2.1 Reagents:
All the recombinant proteins and antibodies used in the study are given in (Supplementary table 1 and 2) with details of dilution used, clone and brand.
2.2 Cells lines:
4T1 cells were provided by Dr. Abhijit De (ACTREC Mumbai). The cells were cultured and maintained in RPMI supplemented with 10% FBS, 1x Penicillin-Streptomycin-Neomycin, at 37°C in a humidified incubator with 5%CO2. Raw264.7 macrophages were cultured and maintained in DMEM supplemented with 10% FBS, 1%Pencillin-Streptomycin-Neomycin, at 37°C in a humidified incubator with 5% CO2.
2.3 Bone marrow derived macrophage (BMDMs):
BMDMs were generated following an already established lab protocol [32][33].Briefly, bone marrow cells were collected from femurs of BALB/c mice and cultured in RMPI-1650 medium supplemented with 10% FBS, 1x sodium pyruvate and HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid) buffer. Cells were cultured in recombinant proteins M-CSF (20ng mL− 1) for macrophage generation (M0). For polarization the cocktail of GM-CSF (20ng mL− 1) and IFNγ (10ng mL− 1) was used for M1 macrophages while M-CSF (20ng mL− 1), IL-4(10ng mL− 1) and IL-10(10ng mL− 1) for M2 macrophages. The cells were plat Extracellular ATP and Adenosine level were quantified by the help of manufacturing Kit. The bone marrow derives macrophages BMDMs was culture with tumor mimicking condition (TMC) for 3 days. The TCM-induced skewing macrophages to M2-like phenotype were confirmed by flow cytometry and ELISA.
2.4 Preparation of tumor mimicking conditioned media and cancer cell lysate:
Crude soluble antigen (CSA) was prepared using freeze-thaw methodology as explained in [32].Confluent 4T1 cells were freeze-thawed for 7 repetitive cycles of -80°C/37°C for 10 min each and centrifuged at 10,000 rpm for 15 minutes. The protein concentration was quantified using Bradford assay. The culture supernatant was harvested from 95% confluent 4T1 cells maintained in RMPI containing 2% FBS in last 24 hrs.Tumor mimicking conditioned media (TMC) was prepared by mixing 10µg mL− 1 of CSA to 200µl of culture supernatant. These concentrations were maintained throughout the study and the TMC was used with fresh medium at 50:50 V/V to ensure nutritional availability [34].
2.5 T cells isolation and macrophages -T cells co-culture:
T cells isolation
Spleen from the naive and tumor bearing mice was collected.T cells purified from the spleen after lysing RBCs using Geiss reagent (Sigma-Aldrich,USA).Total T cells were purified by passing through nylon wool.CD4+ T cells were purified from RBC-lysed splenocytes using an untouched CD4+T cells enrichment kit(eBioscience)using manufacture protocol.CD8+ T cells were purified from RBC-lysed splenocytes using an untouched CD8+ T cells enrichment kit (eBioscience) using manufacturing protocol. [33][35].
Single cells suspension from tumor infiltrating lymph nodes (TILs) were prepared by mincing the lymph nodes between frosted end slides and the purified CD3 cells were used flow cytometric analysis of surface and intracellular marker of T cells.
Macrophages -T cells co-culture:
For macrophage and T-cells co-culture assays was done using the lab-established protocol [33].Briefly, BMDMs was generated in a 96 well plate following lab protocol [32]. After the generation of BMDMs treated with TMC in the absence/presence of doxycycline (20µg mL− 1) for 24 hr. Naive total T-cells/CD4+ T cells were added to the culture at a ratio of 1:10. Naive T cells without any stimulation served as a negative control. T cells were stimulated with CD3 and CD28 activating antibodies to analyse non-specific activation. After 3 days of co-culture in 96 well plates, IL-12, IFN-γ, IL-10 and TGF-β were quantified from the supernatant by ELISA. Simultaneously in a replicate experiment Brefildin-A was added to co-culture after 6hrs and incubated for 48hrs to analyse intracellular marker by flowcytometry or RT-PCR. The proliferation of T cells was measured either by MTT assays or CFSE proliferation assays.
