Preparation of pTRG
Sodium alginate (Sigma Aldrich, St. Louis, MO, USA) was dissolved in deionized water (DW) to obtain a concentration of 40 mg/mL. Collagen (from calf skin, Sigma Aldrich) was dissolved in 0.1 M acetic acid to attain a concentration of 60 mg/mL. To trigger collagen fabrication, the solution was incubated at 37°C for 4 h. To prepare TRG, ICG (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), along with a 5% weight ratio of collagen, was dissolved in DW and added to the solution. pTRG was prepared using the same methods; except that the mixture of ICG and poly was I:C (0.8 mg/mL, HMW; InvivoGen, San Diego, CA), instead of ICG only. The pre-gel solution was treated with 150 mM CaCl2 (Sigma-Aldrich). The difference between the gel and TRG preparation methods was the absence of ICG. For evaluation of the carrier effect of TRGs, the TRG was incorporated with phosphate buffered saline (PBS), 0.8 mg/mL of poly I:C (HMW, InvivoGen), 1.8 mg/mL of stimulator of interferon genes (STING) ligand (2'3'-cGAMP, InvivoGen), 6.3 mg/mL of anti-programmed cell death-1(PD-1) Ab (29F.1A, Bioxcell, Lebanon, NH, USA), or 4.4 mg/mL of anti-programmed cell death-ligand 1 (PD-L1) Ab (29F1A12, Bioxcell), respectively.
Characterization of pTRG
Scanning electron microscopy (SEM) images were obtained using an S-4800 scanning electron microscope (HITACHI, Japan) at the Core Research Support Center for Natural Products and Medical Materials at Yeungnam University, Republic of Korea. The absorption spectra and released poly I:C concentrations were measured using a UV/vis spectrophotometer (Cary 100 Bio, Varian Inc., USA). A fiber-coupled continuous-wave diode laser (808 nm, 10 W) was obtained from Changchun New Industries Optoelectronic Technology Co., Ltd. (China). Thermographic images of the temperature were obtained using the FLIR One Thermal Imaging System (FLIR Systems, Wilsonville, OR, USA).
The murine colon carcinoma cell line CT-26-iRFP (CT26.WT-iRFP-Neo; Imanis Life Sciences, CL091, Rochester, USA) was cultured in DMEM (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich), 1× penicillin/streptomycin (Gibco BRL Ltd, Paisley, Scotland), and 0.4 mg/mL G418 (Thermo Fisher Scientific, Inc.). The murine breast cancer cell line 4T1-iRFP (4T1.WT-iRFP-Neo; Imanis Life Sciences, CL078) were cultured in RPMI (Thermo Fisher Scientific, Inc.), supplemented with 10% FBS (Sigma Aldrich), 1× penicillin/streptomycin (Gibco BRL Ltd, Paisley, Scotland), 0.1 mg/mL G418 (Thermo Fisher Scientific, Inc.), and 2 µg/mL puromycin (InvivoGen). The cell lines were grown at 37°C in a humidified atmosphere containing 5% CO2 and air.
BALB/c mice (6–8 weeks old, female) were obtained from Hyochang Science (Daegu, Republic of Korea). The mice were kept under pathogen-free conditions at the Laboratory Animals Center of Yeungnam University. All experiments were conducted after considering the basic ethical principles of animal experiments and the 3R principles. In addition, the experiment was conducted in compliance with the animal protection law, the law on experimental animals, and the IACUC regulations of Yeungnam University. The Committee on the Ethics of Animal Experiments of Yeungnam University Laboratory Animals Center approved the protocol (mouse protocol number, 2020-039). For ethical reasons, we minimized pain or stress in the animals by euthanizing them with CO2 gas, in accordance with the humanitarian endpoint criteria.
First tumor challenge and PTT
BALB/c mice were subcutaneously injected with 1 × 106 CT-26-iRFP or 4T1-iRFP cells. After 7 days, the mice were randomly separated into six groups: PBS, poly I:C, Gel, pGel, TRG, and pTRG. After intratumorally (i.t.) injection of each sample into the mice, the tumor was irradiated with an NIR laser at 1.5 W/cm2 for 5 min. The elevated temperature was imaged using the FLIR One Thermal Imaging System (FLIR Systems). The tumor volume was monitored on day 28 after tumor challenge and calculated using the formula V = ½ (length × width2).
Anti-IgG1 antibody (Ab), anti-IgG2a Ab, and anti-IgG2b Ab were used as isotype controls and purchased from BioLegend (San Diego, CA, USA). Anti-TCR-β (H57-597), anti-CD4 (GK1.5), anti-CD8α (53 − 6.7), anti- interferon (IFN-γ (B27), anti-CD11c (N418), anti-CD40 (HM40-3), anti-CD80 (16-10A1), anti-CD86 (GL-1), anti-MHC class I (AF6-88.5), and anti-MHC class II (M5/114.15.2) were purchased from BioLegend.
