Mice
C57BL/6, Rag1-/- and Foxp3-GFP-DTR mice were obtained from Jackson Laboratory, USA. All the mice were housed and maintained in a conventional pathogen-free environment at the Small Animal Facility (SAF) located at Translational Health Science and Technology Institute (THSTI). For performing animal experiments prior approval is obtained by Institutional Animal Ethics Committee (IAEC project protocol approval No. IAEC/THSTI/114). All the animal experiments are handled by experienced personnel. The experiments are planned and executed according to the guidelines laid by institutional animal ethics committee of THSTI.
Cell lines
B16F10 and B16F10-ova melanoma cell lines were obtained from American Type Culture Collection (ATCC). The cell lines were maintained in R10 media which comprises of RPMI 1640 (by Invitrogen) complete media containing 10% (v/v) fetal bovine serum (FBS) by Gibco, Penicillin (100 U/ml) by Sigma-Aldrich, Streptomycin (100 μg/ml) by Sigma-Aldrich, L-glutamine (2mM) by Sigma-Aldrich, and HEPES ( working:10mM; stock:0.5M) by Sigma-Aldrich. In this study we have not used any cell lines found in the Register of Misidentified Cell lines which is maintained by International Cell line authentication Committee. (http://iclac.org/databases/cross-contaminations/).
Tumour implantation
B16F10 and/or B16F10-ova cell lines are cultured in sterile R10 medium at 370C incubator maintained with 5% CO2. Six to eight weeks old male mice were used in the study. Each Mice were injected with 0.2x106 cells subcutaneously on the left flank region or injected intravenously particularly for metastasis studies. The mice were kept on normal laboratory diet, high Zn supplement or Zn deficient diet as specified in each experiment. Tumor volume is recorded each day until it reaches 2000 mm3. The formula to measure tumor volume: (mm3) = L x W x W x 0.5, where L=length and W=width of tumor mass (in millimetres). Daily intake of food, water and body weight is measured. Once the tumor volume reaches 2000 mm3 the animals were euthanized and its organs and other samples are collected to use them for various studies. For the survival studies, the animals were kept under observation and the day of mortality of individual animal was recorded to prepare the percent survival curve for 100 days.
Antibodies
Anti mouse: ⍺-CD45.2 (1:700; 104, Biolegend), ⍺-CD3 (1:700; 145-2C11, BioLegend), ⍺-CD4 (1:1000; GK1.5, BioLegend), ⍺-CD8 (1:1000; 53-6.7, BioLegend), ⍺-NK1.1 (1:700; PK136, BioLegend), ⍺-𝛾ẟTCR (1:700; GL3, BioLegend), ⍺-PD1 (1:500; 29F.1A12, BioLegend), ⍺-CTLA-4 (1:500; UC10-4B9, BioLegend), ⍺-TIM3 (1:600; B8.2C12, BioLegend), ⍺-CD107a (1:700; 1D4B, BioLegend), ⍺-CD96 (1:700; 3.3, BioLegend), ⍺-TIGIT (1:700; 1G9, BioLegend), ⍺-Gr1 (1:1500; RB6-8C5, BioLegend), ⍺-CD11b (1:1500; M1/70, BioLegend), ⍺-F4/80 (1:700; BM8, BioLegend), ⍺-CD206 (1:700; C068C2,BioLegend), ⍺-IFN (1:500; XMG1.2, BioLegend), ⍺-TNF (1:500; MP6-XT22, BioLegend), ⍺-IL-17A (1:500; TC11-18H10, BioLegend), ⍺-perforin (1:500; S16009A, BioLegend), and ⍺-Ki67 (1:1000; SolA15, Thermo Fisher Scientific).