2.6 Quantitative real time (RT)-PCR
Gene expression was carried out by stabilised lab protocol [35].Briefly, the total RNA was extracted using TRIzol method and quantified. 2µg of RNA was used for cDNA synthesis by iScript cDNA synthesis kit (Bio-Rad USA).β-Actin was amplified from each sample to ensure equal cDNA input.100 ng of cDNA was used for amplification of mentioned genes in triplicates using gene-specific primers (supplementary table 3).Using power SYBR green 2X Master Mix in applied Bio systems) Step one plus Thermal cycler for 40 cycles and analysed with SDS 2.4 software (Applied Bio systems).Results normalized according to the expression levels of GAPDH mRNA.
2.7 Western Blot:
Protein expression was analysed by western blotting [33]. Briefly Radio immuno precipitation Assay (RIPA) buffer Supplemented with Phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor (PI) acceding to the manufacture’s recommended concentration was used to lysate cells. Each sample was normalized to 50 µg/30µl protein was resolved on an SDS-PAGE gel and electro-blotted onto a PVDF membrane. The membrane was blocked with 5% non-fat milk or 2% BSA in TBS for 2 h at RT. The membrane was probed overnight at 4˚c with following primary antibodies: sheep anti-mouse Arginase-1, Rabbit anti-mouse iNOS and GAPDH at (1:1000) dilutions. GAPDH was used as an internal control. Later, the membrane was probed with respective HRP- conjugated secondary antibody (1:5000) for 1 h at RT followed by the detection with ECL kit.
2.8 Flowcytometry analysis:
Cells were collected and stained for flow cytometry analysis. For In vitro experiment we used BMDMs for macrophage polarization experiment. BMDMs polarized in to M0, M1and M2macrophages [32, 33]and treated with TMC for 24 h followed by doxycycline (20µg/ml) treatment for 24hrs and surface protein was analysed by flow cytometry. The cell were analysed for expression of: CD11b-PE, CD86-FITC and CD206-APC. Details dilution and brand are mentioned in (supplementary table 1).
For In vivo experiment we used Single cell suspensions from tumor infiltrating lymph nodes and tumor tissue, for intracellular staining, Brefeldin-A solution in DMSO had been added 12hrs before collection at a concentration of 1µg/ml. Positive control T cells were activated with CD28/3. Staining with CD44, CD39, CD73, CD11b, F4/80, CD86, CD206, CD3, CD4, CD8, FOXP3, Tbet, IFN-γ, IL-10, TGF-β (250x dilution each) was done using lab optimizing protocol [34]. Details dilution and brand are mentioned in (supplementary table 1).Cells were blocked with Fc block CD16/32 antibody for 30 min before staining as a background staining control. Everything was done on ice. The acquisition was done on Backman Coulter .Cytoflex research flow cytometer. Data were analyzed using CytExpert 2.4 from Beckman Coulter and FlowJo_V10. To quantify the cells recruited, equally weighed tissue was minced in the PBS and cells were collected in equal volumes of PBS. 2.9 ELISA:
BMDM cells were seeded at a density of 105cells/well in triplicates in 96-well plate and were treated was indicated. After24hrs cell free supernatants were analysed for cytokines by ELISA. 96-well plate were coated with the capture antibody in coating buffer (Carbonate buffer PH 9.1) overnight at 4˚C.The wells were washed with washing buffer (PBST with 0.05% Tween)for 5 times and block with blocking buffer (2.5% FBS in washing buffer).The collected cell free supernatants were added to the wells and incubated overnight at 4˚C.Later the wells were washed with buffer for 5 times and incubated with detection antibody (Biotin-labelled) in blocking buffer for 2hrs.After washing the wells were incubated with Streptavidin-HRP for 30 min in dark; following its incubation with TMB substrate for 15 min stop solution (2N orthophosphoric acid) was added to wells and reading were taken at 450nm.All the ELISA experiments were done in triplicates and O.D was used to deduced concentration of cytokines using standard graph[32][33] Details dilution and brand are mentioned in (supplementary table 1).
