Near-infrared photoimmunotherapy with galactosyl serum albumin in a model of diffuse peritoneal disseminated ovarian cancer

Abstract

Near-infrared photoimmunotherapy (NIR-PIT) is a highly cell-selective cancer therapy based on an armed antibody conjugated with a phthalocyanine-based photo-absorber, IRDye700DX (IR700). NIR-PIT can quickly kill target cells that express specific proteins on the cellular membrane but only when the antibody-IR700 conjugate binds to the cell membrane and is then exposed to NIR light. NIR-PIT is highly selective based on the specificity of the antibody. Galactosyl serum albumin (GSA) is composed of albumin decorated with galactose molecules conjugated to the carboxyl groups of albumin. GSA binds to beta-D-galactose receptors, a surface lectin, which are overexpressed on the cell surface of many cancers, including ovarian cancers and is quickly internalized after binding. Here, we demonstrate the feasibility of NIR-PIT in a model of disseminated peritoneal ovarian cancer (SHIN3 cells) using GSA-IR700 that binds to beta-D-galactose receptors. GSA-IR700 bound quickly to SHIN3 cells, then accumulated in the endo-lysosomes. Cell-specific killing was observed in vitro, yet a relatively large dose of NIR light exposure was required for cell killing compared to antibody-IR700 conjugates. To evaluate in vivo therapeutic effects of GSA-IR700 NIR-PIT, peritoneal disseminated SHIN3 tumor-bearing mice were separated into four groups: no treatment; NIR light only; GSA-IR700 only; and GSA-IR700 NIR-PIT. Repeated NIR-PIT showed significant suppression of tumor based on bioluminescence compared to the other groups (p < 0.05). Thus, repeated NIR-PIT using GSA-IR700 can achieve efficient antitumor effects, although GSA-IR700 NIR-PIT was less effective than antibody-IR700 NIR-PIT conjugates likely due to the rapid internalization of GSA-IR700.

Keywords: beta-D-galactose receptor; galactosyl serum albumin; near-infrared photoimmunotherapy; ovarian cancer; peritoneal cancer metastases.

Figure 1

A. Differential interference contrast (DIC) and fluorescence microscopy images of SHIN3 cells after incubation with GSA-IR700 for 1, 3, and 6 h. The small fluorescent dots were seen in the cytoplasm 3 and 6 h after incubation. Necrotic cell death was observed upon excitation with NIR light (after 0, 5 min) regardless of incubation time. Scale bars = 20 μm. B. Dose and C. time-course examination using flow cytometric analysis. Binding affinity of GSA-IR700 to SHIN3 cells was examined. GSA-IR700 showed dose-dependent binding affinity to SHIN3 cells. GSA-IR700 bound to SHIN3 cells by 1 h of incubation, with increasing up to 3 h then plateaued. D. Membrane damage induced by NIR-PIT was measured by a dead cell count using SYTOX Green staining, which increased in a NIR light dose dependent manner (n = 3, *p < 0.05, **p < 0.01 vs. untreated control, by Student's t-test). E. Luciferase activity in SHIN3-luc cells was measured in relative light units (RLU), which decreased in a NIR light dose dependent manner (n = 6, *p < 0.05, **p < 0.01 vs. untreated control, by Student's t-test) F. Bioluminescence imaging (BLI) of a 10 cm dish. Luciferase activity in SHIN3-luc cells decreased in a NIR light dose dependent manner.

Figure 2

A. IR700, RFP fluorescence, and luciferase activity image of extracted mesentery derived from a SHIN3-luc-RFP tumor bearing mouse 3 h after i.p. administration of GSA-IR700. The fluorescence of IR700 was confirmed to be mostly coincident with RFP positive foci. Moreover, the fluorescence of luciferase activity was also confirmed to be mostly coincident with RFP positive foci. B. In vivo serial IR700 fluorescence images of SHIN3-RFP (upper row) and SHIN3-luc (lower row) using tumor-bearing mice with administration of GSA-IR700. The distribution of GSA-IR700 correlated well with the fluorescence of RFP or luciferase activity without evident accumulation in other organs up to 6 h after GSA-IR700 administration.

Figure 3

A. Outline of in vivo NIR-PIT regimen. B. BLI of SHIN3-luc tumor bearing mice was obtained every day up to day 7. Luciferase activity was decreased only in the NIR-PIT group. C. Quantitative analysis of RLU ratio. Significant suppression of increment of RLU ratio was seen in the NIR-PIT group compared to other groups (n = 5, *p < 0.05, **p < 0.01 vs. other groups, Tukey's HSD test with ANOVA).