Immunogenic cell death due to a new photodynamic therapy (PDT) with glycoconjugated chlorin (G-chlorin)

Abstract

Both the pre-apoptotic exposure to calreticulin (CRT) and the post-apoptotic release of high-mobility group box 1 protein (HMGB1) are required for immunogenic cell death. Photodynamic therapy (PDT) uses non-toxic photosensitizers and visible light at a specific wavelength in combination with oxygen to produce cytotoxic reactive oxygen species that kill malignant cells by apoptosis and/or necrosis, shut down the tumor microvasculature, and stimulate the host immune system. We have previously shown that glycoconjugated chlorin (G-chlorin) has superior cancer cell selectivity and effectively suppresses the growth of xenograft tumors. In the present study, we evaluated the immunogenicity of PDT with G-chlorin treatment in colon cancer cells. PDT with G-chlorin suppressed CT26 (mouse colon cancer cells) tumor growth considerably more efficiently in immunocompetent mice (wild-type mice, allograft model) than in immune-deficient mice (nude mice, xenograft model), although control treatments were not different between the two. This treatment also induced CRT translocation and HMGB1 release in cells, as shown by western blot and immunofluorescence staining. To evaluate the use of PDT-treated cells as a tumor vaccine, we employed a syngeneic mouse tumor model (allograft model). Mice inoculated with PDT-treated CT26 cells were significantly protected against a subsequent challenge with live CT26 cells, and this protection was inhibited by siRNA for CRT or HMGB1. In conclusion, PDT with G-chlorin treatment induced immunogenic cell death in a mouse model, where the immunogenicity of this treatment was directed by CRT expression and HMGB1 release.

Keywords: calreticulin (CRT); glycoconjugated chlorin (G-chlorin); high-mobility group box 1 protein (HMGB1); immunogenic cell death (ICD); photodynamic therapy.

Figure 1. Inhibition of tumor growth of PDT with G-chlorin

CT26 cells were inoculated into the dorsal skin of immunocompetent or immunodeficient mice at a concentration of 1 × 10⁶ cells/200 μL in PBS. Tumor-bearing mice were intravenously injected with 6.25 μmol/kg G-chlorin and, after 4 hours, were illuminated with 660-nm LED light (40 J/cm²). Each group comprised five mice. Data are mean ± SD. Significance was determined by Welch's t-test. * P < 0.05, ** P < 0.01 relative to control. (A. immunocompetent mice, B. immunodeficient mice)

Figure 2. Expression of CRT and HMGB1 by PDT

CT26, HT29, or HCT116 cells were loaded with G-chlorin for 4 hours and then irradiated with 16 J/cm² of 660-nm LED light. CT26, HT29, and HCT116 cells were incubated with 1 μM mitoxantrone (MTX) as a positive control. CRT or HMGB1 protein expression was measured by western blotting at 4 hours after treatment.

Figure 3. Translocation of CRT and HMGB1 by PDT

CT26 cells were loaded with G-chlorin for 4 hours and irradiated with 16 J/cm² of 660-nm LED light. Translocation of CRT and HMGB1 was assessed by immunofluorescence staining at 4 hours after treatment with PDT with G-chlorin. Images were obtained using confocal microscopy (original magnification ×1000; scale bar = 10 μm). Data are means of three independent experiments ± SD. (A. CRT, B. HMGB1)

Figure 4. Immunohistochemistry of allograft tumors

CT26 cells were inoculated into the dorsal skin of mice. Tumor-bearing mice were intravenously injected with 1.25 μmol/kg G-chlorin and, after 4 hours, were illuminated with 660-nm LED light (15 J/cm²). The tumors were excised and fixed in formalin for immunohistochemical examination at 3 hours after treatment with PDT plus G-chlorin. Representative immunohistochemical findings for lesions in the tumors of the control and PDT-treated mice (Panel A. original magnification ×400; scale bar = 50 μm). CRT B. and HMGB1 C. labeling indices in tumors. Data are the mean ± SD. Significance was determined by Welch's t-test. ** P < 0.01 relative to control.

Figure 5. Vaccination

A. Knockdown of CRT or HMGB1 by using short interfering RNA (siRNA). CT26 cells were transiently transfected with siRNAs against CRT or HMGB1. CRT or HMGB1 protein expression was analyzed by western blotting at 48 hours after transfection. B, C. CT26 cells treated in vitro with G-chlorin-PDT were inoculated subcutaneously into BALB/c mice. After 7 days, mice were re-challenged with live CT26 cells. The percentages of tumor-free mice were pooled. Each group comprised ten mice. Significance was determined by the log-rank statistic. * P < 0.05, ** P < 0.01 relative to control. (B; CRT, C; HMGB1)

Figure 6. Chemical structure of G-chlorin

Glycoconjugated chlorin; 5, 10, 15, 20-tetrakis (4-(β-D-glucopyranosylthio)-2, 3, 5, 6- tetrafluorophenyl)-2, 3-(methano (N-methyl) iminomethano) chlorine