Mouse lymphoid DC analysis
Tumor-draining lymph nodes (tdLNs) were homogenized using a slide class. Lipids and debris were removed though 100 nm nylon mash. After washing with PBS, the cells were stained with fluorescence-labeled Abs for 30 min. To define DCs, the cells were stained with lineage markers, including anti-B220 (RA3-6B2), anti-CD3 (17A2), anti-CD49b (DX5), anti-Gr1 (RB68C5), anti-Thy1.1 (OX-7), and anti-TER-119 (TER-119). Lineage−CD11c+ cells were further divided into cDC1 and cDC2 cells. Co-stimulator and MHC molecule levels were measured in cDC1 and cDC2 using a NovoCyte flow cytometer (ACEA Biosciences Inc., San Diego, CA, USA).
Flow cytometry analysis
The tdLN cells were stained with unlabelled isotype control Abs and Fc-block Abs (BioLegend) for 15 min to prevent non-specific binding of Abs. After washing with PBS, the cells were stained with fluorescence-conjugated antibodies at 4°C for 30 min. After removing free Abs by washing with PBS, the cells were suspended with 4',6-diamidino-2-phenylindole (Sigma Aldrich) containing flow cytometry buffer (BioLegend) and were analyzed using a NovoCyte flow cytometer (ACEA Biosciences Inc.).
Second tumor challenge
The cured mice from the first challenge of CT-26 or 4T1 were injected intravenously with CT-26-iRFP cells (0.7 × 106/100 µL of PBS) or 4T1-iRFP (0.5 × 106/100 µL of PBS), respectively. On day 10 after the second cancer challenge, the mice were sacrificed, and their lungs and spleen were harvested for further experiments.
In vivo fluorescence imaging
iRFP fluorescence images were captured using the fluorescence in vivo imaging system, FOBI (Cellgentek, Cheongju, Republic of Korea) on the first and second challenges.
As previously described in detail, lung samples were harvested 10 days after the second tumor challenge and fixed with 10% formalin. After rehydration by gradient change of ethanol, the lungs were embedded in paraffin. The lung tissue was sectioned to a thickness of 5 µm using a microtome and attached to a glass slide. The sections were rehydrated and stained with hematoxylin and eosin. Tumor infiltration in the lung was observed using an EVOS M5000 microscope (Invitrogen, Waltham, MA, USA).
Analysis of CT-26 specific T cell immunity
CT-26 cells (1 × 107 CT-26 cells were freeze-dried and thawed three times to obtain a lysate. After centrifugation (10,000 × g, at 4°C for 5 min), the concentration of CT-26 lysate was determined in the supernatant using the Bradford assay. Splenocytes (1 × 106 splenocytes) were treated with 10 µg/mL CT-26 cell lysates. Twenty-four hours after incubation, the cells were stained with surface antibodies (APC/Cy7-anti-TCR-β, PerCP5.5-anti-CD4, and BV785-anti-CD8α) for 20 min. After washing with PBS, the cells were fixed and permeabilized with intracellular staining buffer (BioLegend) at 4°C for 30 min. After washing, the cells were incubated with intracellular antibodies (PE-anti-IFN-γ) for 30 min. IFN-γ-producing CD4 and CD8 T cells were analyzed using NovoCyte (ACEA Biosciences Inc.).
Enzyme-linked immunosorbent assay (ELISA)
Serum concentrations of interleukin (IL)-6, IL-12p70, and tumor necrosis factor (TNF)-α were measured 24 h after the first tumor therapy using ELISA kits (BioLegend). The antigen-specific production of TNF-α, IFN-γ, perforin, and granzyme B in cultured medium was analyzed 24 h after stimulation of splenocytes with 10 µg/mL of CT-26 lysate.
In vivo cytotoxicity assay
Splenocytes were harvested from BLAB/c mice and labeled with 200 nM of CFSE or 10 mM CellTracker™ Orange CMTMR (Life Technologies). The CFSE-labeled splenocytes were coated with 200 nM of CT-26 antigen AH1 (SPSYVYHQF) peptide and the CMTMR-labeled cells were loaded with the control peptide. The CFSE and CMTMR-labeled cells were mixed at a 1:1 ratio, and a total of 10 × 106 labeled cells were transferred to BLAB/c mice, which were treated with TRG and pTRG for the first time. The PBS, poly I:C, and pGel-treated mice were also transferred to labeled cells as controls. Twelve hours after splenocyte transfer, the spleen was harvested, and specific killing was analyzed using NovoCyte (ACEA Biosciences Inc.).
Depletion of CD4 and CD8 cells
After treatment of the first tumor by PTT, the cured mice received 1 mg/kg of anti-CD4 Ab (GK1.5, 1) or 1 mg/kg of anti-CD8 Ab (YTS169.4) (both from BioXcell) every 2 days from 28 days after the 1st tumor challenge (2 days before the 2nd challenge of cancer cells). The depletion efficacy of Ab injection was analyzed using NovoCyte (ACEA Biosciences Inc.) and showed > 98% depletion of CD4 or CD8 cells in the mice.
The data were analyzed using SPSS (Chicago, IL, USA) and expressed as the mean ± standard error of the mean. The values *p < 0.05, and **p < 0.01 were considered statistically significant.