2.10 ATP Determination Assays:
4T1 or BMDM cells were seeded into a 96-well plate at a density of 10,000 cells per well in 10% FBS containing media. The cells were treated with doxycycline 20µg/ml, in the presence of the Real Time-Glo™ Extracellular ATP Assay Reagent (Promega, GA5010-1KT)[36].Luminescence data was collected every 6 hours using a BMG POLAR star® plate reader
2.11 Adenosine Assays:
Adenosine was determined using culture supernatant of 4T1, BMDM and T cells samples by Adenosine Quantification Assay Kit (Sigma, MAK433-1KT) [37].According to the manufacturing recommendations, we have used 20µL of frozen plasma samples to measure adenosine concentration. The plate was read on spectraMaxM2 Spectrofluorometer. The fluorescent product was excited at 535 nm and detected at 587 nm.
2.12 Cytotoxic T-cell assay:
For CTL assay, in order to track proliferation of the 4T1 cells, 4T1 cells were stained with CFSE cell division tracker kit (Biolegend Cat#423801) according to the manufacturer’s protocol, Briefly,4T1 resuspended in PBS 106 cells per mL were stained with 2µl of 10mM CFSE per 106 cells to yield a final CFSE concentration of 1µM.4T1 cells were incubated at 37˚ C for 15 min, centrifuged at 500g for 8min,resuspended in RPMI to neutralize the Dye, and incubated at 37˚C for 30 min. Cells were centrifuged again, reseupended at 106 cells per mL, and stored at 37˚C until plating with the total T-cells. Later total T-cells were purified from splenocytes of different experimental groups using nylon wool column. Total T-cells were co-cultured with polarized macrophage as mentioned earlier.[32][33]. Later the primed T cells, harvested from each experimental group were plated with CFSE stained 4T1 at ratios of 1:100 for 5 days. These cells were centrifuged, the supernatants were stored at -20˚C for analysis of cytokines levels using ELISA and cells were stained with CD44-APC.The cytolytic activity of Tcells against 4T1 cells were analyzs by CFSE proliferation assay.
2.13 4T1 Tumor model
A syngenic orthotropic mouse breast cancer model was established using 4T1 cells as previously reported [38] [39][40]Briefly, 4T1 cells (5*105in PBS) were injected into BALB/C mice (n = 5) through subcutaneously mammary gland. After tumor was palpable, the mice were treated with, Doxycycline (50 mg kg− 1of body weight), ARL67156 (2mg kg− 1of body weight) and AMPCP (20mg kg− 1 of body weight) was injected intraperitoneally in 50µL PBS on days 10, 13, 16, 19, 22, 25, 28, and 31th days. The tumor size was measured every alternate day using Vernier callipers, and the volume was calculated using the formula (length*width*width)/2. The mice were sacrificed on day 34 of tumor inoculation and excised tumors were weighed.
All experiments were conducted in accordance with the guidelines of the Institutional Animal Ethical Committee after obtaining a clearance at Nirma University, Ahmedabad India. Housing and handling of mice was in accordance with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) at Institute of Pharmacy Nirma University, Ahmedabad, India. Protocol No: IP/PCOL/FAC/32/2022/49dated 20/09/2022.
2.14 Statistical tests:
All the data were collected and analysed from minimum of three individual biological experiment replicates including animal experiments. One-way ANOVA with Turkey’s test was used to analyse the significance between the groups. Two-way ANOVA with Turkey’s multiple comparison tests was used to analyse the difference among subgroups and the difference among groups; and longitudinal values. Significance was denoted as p. (*** means p < 000.1; ** means p < 0.005; * means p < 0.01; n.s means not significant). In the figures * to denote the significance difference. These denotations were explained in the figure legend.