Near infrared photoimmunotherapy for lung metastases

Abstract

Lung metastases are a leading cause of cancer related deaths; nonetheless current treatments are limited. Near infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of intravenously injected antibodies that target tumors with the toxicity induced by photosensitizers activated by NIR-light. Herein, we demonstrate the efficacy of NIR-PIT in a mouse model of lung metastases. Experiments were conducted with a HER2, luciferase and GFP expressing cell line (3T3/HER2-luc-GFP). An antibody-photosensitizer conjugate (APC) consisting of trastuzumab and a phthalocyanine dye, IRDye700DX, was synthesized. In vitro NIR-PIT-induced cytotoxicity was light dose dependent. With 3D culture, repeated NIR-PIT could eradicate entire spheroids. In vivo anti-tumor effects of NIR-PIT included significant reductions in both tumor volume (p = 0.0141 vs. APC) and bioluminescence image (BLI) (p = 0.0086 vs. APC) in the flank model, and prolonged survival (p < 0.0001). BLI demonstrated a significant reduction in lung metastases volume (p = 0.0117 vs. APC). Multiple NIR-PIT doses significantly prolonged survival in the lung metastasis model (p < 0.0001). These results suggested that NIR-PIT is a potential new therapy for the local control of lung metastases.

Keywords: Bioluminescence; HER2 receptor; Lung metastases; Near infrared photoimmunotherapy.

Fig. 1. Evaluation of NIR-PIT effect on in vitro 2D culture

(A) 3T3/HER2-luc-GFP cells were incubated with tra-IR700 for 6 hr, and observed with a microscope before and after irradiation of NIR-light (2 J/cm²). Necrotic cell death was observed after exposure to NIR-light (1 hr after PIT). Bar = 50 μm. (B) Membrane damage and necrosis induced by NIR-PIT was measured by dead cell count using PI staining. Cell killing increased in a NIR-light dose-dependent manner. (n = 4, *p < 0.0001, vs. untreated control, Student’s t test) (C) Luciferase activity in 3T3/HER2-luc-GFP cells was measured as relative light unit (RLU), which also decreased in a NIR-light dose-dependent manner. (n = 4, **p < 0.0001, vs. untreated control, Student’s t test) (D) Bioluminescence imaging (BLI) of a 10 cm dish demonstrated that luciferase activity in 3T3/HER2-luc-GFP cells decreased in a NIR-light dose-dependent manner. (E) 3T3/HER2-luc-GFP cells were incubated with tra-IR700 for 6 hr and irradiated with NIR-light (0.5 J/cm²). GFP-fluorescence intensity decreased in dead cells (*) stained by PI but was unchanged in living cells at 1 hr after NIR-PIT. Bar = 200 μm. (F) GFP fluorescence intensity decreased after NIR-PIT in a NIR-light dose-dependent manner as measured by FACS. (n = 4, ***p < 0.0001, vs. untreated control, Student’s t test) (G) Diminishing GFP-fluorescence intensity at 1 hr after PIT occurred in a manner dependent on the light dose (total pixel of GFP fluorescence in the same)(n = 12 fields)(*p < 0.0001, vs. untreated control, Student’s t test).

Fig. 2. Characterization and evaluation of NIR-PIT in vitro 3D culture

(A) 3D spheroid at day 7 after 6hr incubation with tra-IR700, before and after irradiation of NIR-light (0.5 J/cm²). Necrotic cell death was observed in the layer of IR700 1hr after NIR-light. Bar = 200 μm. (B) Bioluminescence in 3T3/HER2-luc-GFP 3D spheroids was measured as RLU, and decreased in a NIR-light dose-dependent manner. (n = 5, *p < 0.0001, vs. untreated control, Student’s t test) (C) The PIT regimen incorporating repeated NIR-light exposures is shown. (D) Day 7 3T3/HER2-luc-GFP 3D spheroids were divided into 4 groups as shown. Bar = 200 μm. (E) BLI of each group demonstrated that luciferase activity decreased after repeated PIT. Bar = 5 mm. (F) Luciferase activity in 3D spheroids gradually decreased after repeated NIR-PIT leading to complete killing of cells in the spheroid (n = 8). (**p = 0.0075 < 0.01, ***p < 0.0001 vs APC, Tukey’s test with ANOVA).

Fig. 3. Characterization the lung metastasis tumor model

(A) In vivo BLI and fluorescence (GFP/IR700/IR800) imaging of 3T3/HER2-luc-GFP lung metastasis model are shown and indicated colocalization of signal. To avoid auto-fluorescence, tra-IR800 was used as well as tra-IR700 for imaging. Fluorescence thoracoscopy was indicated lung metastasis tumors had both IR700 and GFP fluorescence signals. APCs were intravenously injected 1 day before the imaging. (B) CT image confirmed the multiple metastasis inside lung.

Fig. 4. Evaluation of NIR-PIT effects on lung metastasis model

(A) The regimen of NIR-PIT is shown. Images were obtained at each time point as indicated. (B) In vivo BLI and IR700 fluorescence imaging of lung metastasis model. Prior to treatment mice exhibiting approximately the same luciferase activity in the chest were selected. (C) Quantitative RLU in the lung metastasis model showed a significant decrease in the PIT (n = 8 mice in each group)(*p = 0.0117 < 0.05, vs APC, Tukey’s test with ANOVA). (D) NIR-PIT led no significant prolonged survival in 3T3/HER2-luc-GFP lung metastasis model. (n = 8 mice in each group)(**p = 0.2674, ns, Long-rank test and Wilcoxon test). (E) BW ratio of tumor bearing mice in response to NIR-PIT. No significant difference was detected among the groups (n = 8).

Fig. 5. Evaluation of multiple NIR-PIT effects on lung metastasis model

(A) The regimen of multiple NIR-PIT is indicated. Images were obtained at each time point as indicated. (B) In vivo BLI and fluorescence imaging of lung metastasis model in response to multiple NIR-PIT. (C) Quantitative RLU in the lung metastasis model showed a significant decrease in the multiple PIT (n = 8 mice in each group)(*p = 0.0491 < 0.05, **p = 0.0376 < 0.05 vs. APC, Tukey’s test with ANOVA). (D) Multiple NIR-PIT significantly prolonged survival in lung metastasis model. (n = 9 mice in each group)(***p < 0.0001, Long-rank test and Wilcoxon test). (E) BW ratio of tumor bearing mice in response to multiple NIR-PIT. No significant difference was detected among the groups (n = 8 mice in each group)(****p = 0.0146 < 0.05 vs. APC, Tukey’s test with ANOVA).

Fig. 6. NIR-PIT effects on flank or lung metastasis model by GFP fluorescence imaging

(A) The regimen of NIR-PIT is shown. Images were obtained at each time point as indicated. (B) In vivo BLI and ex vivo fluorescence imaging of lung metastasis model in response to NIR-PIT. Both bioluminescence and IR700 fluorescence intensity was decreased by NIR-PIT. GFP fluorescence of ex vivo indicated the decrease with NIR-PIT compared to APC mouse. (C) Histological specimens of lung with metastatic tumors, which were treated with PIT at 0, 100 J/cm², are shown. All specimens are stained with Hematoxylin and Eosin. Well-packed metastatic tumor nodules containing scattered clusters of necrotic cells surrounded by layers of necrotic tussue were shown after PIT with 100 J/cm², while the intact structure of alveolar was sustained. Necrotic tissues especially located around tumor nodules were not found without PIT. Bar indicates 50 μm.