Oligonucleotides and antibodies
TASO, also known as trabedersen, was provided by Autotelic Bio. Inc. (Cheongju, Korea). Antibodies used in flow cytometry analysis included PE-conjugated anti-human CD45 (clone 2D1), FITC-conjugated anti-human CD3 (clone HIT 3a), APC-conjugated anti-human CD8 (clone SK1), PE-conjugated anti-human CD4 (clone SK3), APC-conjugated anti-human CD25 (clone BC 96), and FITC-conjugated anti-human Foxp3 (clone 206D), and were procured from BioLegend (BioLegend, San Diego, CA, USA). Rabbit monoclonal antibodies against human CD8 (clone D8A8Y), Foxp3 (clone D2W8E), granzyme B (clone D6E9W), p-SMAD2/3 (clone D27F4), and rabbit polyclonal antibody against SMAD2/3 were purchased from Cell Signaling Technology (Cell Signaling Technology, Inc., Danvers, MA, USA). Rabbit polyclonal antibody against human TGF-β2, CCL-22 and horseradish peroxidase (HRP)-conjugated goat polyclonal antibody to rabbit IgG were purchased from Abcam (Abcam, Cambridge, UK). Mouse monoclonal antibody against β-actin and HRP-conjugated goat polyclonal antibody to mouse IgG were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Four-week old female NOD/scid/IL-2Rγ−/− (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ, NSG) mice were purchased from Charles River Japan, and acclimated for 1 week. All procedures involving experimental animals complied with regulations for the care and use of laboratory animals of the animal ethics committee of Chungbuk National University (CBNUA-1235-18-01). The mice were maintained under specific pathogen free conditions, according to guidelines of the National Institutes of Health for the care and use of laboratory animals.
Preparation and transplantation of human peripheral blood mononuclear cells (PBMCs)
The human cryopreserved PBMCs obtained from Zen-Bio (Zen-Bio, INC., Research Triangle, NC, USA) were quickly thawed and washed once. Human PBMCs used in this study were isolated from a healthy single donor, the information of PBMCs such as HLA types, viability and population distribution was provided by Zen-Bio (Zen-Bio, INC., Research Triangle, NC, USA). PBMCs were incubated in the lymphocyte specific RPMI1640 medium supplemented with 20% FBS and DNAse I (LYMPH-1; Zen-Bio, INC., Research Triangle, NC, USA) for 1h; 1×107 cells were subsequently resuspended in PBS and injected into the lateral side of tail vein of mice. Preliminary experiment was performed to evaluate appearance of human PBMCs in mouse blood and onsets of graft versus host disease (GvHD).
Establishment of xenografts
The MDA-MB-231 TNBC cell line procured from the American Type Culture Collection was cultured in RPMI1640 supplemented with 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin, in a humidified chamber with 5% CO2 at 37℃. A total of 1×107 MDA-MB-231 tumor cells suspended in PBS were implanted subcutaneously in the right flanks of NSG mice (this is day 0 of the experiment), 6 days after human PBMC engraftment. Mice were intra-peritoneally administered with TASO every alternate day, starting from day 3. From day 4, tumor volume was measured every alternate day using a caliper, and calculated using the formula (length) × (width)2/2. Mice were sacrificed on day 19, and the implanted tumors were harvested, weighed, and processed for analysis (Fig. 2A).
Fluorescence activated cell sorting (FACS)
Using a capillary tube, mouse blood was collected from the orbital venous plexus into an EDTA tube on days 17 and 19. RBCs were eliminated by incubating 100 μl blood with RBC lysis buffer (BioLegend, San Diego, CA, USA), after which the cells were washed with cell staining buffer (BioLegend, San Diego, CA, USA). Washed cells were subsequently incubated with anti-CD16/32 for 5 min at 4℃, to block the Fc receptors and reduce non-specific binding. On day 17, cells were subjected to PE-conjugated anti-human CD4, APC-conjugated anti-human CD25 for cell surface staining, and FITC-conjugated anti-human Foxp3 for intracellular staining, after fixing/permeabilization with True-NuclearTM Transcription factor buffer set (BioLegend, San Diego, CA, USA). And PE-conjugated anti-human CD45, FITC-conjugated anti-human CD3, and APC-conjugated anti-human CD8 were used for cell surface staining on day 19. Staining with antibodies was performed for 30 min at 4℃ in the dark. Stained cells were washed with cell staining buffer, followed by 4% paraformaldehyde fixation. The fluorescence of stained cells was measured by FACS Aria II (BD Bioscience, San Diego, CA, USA), and data were analyzed using the FlowJo software (TreeStar, San Carlos, CA, USA).
