Cell culture
B16 melanoma cells (American Type Culture Collection (ATCC), Manassas, VA, USA) and Chinese hamster ovary-K1 (CHO, ATCC) cells were cultured in Dulbecco's modified Eagle's medium (DMEM, ThermoFisher, Carlsbad, CA, U.S.A.) containing 10% fetal bovine serum (Carpricorn Scientific, Ebsdorfergrund, Germany), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco BRL, Dublin, Ireland). CT-26 cells (provided by Dr. Kwon, Ajou University) were cultured in Roswell Park Memorial Institute (RPMI) 1640 media (GIBCO) containing 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin. HEK-293 T cells (provided by Dr. Kwon, Ajou University) were cultured in FreeStyleTM 293 expression medium (Gibco).
Construction of expression vectors for Tim-3 blocking molecules
IgV domain of Tim-3 was amplified by PCR using primers (forward primer: 5’-CGG GGT ACC GAT TGG AAA ATG CTT ATG TGT TTG AG and reverse primer: 5’-GAA TTC TGC TTT GAT GTC TAA TTT CAG TTC) and plasmid pIRES2-EGFP-Tim3SVMhIg [19]. The Tim3 V-domain DNA segment was inserted into the pSecTag2C vector (ThermoFisher Scientific, Waltham, MA, USA) containing mouse immunoglobulin (mIgG2a) CH2CH3 with and without hinge region, and named pSecTag2C-Tim3VdIg and pSecTag2C-Tim3VmIg, respectively.
Western blotting for detection of Tim3VdIg protein
CHO cells or HEK-293 T cells were transfected with a Tim3 expression vector using Polyethylenimine (Polyscience, PA USA). After 2 days, culture supernatant was collected and loaded on the sodium dodecyl sulfate polyacrylamide gel for electrophoresis (PAGE) or non-denatured PAGE followed by transfer onto polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). The membrane was incubated with anti-mouse IgG Ab conjugated with horseradish peroxidase (ZYMED® Laboratories, Invitrogen, Carlsbad, CA, USA) and then developed using an enhanced chemiluminescence kit (GE Healthcare, Little Chalfont, UK).
Production and purification of Tim3VdIg protein
HEK-293T cells (2x106/mL) were transfected with pSecTag2C-Tim-3VdIg using polyethylenimine. After 7 days, the supernatant was harvested from the culture and then Tim3VdIg protein was purified using Protein A beads (GE Healthcare, Little Chalfont, UK).
Evaluation of tumor growth
Tumor growth experiments in mice were approved by the Institutional Animal Care and Use Committee, Ajou University Medical Center (IACUC protocol #2016-0003). Six-week-old male C57BL/6 and BALB/c mice were purchased form OrientBio (Gyeonggido, Korea). Mice were maintained in specific pathogen free conditions and in separate cages according to strain and treatment. Antibiotics mixture containing 900 mg/L for ampicillin (Gold Biotechnology, Saint Louis, MO, USA), 900 mg/L for neomycin (BioVision, Milpitas, CA, USA), 900 mg/L metronidazole (BioVision, Milpitas, CA, USA), and 300 mg/L for vancomycin (Gold Biotechnology, Saint Louis, MO, USA) was provided to mice via drinking water. Drinking water was replaced every third day. Three weeks later, all mice were challenged subcutaneously with 100 µL tumor cells (3x106 cells/mL) which were B16 cells for C57LL/6 mice and CT-26 for BALB/c mice. Tim-3VdIg protein (60 µg/mouse) was intraperitoneally injected on every second day for 12 days after tumor challenge. Tumor progression was assessed every second day by determining tumor volume using the formula: tumor volume = 0.523 x tumor length x (tumor width)2.
Oral administration of bacteria to mice
Fecal bacteria stock was prepared by collecting feces from the large intestine of eight-week-old BALB/c and C57BL/6 mice under anaerobic condition. Feces were suspended in phosphate buffered saline (PBS) at concentration of 60 mg/mL and followed by centrifugation at 800 × g for 3 min. The supernatant was aliquoted for storage at -70oC until it was administered orally at an amount of 100 µL per mouse seven times in total, once every third day starting seven days before tumor challenge to a mouse. Lactobacillus johnsonii (Korean Culture Center of Microorganisms) and Enterococcus hirae (Korean Culture Center of Microorganisms) were cultured at 37oC in Brain Heart Infusion media and De Man, Rogosa, and Sharpe (MRS) agar, respectively. The bacteria were cultured to an OD of 1.8 measured at 600 nm (corresponding to 109 CFU/mL) and then aliquoted and cryopreserved in 15% glycerol. Each bacterial suspension (100 µL/head) was administered to a mouse seven times in total, every third day.
Collection of gut microbiota samples and bacterial DNA sequencing
Mice were sacrificed on eighth day after tumor cell challenge to collect cecal content, which was immediately frozen at -70oC for microbiome analysis. DNA was extracted from cecal samples using DNeasyPowerSoil Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA quality was assessed by gel electrophoresis and fluorometry. Sequencing libraries were constructed according to the Illumina 16S Metagenomic Sequencing Library protocols using Herculase II fusion DNA polymerase (Agilent Technologies, Santa Clara, CA) and the universal primer pair specific for the V3-V4 region of the 16S rRNA gene with Illumina adapter overhang sequences. The purified PCR product was quantified according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, Waldbronn, Germany). Paired-end sequencing was performed with Macrogen (Seoul, Korea) using the MiSeq™ platform (Illumina, San Diego, USA).
Sequence processing and taxonomic assignment
The FLASH (Fast Length Adjustment of SHort reads, 1.2.11) program was employed to merge paired-end reads [27]. Open reference operational taxonomy unit (OTU) picking was utilized using QIIME-UCLUST and NCBI databases.
Statistics
The statistical significance of differences in tumor growth between groups was analyzed using the Student’s t-test or ANOVA with Bonferroni multiple comparison test. p< 0.05 indicated statistical significance. Significant differences in alpha diversity were computed using Kruskal-Wallis test with Dunn’s multiple comparison test. Significant differences in beta diversity were computed using PERMANOVA and ANOSIM. Significant differences in dispersion were determined by permDISP.