Concanavalin A (ConA)-stimulated splenocytes.
Supernatants obtained from spleen cells (harvested on days 14 and 28) stimulated with LPS (A) and ConA (B). Plasma from mice harvested on days 14 or 21 and 28 or 33 (C). Samples were analyzed with ELISA tests. Number of samples analyzed was 2-6 per group. Data are presented as mean ± SD and individual sample results. Real-time PCR analysis of four selected genes. (A) Ceruloplasmin (Cp), (B) Coagulation factor II, thrombin (F2), (C) Fibrinogen alpha chain (Fga), and (D) Serum amyloid A (Saa). Real-time PCR reaction was performed using specific primers coding following genes: Cp (Mm01289313_m1), F2 (Mm00438843_m1), Fga (Mm00802584_m1), and Saa (Mm04208126_m1). Briefly, 50 ng of cDNA was used for a single reaction and each sample was performed in triplicate in a single experiment. Data were analyzed using comparative ΔΔCt method by DataAssist 3.01 software in comparison to endogenous control: hypoxanthine phosphoribosyltransferase 1 (Hprt1, Mm00446968_m1). Mice were orthotopically inoculated with 4T1 cells on day 0. From day 7 (7 days after tumor inoculation), vitamin D analogs were administered subcutaneously (s.c.) thrice a week. A single dose of compounds were as follows: calcitriol, 0.5 µg/kg; PRI-2191, 1.0 µg/kg; and PRI-2205, 10.0 µg/kg. Number of mice: 9-12 per group. Data are presented as mean ± SD. Statistical analysis: Kruskal-Wallis multiple comparison test. *p<0.05.

Supplementary
(A) T lymphocytes CD3e + . (B) B lymphocytes CD19 + . (C) NK cells CD335 + . (D) TCD4 + lymphocytes. (E) TCD4 + CD25 + lymphocytes. (F) TCD8 + lymphocytes. (G) Representative dot plots of selected analysis performed on day 28. Six samples per group was analyzed, except: D0 = 2 and D7 = 3. Data analysis was performed using Becton Dickinson FACS Fortessa cytometer with FACSDiva software. Shown data represent mean ± SD. Statistical analysis: Kruskal-Wallis multiple comparison test. *p<0.05. Tables   Table S1. The fold change values of genes associated with precursor T cells differentiation in splenocytes samples from 4T1 tumor bearing mice treated with calcitriol or its analogs. Spleen specimens were collected on the days 21 and 28 (after inoculation with 4T1 cells) from mice treated with calcitriol or its analogs and control group receiving vehicle. Real-time PCR screening was performed using Mouse T Helper Cell Differentiation RT 2 Profiler Array (Qiagen, Hilden, Germany) including 84 key genes and 5 housekeeping genes in the set. Data shows a mean relative quantification (RQ) values. Fold-change (RQ) of target genes was defined using double delta Ct method in reference to actin, beta (Actb) and beta-2 microglobulin (B2m) for splenocytes samples. Then the results were adjusted to the values obtained for the control group within the day 21 or 28 th of the experiment for each treatment group. Data analysis was acquired using Qiagen online software suitable for purchased kit (Qiagen, Hilden, Germany). PCR amplification cycles were as follows 95 °C for 10 s and 58 °C for 45 s (50 cycles). We used 0,5 µg of cDNA (6 mice pooled per group) for a single reaction.

Table S4. Pixel densities of cytokines contained in the supernatants from stimulated with Concanavalin A (ConA) splenocytes obtained using Mouse Cytokine Array Panel A(A-E).
A. Spleen specimens were collected on the days 0, 7, 21 and 33 (after inoculation with 4T1 cells) from mice treated with calcitriol or its analogs and control group receiving vehicle and stimulated with Concanavalin A (ConA). Results were obtained using Proteome Profiler Mouse Cytokine Array Kit. Panel A (R&D alp Systems. Inc. USA) according to the enclosed instruction. This array detects 40 mouse cytokines, chemokines, and acute phase proteins simultaneously. Pixel densities on developed X-ray film were collected using a multifunctional scanning device (Samsung SLC460) or Image Station 4000MM PRO (Carestream Health. Rochester. New York. USA) and image analysis software (ImageJ 1.48v). For each spot the final optical density level was determined as a factor acquired by subtracting the background optical level and dividing by values obtained from the untreated mice (Day 0). Lymph node samples were collected from mice treated with calcitriol or its analogs and control group receiving vehicle on the days 21 and 28 (after inoculation with 4T1 cells). Real-time PCR screening was performed using Mouse T Helper Cell Differentiation RT 2 Profiler Array (Qiagen. Hilden. Germany) including 84 key genes and 5 housekeeping genes in the set (Table S1). Data presented as a mean relative quantification (RQ) values. Foldchange (RQ) of target cDNA was defined using double delta Ct method in reference to beta-2 microglobulin (B2m), glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and heat shock protein 90 alpha, class B member 1 (Hsp90ab1). Next the results were adjusted to the values obtained for the control group within the day 14 or 28 th of the experiment for each treatment group. Data analysis was acquired using Qiagen online software suitable for purchased kit (Qiagen. Hilden. Germany). PCR amplification cycles were as follows 95 °C for 10 s and 58 °C for 45 s (50 cycles). We used 0.5 µg of cDNA (6 mice pooled per group) for a single reaction. Lung specimens were collected on the days 14 and 28 (after inoculation with 4T1 cells) from mice treated with calcitriol or its analogs and control group receiving vehicle. Real-time PCR screening was performed using the mouse tumor invasion/metastasis PCR array library (MTIM-1). From 88 genes available in this array (Table S2) the expression for 45 genes was not detected in 4T1 lung tissue. Data presented as a mean relative quantification (RQ) values (calculated from duplicate). Fold-change (RQ) of target cDNA was determined by calculating the differences in ΔΔCT values in reference to ribosomal protein L13A (Rpl13a) and adjusted to the values obtained for the untreated mice (named D0) for each treatment group. Data analysis was performed by DataAssist v 3.01 software. All PCR amplification cycles were performed at 95 °C for 10 s and 58 °C for 45 s (50 cycles). We used 25 ng of cDNA for a single reaction and each test was performed in duplicate.