DIM (purity > 98%), dimethylsulfoxide (DMSO), 2-hydroxypropyl-β-cyclodextrin and Con A were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse GM-CSF and IL-6 were purchased from PeproTech (Rocky Hill, NJ, USA). MiR-21 precursors (pre-miR-21), miR-21 inhibitors (anti-miR-21) and the corresponding controls (pre-scramble or anti-scramble) were purchased from Gene Pharma (Shanghai, China). All primers used in the experiments were synthesized by Real Gene (Nanjing, China) and were shown in Additional file 2: Table S1. Mouse PD-1 antibody was obtained from Bioxcell (New Hampshire, USA). Fluorescence labeled antibodies used for flow cytometry were purchased from BioLegend (San Diego, CA, USA). Information of the antibodies used for Western blot in this study is listed in Additional file 2: Table S2. DIM was dissolved in DMSO for cell treatments. Due to the poor water solubility of DIM, it was formulated in 2-hydroxypropyl-β-cyclodextrin to increase its water solubility and to avoid the clearance of the mononuclear phagocytic system when used in vivo . Briefly, 20 mg DIM was dissolved in 1 mL of 2-hydroxypropyl-β-cyclodextrin solution (molar ratio 1:10). The stock solution (20 mg/ml) were serially diluted in sterile PBS on the day of use to get certain concentrations according to our previous study .
Animals and cell lines
Wild-type (WT) C57BL/6J and BALB/c mice were purchased from the Laboratory Animal Center of Nanjing University (Nanjing, China). MiR-21loxp/loxp and Cmv-Cre mice in the C57BL/6 background were purchased from Biomodel Organism (Shanghai, China). MiR-21loxp/loxp mice crossed with Cmv-Cre mice to generate miR-21−/− mice. Then, they backcrossed to WT BABL/c mice for 7 generations to obtain miR-21−/− mice with BALB/c background. miR-21−/− mice were determined via PCR assay of tail genomic DNA. The primers used to distinguish WT and miR-21 knockout mice were shown in Additional file 2: Table S1. PCR procedures were as follows: 94 °C for 5 min, 94 °C for 30 s, 60 °C for 45 s and 72 °C for 45 s, for 30 cycles. Agarose gel electrophoresis was performed to examine the length of PCR products: WT (758 bp length) and miR-21 deficiency (480 bp length).
Experimental mice were housed at a specific pathogen free (SPF) environment under a ventilated, temperature-controlled room (23 °C) with a 12 h light/12 h dark cycle. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Nanjing University and approved by the Animal Ethics Committee of Nanjing University.
According to the manufacturer’s instructions, we used cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) to obtain primary MDSCs from bone marrow (BM) and spleen. The primary CD4+ and CD8+ T cells from the spleen were isolated using BD IMag Anti-Mouse CD4 Magnetic Particles-DM and BD IMag Anti-Mouse CD8 Particles-DM (BD Biosciences, San Jose, CA, USA). The murine breast cancer 4T1 cells (ATCC, Manassas, Virginia, USA) and primary T cells were cultured in RPMI 1640 containing 10% FBS (Life Technologies, Grand Island, NY, USA). The murine melanoma B16-F10 cells (ATCC) were cultured in DMEM containing 10% FBS. The cells were incubated at 37 °C with 5% CO2.
To induce BM-derived MDSCs, BM cells from WT mice (female, 5 weeks old) and miR-21−/− mice (female, 5 weeks old) were stimulated with 40 ng/ml GM-CSF and 40 ng/ml IL-6 for 4 days in RPMI 1640 containing 10% FBS. Under some circumstances, different concentrations of DIM (10, 20, 40 or 80 µM) were added. Pre-miR-21, anti-miR-21, PIAS3 or PTEN expressing plasmid (VectorBuilder Inc., Guangzhou, China) and their corresponding controls were transfected into different treated MDSCs. Then part of the MDSCs was harvested to examine the expansion states by flow cytometry, and others were used to detect the miR-21 level, the mRNA levels of Arg1 and iNOS via qRT-PCR or the expression levels of potential miR-21 targeting genes by Western blot. For T cell proliferation assay, the CFSE-labeled CD4+ and CD8+ T cells were stimulated with 2 µg/ml Con A and cocultured with differently treated MDSCs at the ratio of 1:0.5 or 1:1 in 48-well plates for 3 days. Then T cell proliferation was analyzed by flow cytometry.
