Low dose of GRP78-targeting subtilase cytotoxin improves the efficacy of photodynamic therapy in vivo

Abstract

Photodynamic therapy (PDT) exerts direct cytotoxic effects on tumor cells, destroys tumor blood and lymphatic vessels and induces local inflammation. Although PDT triggers the release of immunogenic antigens from tumor cells, the degree of immune stimulation is regimen-dependent. The highest immunogenicity is achieved at sub-lethal doses, which at the same time trigger cytoprotective responses, that include increased expression of glucose-regulated protein 78 (GRP78). To mitigate the cytoprotective effects of GRP78 and preserve the immunoregulatory activity of PDT, we investigated the in vivo efficacy of PDT in combination with EGF-SubA cytotoxin that was shown to potentiate in vitro PDT cytotoxicity by inactivating GRP78. Treatment of immunocompetent BALB/c mice with EGF-SubA improved the efficacy of PDT but only when mice were treated with a dose of EGF-SubA that exerted less pronounced effects on the number of T and B lymphocytes as well as dendritic cells in mouse spleens. The observed antitumor effects were critically dependent on CD8+ T cells and were completely abrogated in immunodeficient SCID mice. All these results suggest that GRP78 targeting improves in vivo PDT efficacy provided intact T-cell immune system.

Figure 1

EGF-SubA exerts cytostatic/cytotoxic effects on CT26 cells expressing human EGFR. (A) CT26 cells were infected with retroviruses containing human EGFR and sorted for high expression of surface EGFR. The level of expression was confirmed by flow cytometry. (B) CT26-EGFR cells were plated onto 60-mm dishes and incubated for 48 h with different concentrations of EGF-SubA. The amounts of GRP78 protein and the 46-kDa GRP78 cleavage product (cl-GRP78) were evaluated by western blotting. Actin was used as a loading control. (C) CT26-EGFR and CT26 WT cells were plated onto a 96-well plate and incubated for 48 h with increasing concentrations of EGF-SubA. The viability of the cells was determined by crystal violet staining.

Figure 2

EGF-SubA affects immune cells. EGF-SubA at a dose of 25 or 50 µg/kg was administered i.p. for three consecutive days. Twenty-four hours after the last injection, mouse spleens were collected, total numbers of splenocytes were counted and analyzed by flow cytometry. Total numbers of cytotoxic (A) and helper (B) T cells, dendritic cells (C), B cells (D), and total leukocytes (E) were calculated. n=7 (control group), n=8 (25 and 50 µg/kg). ^*P<0.05, ^**P<0.01, ^***P<0.005 compared with the control group (Mann-Whitney test).

Figure 3

EGF-SubA causes pathological changes in murine livers. Hematoxylin and eosin-stained liver sections from control and 50 µg/kg EGF-SubA-treated mice, scale bars A, C, E and G = 250 µm; B, D, F and H = 125 µm. (A and B) Periportal area (centrilobular area) of control liver. (C) Hepatocytes with various densities of cytoplasm; the white arrow indicates the area around a central vein; the black arrow indicates the periportal area. (D) A higher magnification of the area around a central vein. (E) An area of hemorrhagic foci and necrosis are indicated with white and black arrows, respectively. (F) Dispersed lymphocytic infiltration. (G and H) Necrosis with erythrocytes, marked with arrows.

Figure 4

EGF-SubA at a dose of 25 µg/kg potentiates the antitumor effects of PDT in BALB/c mice injected with CT26-EGFR cells. (A) Mice were inoculated with 3×10⁵ CT26-EGFR cells. On days 6–8 mice were treated with EGF-SubA at a dose of 25 µg/kg (B and C) or 50 µg/kg (D and E). Photofrin was administered i.p. at a dose of 10 mg/kg on day 6. Twenty-four hours later, the tumor site was illuminated with laser light at a fluence of 50 J/cm². (B and D) Mean tumor volumes (±SE). Experiments were performed twice (EGF-SubA 25 µg/kg) or once (EGF-SubA 50 µg/kg). One representative result is shown for EGF-SubA 25 µg/kg (n=7–8 for all experimental groups). (C and E) Kaplan-Meyer plots of the survival of the mice bearing CT26-EGFR tumors. The overall survival from two combined independent experiments is shown for EGF-SubA 25 µg/kg (n=12). P=0.067 (PDT + EGF-SubA compared to all other groups, log-rank test). n=5–6 in all experimental groups in the EGF-SubA 50 µg/kg group.

Figure 5

The efficacy of the combination treatment is dependent on CD8⁺ T cells. (A) BALB/c mice were inoculated with 3×10⁵ CT26-EGFR cells. On days 6–8, the mice were treated with EGF-SubA at a dose of 25 µg/kg. Photofrin was administered i.p. at a dose of 10 mg/kg on day 6. Twenty-four hours later, the tumor site was illuminated with laser light at fluence of 50 J/cm². On days 5 and 11, the mice were injected i.p. with 100 µg of anti-CD8 (YTS169) mAbs or a control antibody. (B) Mean tumor volumes (±SE). The experiment was repeated twice and a representative result is shown. Depl (depletion) describes group of mice treated with depleting mAbs and iso refers to the group treated with isotype control mAbs (n=6 for control iso and control depl, n=5 for PDT + EGF-SubA iso and n=8 for PDT + EGF-SubA depl). (C) Kaplan-Meyer plots of the survival of the mice bearing CT26-EGFR tumors. The overall survival from two combined independent experiments is shown (n=12–13). ^*P<0.05 compared with all experimental groups (Mann-Whitney test). ^#P<0.001 compared with all experimental groups (log-rank test).

Figure 6

EGF-SubA + PDT treatment is ineffective in immunocompromised mice. (A) SCID mice were inoculated with 2×10⁶ of DU-145 cells. On days 7–9, the mice were treated i.p with EGF-SubA (25 µg/kg) in PBS. Photofrin was administered i.p. at a dose of 10 mg/kg on day 7. Twenty-four hours later, the tumor site was illuminated with laser light at fluence of 43 J/cm². (B) Mean tumor volumes (±SE). The experiment was performed once. (C) Kaplan-Meyer plots of the survival of the mice bearing DU-145 tumors (n=7–8).