Flow Cytometry and intracellular cytokine staining
Organs (Spleen, lymph nodes and tumor) are processed and tissue infiltrated lymphocytes are isolated and stained for surface markers by fluorescence-tagged antibodies. Staining for surface markers should be done in fluorescence-activated cell sorting (FACS) buffer (1X PBS and 2% FBS). For intracellular cytokine staining, the cells are stimulated for cytokine production with phorbol 12-myristate13-aceate (PMA; 50 ng/ml; Sigma-Aldrich), ionomycin (1 g/ml; Sigma-Aldrich) and monensin (#554724 GolgiStop, BD Biosciences) for 4 hr at 370C and 5% CO2. Suraface staining (10 staining) is performed with antibodies diluted in FACS buffer as indicated above and incubated for 30 min at 40C in dark. Then the cells were fixed in Cytofix and permeabilized with Perm/Wash buffer using the Fixation Permeabilization Solution kit (#554714,BD Biosciences). The secondary staining is then performed to stain the intracellular cytokines by diluting the antibodies in permeabilization buffer and incubated for 30 min at 40C in dark. The cells were then washed and proceeded for flow cytometry (Canto II, BD Biosciences). Data analysis was performed using FlowJo software (TreeStar).
Quantitative polymerase chain reaction
The total RNA was extracted from the samples using an RNeasy kit (#74104, Qiagen) and complementary DNA (cDNA) were synthesized using an iScript cDNA Synthesis kit (#1708891,Bio-Rad) according to manufacturer’s instructions.Real-time qPCR was performed using SyBr green gene expression assay and the Fast 7500 Dx qPCR System (Applied Biosystems) using the following parameters: 950C for 15 min, 40 cycles of 940C for 15 s, 580C for 30 s, and 720C for 30 s. The Ct values for the individual samples (genes) was then normalized to the endogenous control GAPDH [ꞵ-actin/glyceraldehyde-3-phosphate dehydrogenase] gene expression. To determine the Ct value change (ΔCt), the Ct value of the endogenous control gene was subtracted from the Ct value of each target gene. Then the fold change is calculated to determine the relative expression of each gene using the formula [POWER (2, -ΔCt) × 10,000]74. All the primer sets were purchased from Sigma-Aldrich.The following primer sets were used: IFN𝛾, TNF⍺,T-bet, IL2, TGFꞵ, Foxp3, NFAT1, PD1, FOXO1, SIRT1, GAPDH. The results were analysed using SDS 2.1 software.
Statistical analysis
Statistical analysis was carried out with Graph Pad Prism 7.0 software. FACS and qPCR data were compared and analysed by using one-way ANOVA or Student’s t test for n = 7 mice per group. Graphs are depicted as means with SEM. P value of less than 0.05 was considered as statistically significant.
Lung metastasis
B16F10 cell lines are cultured in sterile R10 medium at 370C incubator maintained with 5% CO2. Six to eight weeks old male mice were injected with 0.2x106 cells intravenously into the tail vein. The mice were then kept on normal laboratory diet, high Zn supplement or Zn deficient diet. The lungs were isolated from the mice at different time points starting from the day 15th. The number of foci were counted and graphically represented. The metastasis mice were also monitored for over 100 days for survival study where the mortality of individual mice was recorded and represented graphically.
Histology
The tissues used for histology were fixed in 10% (v/v) formalin until the samples are sent for analysis. The tissue samples were processed and stained with hematoxylin and eosin to measure the number of pus zones. Masson’s Fontana stain to determine the melanin production and Immunohistochemistry using ⍺-Ki67 antibody to determine the level of Ki67 expression75. A certified histologist through blind sampling carried out the assessment of stained slides and gave the histological score on the scale of 0 to 5 (where 0 meant no staining).
Isolation of tumor-infiltrating immune cells
At the end of each study the tumors were excised and tumor infiltrated immune cells were isolated using Percoll density gradient method76. As the first step, tumor is chopped into smaller pieces and mechanically dissociated in the presence of complete IMDM media in a C-tube using a magnetic cell sorting dissociator (Miltenyi Biotec). The cells were pelleted by centrifugation at 2000 rpm for 5 min. The pellet is then subjected to enzymatic dissociation in the HBSS media or complete IMDM media that contains collagenase type IV (100 U/ml), deoxyribonuclease I (100 g/ml) and CaCl2 / MgCl2 (2.5mg/ml) (stock: 50mg/ml) to make up the volume to 3 ml and then incubated for 30 min at 370C shaker incubator. After the incubation, the dissociated tissue is passed through 70μm cell strainer and centrifuged at 2000 rpm for 5 min at room temperature. To the pellet add 5 ml of 63% percoll and slowly layer it with 3 ml of 47% percoll and finally layer on the top with 2 ml of 33% percoll. Centrifuge the tubes at 2000 rpm for 30 min at 40C without de-acceleration. After centrifuge, carefully isolate the tumor infiltrated lymphocytes obtained as a faint layer just above 63% gradient and suspended them in complete IMDM media and used for further analysis.
