2.1 Cell culture
The human colon cancer cell line HCT-116 (KAC Co., Ltd., Kyoto, Japan, Cat. No.: EC91091005-F0) was used for in vitro and in vivo experiments. The cell line was confirmed to be negative for mycoplasma. Cells were maintained and cultured in accordance with international guidelines for proper cell culture practices. Cells were cultured in RPMI 1640 medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in a 37°C humidified incubator with 5% CO2.
2.2 Preparation of conditioned medium (CM) and TAMs
To obtain CM, HCT-116 cells were cultured in 100 mm culture dishes until 80% confluent. The cells were washed twice with pre-warmed phosphate-buffered saline (PBS) and incubated in fresh medium without FBS. After incubation at 37°C for 48 hours, the supernatant was collected, centrifuged at 800´g for 5 minutes at 22°C, and filtered through a 0.2-µm sterile filter.
To obtain TAMs, M0 macrophages were treated with CM derived from HCT-116 cells. CM was added to the culture medium at a 1:1 ratio and cells were incubated at 37°C for 48 hours.
To obtain TAM-CM, the TAMs were treated with blue LED light (465 nm, 30 mW/cm2, 30 minutes) (irradiation group) or left untreated. The medium was changed once and the supernatant (TAM-CM) was collected. A schematic for these processes is shown in Figure 1.
2.3 Animals
Four-week-old female BALB/c mice were purchased from Charles River Japan, Inc. (Kanagawa, Japan). Animals were caged under controlled conditions and provided water and standard laboratory rations for at least seven days prior to use. Before and after surgery, the animals had free access to tap water and food. The experiments and procedures in this study were approved by the Animal Welfare and Use Committee of the University of Tokushima (Approval No. T2021-42). All animal experiments were performed in accordance with the U.K. Animals (Scientific Procedures) Act 1986 and associated guidelines, the EU Directive 2010/63/EU for animal experiments, and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978).
2.4 Orthotopic model and LED irradiation
CT-29 cells were harvested with 1 mM ethylenediaminetetraacetic acid (EDTA) in PBS, washed three times with PBS, and resuspended at a density of 1×107 cells/mL in PBS containing 500 mg/mL of Matrigel (Becton Dickinson Labware, Bedford, MA, USA). Animals (n=8) were anesthetized with isoflurane (3% to 4% for induction and 1% to 2% for maintenance). A 7-mm incision was made in the anterior rectal wall to prevent colonic obstruction caused by rectal tumor progression. CT-29 cells (1×106) were injected into the submucosa of the posterior wall of the rectum using a 29-gauge needle.
Seven days after cell implantation, the mice developed rectal cancer. The mice were randomized into two groups (n=4 mice per group) and treated as follows: (i) the LED irradiation group (465 nm, 30 mW/cm2, 30 minutes, once) and (ii) the control group (untreated). The mice were sacrificed by cervical spinal cord dissection at 2 weeks after cell implantation. A humane endpoint was set at the stage when the tumor size increased by more than 10% of the mice’s body weight, but this did not apply to any of the mice.
A 465-nm blue LED (NCSB119, NICHIA Corporation, Tokushima, Japan) was used as a light source. An LED irradiation device (Department of Electrical and Electronic Engineering, Faculty of Engineering, The University of Tokushima, Tokushima, Japan) was used for irradiation experiments.
2.5 Immunohistochemistry (IHC)
The tissues resected from the orthotopic model or cultured cells were fixed in 10% formalin (Wako 066-03847) for 24 hours at room temperature, paraffin-embedded, and sliced into 4-μm-thick sections. The samples were blocked in 3% BSA (MACS®︎ BSA Stock Solution 130-091-376) for 1 hour at room temperature. An anti-F4/80 antibody (15361-1-AP; Proteintech, Rosemont, IL, USA), anti-CD163 antibody (SAB2700986; Sigma-Aldrich), and anti-PD-L1 antibody (17952-1-AP; Proteintech) were used for IHC staining following the manufacturer’s instructions. The fluorescence area was measured using Image J software (ver. 1.53, National Institutes of Health, Bethesda, MD, USA).
2.6 Quantitative real-time PCR (qRT-PCR) analysis
Total RNA was isolated from samples using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Tokyo, Japan). Then, qRT-PCR was performed using a 7500 real-time PCR system, TaqMan gene expression assay on demand, and TaqMan Universal Master Mix (Applied Biosystems). The following TaqMan assays were used: Opn3 (Hs00173892_m1), CD163 (Hs00174705_m1), CD206 (Hs00267207_m1), and PD-L1 (Hs00204257_m1). GAPDH (4326317E) was used as an internal control for mRNA expression. The thermal cycler conditions were as follows: 2 minutes at 50°C, 10 minutes at 95°C, and then 40 cycles of 15 s at 95°C and 1 minute at 60°C. Amplification data were analyzed using the Prism 7500 Sequence Detection System ver. 1.3.1 (Applied Biosystems). qRT-PCR was performed using a Step One Plus Real-Time PCR System (Applied Biosystems). The relative target transcript expression levels were assessed and normalized to the expression levels of GAPDH mRNA as an internal control using the 2-ΔΔCq method (15).
2.7 Migration and scratch assays
For migration assays, Transwell inserts (Corning, Corning, NY, USA) with a pore size of 8 µm were used. CT-116 cells (2´104) were seeded into the upper chamber. After cell attachment, the medium was discarded and fresh medium containing 1% FBS was added to the upper chamber. CM containing 10% FBS was added to the lower chamber. After incubation for 24 hours, cells at the bottom of the Transwell inserts were fixed in 4% paraformaldehyde, then stained with 0.2% crystal violet. The stained cells in three random microscopic fields (100×) were counted.
For scratch assays, HCT-116 cells were seeded at a density of 2×104 cells/well in 6-well plates. After the cells became 100% confluent, a plastic pipette tip was used to scrape the center of the well, creating a 1-mm-wide scratch. The medium was discarded and fresh DMEM containing 1% FBS was added, followed by CM at a 1:1 ratio. The final FBS concentration was 0.5%. The cells were then incubated at 37°C for 24 hours. Images of the wound were taken using a phase-contrast microscope (40× magnification; DP22-CU; Olympus Corporation) at 0 and 24 hours after scratching.
2.8 Statistical analysis
All statistical analyses were performed using Stat View version 5.0 software (SAS Institute, Cary, NC, USA). The Mann-Whitney U-test and Wilcoxon signed-rank test were used for statistical comparisons. P-values less than 0.05 were considered statistically significant.