A total of 1 × 106 4T1 cells were injected into the fat pad of a mammary gland of WT BALB/c or miR-21−/− mice with BALB/c background (female, 6–8 weeks old, 18–22 g) to establish mouse breast cancer model and a total of 1 × 106 B16-F10 cells were injected subcutaneously into the left armpit of C57BL/6 mice (female, 6–8 weeks old, 18–22 g) to construct mouse melanoma cancer model.
Tumor-bearing mice were randomly assigned one week after tumor implantation and each group contained 8 mice. 4T1 Tumor-bearing mice were intraperitoneally injected with various dose of DIM (2, 5, 10 mg/kg DIM) at a volume of 100 µl three times a week from day 8 after tumor cell inoculation. Control mice were received with the same volume of 2-hydroxypropyl-β-cyclodextrin solution. To investigate the inhibitory effect of DIM on tumor growth through reducing MDSCs, DIM-treated 4T1 tumor-bearing mice were intravenously injected with 5 × 106 of purified BM-MDSCs on day 13, 16, 19 after tumor cell inoculation. To examine whether DIM could improve the anti-tumor effect of anti-PD-1 mAb, breast cancer and melanoma tumor-bearing mice were intraperitoneally administrated with 10 mg/kg DIM three times a week from day 8 after tumor cell inoculation and intraperitoneally injected with anti-PD-1 mAb (0.25 mg/mouse) on day 11, 13, 15, 17, 19 after tumor cell inoculation. We measured the tumor size with a caliper every three days. On day 21, the mice were anaesthetized with pentobarbital sodium (80 mg/kg, i.p.) and blood samples were collected on ethylenediaminetetraacetic acid (EDTA) by cardiac puncture. Finally, the animals were killed by cervical dislocation. MDSCs from bone marrow, blood, spleen and tumor as well as T cells from blood and spleen were harvested for flow cytometry. Besides, IFN-γ level in the tumors from differently treated mice was measured by ELISA (eBioscience, San Diego, CA, USA).
Flow cytometry analysis
The collected cells were blocked in 100 µl 1% bovine serum albumin for 30 min on ice and then stained with FITC-Gr-1, APC-CD11b, FITC-Ly-6 C, PE-Ly-6G, FITC-CD4, or APC-CD8 for another 30 min on ice. Flow cytometry was performed on a FACSCalibur device (Becton Dickinson, Franklin Lakes, NJ, USA) and the results were analyzed via using FCS Express V3 (DeNovo Software, Los Angeles, CA, USA).
According to the manufacturer’s protocol, total RNA was isolated from cells by the Trizol reagent. For mRNA detection, 500 ng of total RNA were used for complementary DNA synthesis (One Step SYBR PrimeScript™ RT-PCR Kit, Takara, Shiga, Japan), according to the manufacturer’ instructions. For miR-21 detection, 2 µg of total RNA was used for first-strand DNA synthesis using AMV reverse transcriptase (Takara) and a stem-loop RT-primer (Life Technology). Real-time q-PCR was performed by 7300 real time PCR System (Applied Bio-systems, Foster City, CA) according to the manufacturer’s protocol. mRNA level was normalized to β-actin, while miR-21 expression was normalized to small nuclear RNA U6.
Total protein of cells was extracted with lysis buffer. Then the protein concentration was measured via Protein Quantitative Analysis Kit (shenergy Bio-color, Shanghai). 60 µg protein lysate was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred onto polyvinylidene difluoride (PVDF) membrane (Immobilon P, Millipore, Milford, MA). Membranes were blocked for 1 h using 5% skim milk and incubated with diluted primary antibody at 4 °C gently shaking overnight. Then the membrane was washed five times using PBST and incubated for 1 h at room temperature with secondary HRP-conjugated Antibody. The bound antibody was detected by ECL (Cell Signal Technology).
Results are expressed as the mean ± standard error (SEM). Data was statistically analyzed using GraphPad Prism 8.0.2 (GraphPad Software Inc. La Jolla, CA, USA) and assessed for normality or homogeneity of variance. Significant differences between two groups were evaluated using two-tailed Student’s t-test. Differences between multiple groups were compared using one-way ANOVA with Dunnett’s tests or, if appropriate, using one-way ANOVA with post-hoc Bonferroni correction. The difference was considered significant when p < 0.05; ns = not significant.