Immune checkpoint inhibition by anti-PD1 therapy
Anti-PD1 monoclonal antibody (RMP1-14, BioXcell) was used for immune checkpoint inhibition therapy. We have used both sub-optimal (5 mg/kg) and optimal (10 mg/kg) doses of ⍺-PD1 mAb for therapeutic purpose. Doses are given intra-peritoneously, two times a week starting from day when tumor becomes palpable77,78. Mice given ⍺-PD1 mAb doses were kept either on normal laboratory diet, Zn supplement or Zn deficient diet.
Zn chelation as a therapy model using Clioquinol (CQ)
Clioquinol (Cat. No. 33931, Sigma) is a tissue Zn chelator in mice. It removes only chelatable Zn pools, not the Zn that are bound to proteins. CQ doses are given intra-peritoneously as oral administration will not have any effect. Once injected CQ is absorbed rapidly and metabolised and will cause Zn chelation within 2 hr. We have used 25 mg/kg as the optimal dose and 10 mg/kg as the suboptimal dose, injected as a therapeutic dose once the tumor becomes palpable and also from the day of tumor implantation till endpoint.
Sorting of CD4 T cells, CD8 T cells and NK cells
Sorting of CD4 T cells, CD8 T cells and NK cells from isolated tumor-infiltrating immune cells is carried out on FACS Aria III (BD Biosciences) based on the surface markers CD3+, NK1.1+, CD4+, CD62L+, CD8+ according to the gating strategy. The purity of the sorted cells was ~85-95% in post-sort analysis. Sorted cells were then invitro activated and differentiated in the presence and absence of Zn to do the further analysis. The cells are used for staining followed by FACS and also for qPCR analysis.
AIMD mice
Antibiotic induced microbial depletion is carried out in C57BL/6 mice according to the earlier published protocol79. For the initial 3 days Amphotericin-B (0.1 mg/ml; Sigma-Aldrich) was administered orally two times a day. From day 3 onwards mice were kept on ampicillin water (1 g/litre; Sigma-Aldrich) and a cocktail comprising of vancomycin (5 mg/ml; Sigma-Aldrich), metronidazole (10 mg/ml; Sigma-Aldrich), and neomycin (10 mg/ml; Sigma-Aldrich) given through oral gavage twice daily. As the mice show caprophagy behaviour they were housed in separate cages once the antibiotic treatment is commenced throughout until endpoint. Depletion of microbiota was confirmed by absence of colony formation upon culturing of stool samples.
In Vitro stimulation
For invitro activation and stimulation freshly sorted cells, the 96-well plates were initially coated with ⍺CD3/⍺CD28 (2 μg/ml) and incubated overnight at 40C. The next day plates are centrifuged and supernatant is aspirated. Then the cells were seeded (0.1x106 cells per well) in the 96-well plate along with the respective cytokines in the IMDM media and incubated for 48-72 hr at 370C incubator maintained with 5% CO2. Naive CD4 T cells were differentiated into Th1 cells using IL12 (15 ng/ml), Th2 cells using IL4 (30 ng/ml), Th9 cells using TGFꞵ (2.5 ng/ml) and IL4 (30 ng/ml), Th17 cells using TGFꞵ (2.5 ng/ml) and IL6 (25 ng/ml) and iTreg cells using TGFꞵ (2.5 ng/ml). Activation of NK cells were done by culturing the cells in the media containing IL-2 (1000 U) and IL-12 (10 ng/ml) for 48-72 hr80,81. To determine the effect of Zn, we have carried out the activation and differentiation of the cells with or without 50μM Zn supplement (ZnSO4.7H2O, Sigma).