Mouse blood was collected from the caudal vena cava on day 19, and serum was isolated for cytokine analysis. Levels of human IL-10, IFN-γ, and TGF-β2 in mouse serum were measured by applying the human magnetic luminex assay kit (R&D systems, Abingdon, UK), according to the manufacturer’s instruction. Measurement was performed on the Luminex 200TM (Luminex, Austain, TX, USA), and analyzed using the Masterplex QT 2010 Software (MiraiBio, Hitachi, CA, USA).
Paraffin-embedded spleen and implanted tumors were cut into 4 μm thick sections, deparaffinized, and subsequently rehydrated. After rehydration, antigen retrieval was performed by incubating with 10 mM sodium citrate buffer (pH 6.0) at 100℃ for 10 min. The tissue sections were then incubated with 3% hydrogen peroxide in PBS to block the endogenous peroxidase activity, followed by incubation with 5% BSA in PBS to reduce non-specific binding. The slides were subsequently incubated overnight with primary antibodies against human TGF-β2 (1:100), CCL22 (1:200), Foxp3 (1:200), CD8 (1:200), and granzyme B (1:200). The slides were subsequently incubated with a biotinylated secondary antibody for 1 h, and signals were amplified by avidin-biotin peroxidase complexes (ABC Elite kit; Vector Labs, Burlingame, CA, USA) for 30 min. Peroxidase activity was visualized using the DAB kit (Vector Labs, Burlingame, CA, USA), followed by counterstaining with hematoxylin. Images were captured with Axio Imager A2 microscope (Carl Zeiss, Jena, Germany) and DAB intensity was semi-quantified using Image J Fiji software (version 1.53c; WS Rasband, National Institute of Health, Bathesda, MD, USA) as described previously .
After sacrifice on day 19, tumor tissues were harvested and homogenized with pro-prep buffer (iNtRON Biotechnology, Inc., Seongnam, Korea) to extract the total proteins. Protein concentration was measured by the modified Bradford method, using the pro-measure kit (iNtRON Biotechnology, Inc., Seongnam, Korea) at 595 nm. Approximately 40 μg of total protein was loaded and resolved on 12.5% SDS-PAGE, followed by transferring to a PVDF membrane (Perkin Elmer Co., Waltham, MA, USA). The membrane was subsequently incubated with rabbit monoclonal antibodies against p-SMAD 2/3, SMAD 2/3 (1:1000) and mouse monoclonal antibody against β-actin (1:2000), followed by HRP conjugated secondary antibodies against anti-rabbit or anti-mouse (1:3000). Immunoreactive bands were detected using a chemiluminescent detection reagent (Thermo Fisher Scientific Inc., Waltham, MA USA), which is an HRP chemiluminescent substrate. The luminescence intensity of the target protein bands was detected using the Lumino Graph 2 (ATTO Corporation, Tokyo, Japan), and adjusted by the Image Saver 6 (ATTO Corporation, Tokyo, Japan). Intensity levels of images were quantified using Image J analysis software (version 1.53e; WS Rasband, National Institute of Health, Batheda, MD, USA).
All data are presented as means ± standard errors of the mean (S.E.M.), and statistical significance was analyzed by one-way analysis of variance (ANOVA) followed by a post hoc Dunnett's test using the GraphPad Prism 5.01 software (GraphPad Software Inc., San Diego, CA, USA). A p-value < 0.05 is considered statistically significant.