Photosensitizers
M-chlorin e6 (methyl(7S,8S)-18-ethyl-5-(2-methoxy-2-oxoethyl)-7-(3-methoxy-3-oxopropyl)-2,8,12,17-tetramethyl-13-(1-(3-(((2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)propoxy)ethyl)-7H,8H-porphyrin-3-carboxylate) and G-chlorin e6 (methyl(7S,8S)-18-ethyl-5-(2-methoxy-2-oxoethyl)-7-(3-methoxy-3-oxopropyl)-2,8,12,17-tetramethyl-13-(1-(3-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)propoxy)ethyl)-7H,8H-porphyrin-3-carboxylate) were synthesized and provided by the laboratory of Osaka Prefecture University (Osaka, Japan). M-chlorin e6 was synthesized as described previously [7].
Cell culture
CT26 mouse colon cancer cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured at 37°C in a 5% CO2, 95% air environment. CT26 cells were grown in low-glucose (1000 mg/L) Dulbecco’s modified Eagle’s medium (DMEM, Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. RAW264.7 mouse macrophages were purchased from the ATCC and cultured at 37°C in a 5% CO2, 95% air environment. RAW264.7 cells were grown in (1000 mg/L) RPMI-1640 (Wako) medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin.
Flow cytometric analysis of CRT
CT26 cells (n=1×106) were seeded in 10-cm dishes and incubated for 24 h at 37°C with 5% CO2 and divided into a control group and PDT group. In the control group, the medium was changed to fresh DMEM low glucose for another 24 h. In the PDT group, the medium was changed to fresh DMEM low glucose containing M-chlorin e6 (120 nM) and incubated for another 24 h, after which the medium was replaced with phosphate-buffered saline (PBS), and the cells were irradiated with LED light (660 nm, 16 J/cm2) using an LEDR-660DL (Opto Code, Tokyo, Japan). In both groups, the medium was then changed again to fresh DMEM low glucose (without M-chlorin e6) and incubated for 1 h, after which the cells were detached using TrypLETM Express Enzyme (1×) without phenol red (Thermo Fisher Scientific, San Diego, CA, USA). The cells were counted and then aliquoted at 1×106 cells per sample, after which primary antibody (Calreticulin [D3E6] XP® Rabbit mAb; Cell Signaling Technology, Danvers, MA, USA) was added, and the cells were placed on ice for 30 min. The secondary antibody (Alexa FluorTM 488 F[ab']2 fragment of goat anti-rabbit IgG [H+L]; Invitrogen, Waltham, MA, USA) was then added, and the cells were kept on ice for 30 min. The cells were then washed with PBS, suspended in Stain Buffer (BD Biosciences, San Jose, CA, USA), and 7-AAD (BD Biosciences) was added. Finally, the cells were analyzed using a FACS VerseTM flow cytometer (BD Biosciences).
Phagocytosis assay
CT26 cells (n=3×105) were seeded in 3.5-cm glass-bottom dishes and incubated for 24 h at 37°C with 5% CO2 and then divided into a control group and PDT group. In the control group, the medium was changed to fresh DMEM low glucose containing M-chlorin e6 (120 nM), and the cells were incubated for another 24 h. In the PDT group, the medium was changed to fresh DMEM low glucose containing M-chlorin e6 (120 nM), and the cells were incubated for another 24 h, after which the medium was replaced with PBS, and the cells were irradiated with LED light (660 nm, 16 J/cm2) using an LEDR-660DL. In both groups, the medium was changed again to DMEM low glucose (without M-chlorin e6), and the cells were incubated for 1 h, after which the medium was changed to RPMI-1640 medium containing suspended RAW264.7 cells (n=1×105) labeled with BCECF-AM special packaging (Dojindo, Kumamoto, Japan), and the cells were incubated for 3-24 h. Phagocytosis was evaluated using an LSM800 confocal microscope (Carl Zeiss, Oberkochen, Germany).
Flow cytometric analysis of CD80 and CD86
CT26 cells (n=1×106) were seeded in 10-cm dishes and cultured for 24 h at 37°C with 5% CO2 and then divided into a control group and PDT group. In the control group, the medium was changed to fresh DMEM low glucose, and the cells were incubated for another 24 h. In the PDT group, the medium was changed to fresh DMEM low glucose containing M-chlorin e6 (120 nM), and the cells were incubated for another 24 h, after which the medium was replaced with PBS, and the cells were irradiated with LED light (660 nm, 16 J/cm2) using an LEDR-660DL. In both groups, the medium was again changed to DMEM low glucose (without M-chlorin e6), and the cells were incubated for 1 h, after which the medium was changed to RPMI-1640 containing suspended RAW264.7 cells (n=1×106), and the cells were incubated for another 24 h. The cells were then detached using TrypLETM Express Enzyme (1×) without phenol red and counted. The cells were then aliquoted at 1×106 cells per sample, Mouse BD Fc blockTM was added, and the cells were placed on ice for 10 min. Finally, antibody (PE hamster anti-mouse CD80, PE-Cy7 rat anti-mouse CD86 [BD Biosciences]) or isotype control (PE hamster IgG2 kappa isotype control, PE-Cy 7 rat IgG2a kappa isotype control [BD Biosciences]) was added, and the cells were kept on ice for 30 min. The cells were washed with PBS, suspended in Stain Buffer, and 7-AAD was added. The cells were analyzed using a FACS VerseTM flow cytometer (BD Biosciences).
Statistical analyses
All statistical analyses were performed using EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R. More precisely, EZR is a modified version of R Commander designed to add statistical functions frequently used in biostatistics [12]. The two-tailed non-paired Student's t-test was used for comparisons between two groups. Data are presented as the mean ± standard error. A p value of <0.05 was considered statistically significant.