Preparation of pTRG
The TRG was formulated from alginate/collagen, as described in a previous study [34,35,36]. Briefly, sodium alginate (Sigma Aldrich, St. Louis, MO, USA) was dissolved in deionized water (DW) at a concentration of 40 mg/mL. Collagen (from calf skin, Sigma Aldrich) was dissolved in 0.1 M acetic acid at a concentration of 60 mg/mL. The alginate solution (40 mg/mL) was mixed with collagen solution (60 mg/mL) at a 1:1 volumetric ratio. To prepare the 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. The pre-gel solution was treated with 150 mM calcium chloride (CaCl2; Sigma Aldrich). For the evaluation of the carrier effect of TRGs, the TRG was incorporated with 0.8 mg/mL of poly I:C (HMW, InvivoGen, San Diego, CA), 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) antibody (Ab) (29F.1A, BioXcell, Lebanon, NH, USA), or 4.4 mg/mL of the 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). Released poly I:C, STING ligand, anti-PD-1 Ab, and anti-PD-L1 Ab from TRG after 808-nm laser irradiation were quantified by obtaining the difference between the total quantity and the supernatant quantity. To determine the concentration of poly I:C released from the TRG, absorbance using a UV/vis spectrophotometer (Cary 100 Bio, Varian Inc., Palo Alto, CA, USA) was measured at 280 nm. The released concentration of STING ligand was measured using the 2′3′-cGAMP enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemical Company, Ann Arbor, MI, USA). In addition, the concentrations of the released anti-PD-1 and anti-PD-L1 Abs were determined using indirect ELISA. The recombinant murine PD-1 or PD-L1 were precoated in the plate; then, we added the released Ab-containing buffer. After washing with PBS, HRP-conjugated secondary Abs were added, and the concentration of the released Abs was determined using a UV/vis spectrophotometer (Cary 100 Bio, Varian Inc.). A fiber-coupled continuous-wave diode laser (808 nm, 10 W) was obtained from Changchun New Industries Optoelectronic Technology Co., Ltd. (China). Thermographic images were obtained using the FLIR One Thermal Imaging System (FLIR Systems, Wilsonville, OR, USA). Rheological properties of TRG were characterized using a rotational rheometer (MCR 302, Anton Paar, Austria). A 4.7-mL-volume coaxial cylinder (CC17) was used for the rheological measurement. The dynamic frequency sweeping was carried out at 25 °C at a 5% strain level over an angular frequency range of 0.05–200 rad s−1. The thermally responsive gel-sol transition was assessed by the temperature sweeping test. The heating measurement was conducted at an angular frequency and strain level of 1 rad s−1 and 5%, respectively, and the temperature was ramped at a heating rate of 5 °C min−1 over the range of 25–70 °C.
The murine colon carcinoma cell line CT-26-iRFP (CT26.WT-iRFP-Neo; Imanis Life Sciences, CL091, Rochester, USA) was cultured in Dulbecco’s modified Eagle’s medium (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) was cultured in Roswell Park Memorial Institute medium (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 cultured 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 maintained under pathogen-free conditions at the Laboratory Animals Center of Yeungnam University. All experiments were conducted in consideration of the basic ethical principles of animal experiments and the 3R principles. In addition, the experiments were 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 the pain or stress experienced by the animals by euthanizing them with CO2 gas, following 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 distributed into six groups as follows: PBS, poly I:C, Gel, pGel, TRG, and pTRG. After an intratumoral (i.t.) injection of 50 µL of the sample using a 21-gauge needle, the tumor was irradiated with an 808-nm 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 the 28th day after the tumor challenge and calculated using the formula V = ½ (length × width2).
Anti-IgG1, anti-IgG2a, and anti-IgG2b Abs were used as isotype controls and purchased from BioLegend (San Diego, CA, USA). Allophycocyanin (APC)-Cy7-anti-T cell receptor (TCR)-β (H57-597), PerCP5.5-anti-CD4 (GK1.5), BV785-anti-CD8α (53–6.7), phycoerythrin (PE)/cy7-anti-interferon-gamma (IFN-γ) (B27), BV 785-anti-CD11c (N418), Alexa Fluor 647-anti-CD40 (HM40-3), BV 605-anti-CD80 (16-10A1), PE/Cy7-anti-CD86 (GL-1), PerCP5.5-anti-MHC class I (AF6-88.5), and PE-anti-MHC class II (M5/114.15.2) were obtained from BioLegend.
Mouse lymphoid DC analysis
Tumor-draining lymph nodes (tdLNs) were homogenized using a glass slide. Lipids and debris were removed using a 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 unlabeled isotype control Abs and Fc-block Abs (BioLegend) for 15 min to prevent any non-specific binding. After washing with PBS, the cells were stained with fluorescence-conjugated Abs at 4 °C for 30 min. After removing the free Abs by washing with PBS, the cells were suspended in a flow cytometry buffer (BioLegend) containing 4′,6-diamidino-2-phenylindole (Sigma Aldrich) and were analyzed using a NovoCyte flow cytometer (ACEA Biosciences Inc.).
Second tumor challenge
The mice cured of the CT-26 or 4T1 tumor were intravenously (i.v.) injected with CT-26-iRFP (0.7 × 106/100 µL of PBS) or 4T1-iRFP (0.5 × 106/100 µL of PBS) cells, respectively. On the 10th day after the second cancer challenge, the mice were euthanized, 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) after 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 with ethanol gradient solutions, the lungs were embedded in paraffin and sliced into 5-μm thick sections using a microtome. The sections were attached to a glass slide, rehydrated, and stained with hematoxylin and eosin. Any 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) were freeze-dried and thawed thrice to obtain a lysate. After centrifugation (10,000g at 4 °C for 5 min), the protein concentration of the CT-26 supernatant was determined using the Bradford assay. Splenocytes (1 × 106 splenocytes) were treated with 10 μg/mL CT-26 cell lysate. Twenty-four hours after incubation, the cells were stained with surface Abs (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 an intracellular staining buffer (BioLegend) at 4 °C for 30 min. After washing, the cells were incubated with intracellular Abs (PE-conjugated anti-IFN-γ) for 30 min. IFN-γ-producing CD4 and CD8 T cells were analyzed using NovoCyte (ACEA Biosciences Inc.).
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). Antigen-specific production of TNF-α, IFN-γ, perforin, and granzyme B in the 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 BALB/c mice and labeled with 200 nM of carboxyfluorescein succinimidyl ester (CFSE) or 10 mM CellTracker™ Orange CMTMR (Life Technologies). 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. CFSE and CMTMR-labeled cells were mixed at a 1:1 ratio, and a total of 10 × 106 labeled cells were transferred to BALB/c mice, which were treated with TRG and pTRG for the first time. The mice treated with PBS, poly I:C and pGel were also exposed to labeled cells as controls. Twelve hours after the splenocyte transfer, the spleen was harvested, and specific killing was analyzed using NovoCyte (ACEA Biosciences Inc.).
Depletion of CD4 and CD8 T 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 the 28th day after the first tumor challenge (2 days before the second challenge of cancer cells). The depletion efficacy of Ab injection was analyzed using NovoCyte (ACEA Biosciences Inc.); we observed > 98% depletion of CD4 or CD8 cells in the mice.
Data were analyzed using SPSS (Chicago, IL, USA) and expressed as the mean ± standard error. The values *p < 0.05, and **p < 0.01 were considered statistically significant.