Molecular targeted photoimmunotherapy for HER2-positive human gastric cancer in combination with chemotherapy results in improved treatment outcomes through different cytotoxic mechanisms

Abstract

Background: Photoimmunotherapy (PIT) is a novel type of molecular optical imaging-guided cancer phototherapy based on a monoclonal antibody conjugated to a photosensitizer, IR700, in combination with near-infrared (NIR) light. PIT rapidly causes target-specific cell death by inducing cell membrane damages and appears to be highly effective; however, we have previously demonstrated that tumor recurrences were eventually seen in PIT-treated mice, likely owing to inhomogeneous mAb-IR700 conjugate distribution in the tumor, thus limiting the effectiveness of PIT as a monotherapy. Here, we examined the effects of human epidermal growth factor-2 (HER2)-targeted PIT in combination with 5-fluorouracil (5-FU) compared to PIT alone for HER2-expressing human gastric cancer cells.

Methods: NCI-N87 cells, HER2-positive human gastric cancer cells, were used for the experiments. Trastuzumab, a monoclonal antibody directed against HER2, was conjugated to IR700. To assess the short-term cytotoxicity and examine the apoptotic effects upon addition of 5-FU in vitro, we performed LIVE/DEAD and caspase-3 activity assays. Additionally, to explore the effects on long-term growth inhibition, trypan blue dye exclusion assay was performed. NCI-N87 tumor xenograft models were prepared for in vivo treatment studies and the tumor-bearing mice were randomized into various treatment groups.

Results: Compared to PIT alone, the combination of HER2-targeted PIT and 5-FU rapidly induced significant cytotoxicity in both the short-term and long-term cytotoxicity assays. While both 5-FU and/or trastuzumab-IR700 conjugate treatment induced an increase in caspase-3 activity, there was no additional increase in caspase-3 activity upon NIR light irradiation after incubation with 5-FU and/or trastuzumab-IR700. The combination of HER2-targeted PIT and 5-FU resulted in greater and longer tumor growth inhibition than PIT monotherapy in vivo. This combined effect of PIT and 5-FU is likely owing to their different mechanisms of inducing tumor cell death, namely necrotic membrane damage by PIT and apoptotic cell death by 5-FU and trastuzumab.

Conclusions: PIT in combination with 5-FU resulted in enhanced antitumor effects compared to PIT alone for HER2-expressing human gastric cancer in vitro and in vivo. This combination photoimmunochemotherapy represents a practical method for treating human gastric cancer and should be investigated further in the clinical setting.

Fig. 1

In vitro human epidermal growth factor receptor 2 (HER2) expression in human gastric cancer cells. a, b Flow cytometry revealed strong HER2-specific signals in NCI-N87 cells and very weak signals in MKN-45 cells. Specific binding of trastuzumab-IR700 was demonstrated by excess trastuzumab blocking. c, d, e Fluorescence microscopy showed HER2-specific IR700 localization in NCI-N87 cells. Conversely, IR700 fluorescence was not detected in MKN-45 cells or in NCI-N87 cells pre-treated with an excess volume of trastuzumab. Cellular swelling and bleb formation were rapidly induced after excitation light in NCI-N87 cells but not in MKN-45 cells (lower right). Scale bar, 50 μm. DIC: differential interference contrast, NIR: near-infrared

Fig. 2

In vitro photoimmunochemotherapy for human gastric cancer cells. a, b Human epidermal growth factor receptor 2 (HER2)-targeting photoimmunotherapy (PIT) induced near-infrared (NIR) light dose-dependent cell death in NCI-N87 cells but not in MKN-45 cells. The percentage of cell death was significantly increased by PIT treatment in combination with 5-fluorouracil (5-FU) compared to PIT alone. Significant cytotoxic effects of 5-FU were observed in both NCI-N87 and MKN-45 cells. Spotted bar: no treatment, oblique line bar: trastuzumab-IR700 (Tra-IR700) (10 μg/ml) alone, unfilled bar: Tra-IR700 (10 μg/ml) followed by NIR light irradiation (PIT alone), filled bar: addition of 5-FU (3 μM). Data are presented as means ± SEM (n = 3, *P < 0.05, **P < 0.01, Student’s t test). c Significant long-term growth inhibition was observed in cells treated by PIT and 5-FU, as determined by the trypan blue exclusion assay. Data are presented as means ± SEM (n = 3, *P < 0.05, ***P < 0.001 vs. treated with PIT alone, Student’s t test). D While 5-FU and/or Tra-IR700 treatment induced an increase in caspase-3 activity, NIR light irradiation (PIT treatment) did not further increase the caspase-3 activity in NCI-N87 cells. Data are presented as means ± SEM (n = 3, *P < 0.05, ***P < 0.001, Student’s t test). Spotted bar: no treatment, oblique line bar: Tra-IR700 (10 μg/ml) alone, unfilled bar: Tra-IR700 (10 μg/ml) followed by NIR light irradiation (PIT alone), filled bar: addition of 5-FU (3 μM)

Fig. 3

Human epidermal growth factor receptor 2 (HER2)-specific accumulation of trastuzumab-IR700 conjugate (Tra-IR700) in vivo. a Tra-IR700 distribution over time was assessed using an in vivo imaging system. NCI-N87 tumors (right dorsum) were clearly and selectively visualized by IR700 fluorescence as early as 1 day after Tra-IR700 injection. Conversely, MKN-45 tumors (left dorsum) were only weakly visualized by IR700 fluorescence, and the signals gradually attenuated over time (n = 5 mice). b Fluorescence intensity of IR700 in NCI-N87 tumors, MKN-45 tumors, and background. Data are presented as means ± SEM (n = 5 mice, ** P < 0.01 vs. MKN-45 tumors, Student’s t test). c Tumor-to-background ratios of IR700 fluorescence intensity in NCI-N87 and MKN-45 tumors. Data are presented as means ± SEM (n = 5 mice, ** P < 0.01, *** P < 0.001 vs. MKN-45 tumors, Student’s t test)

Fig. 4

In vivo photoimmunochemotherapy for human gastric cancer xenografts. a Experimental protocol of photoimmunochemotherapy in vivo. (a) No treatment, (b) 30 μg/g/day of 5-fluorouracil (5-FU) i.p. without near-infrared (NIR) light irradiation, (c) 300 μg of trastuzumab-IR700 conjugate (Tra-IR700) i.v. without NIR light irradiation, (d) NIR light irradiation (100 J/cm²) without 5-FU or Tra-IR700 injection, (e) 30 μg/g/day of 5-FU i.p. and 300 μg of Tra-IR700 i.v. without NIR light irradiation, (f) 30 μg/g/day of 5-FU i.p. followed by NIR light irradiation (100 J/cm²), (g) 300 μg of Tra-IR700 i.v. followed by NIR light irradiation (100 J/cm²), and (h) 30 μg/g/day of 5-FU i.p. and 300 μg of Tra-IR700 i.v. followed by NIR light irradiation (100 J/cm²). Tra-IR700 injection was performed at the time of mouse randomization (Day 0), and NIR light irradiation was performed 24 h after Tra-IR700 injection (Day 1). Injection of 5-FU was performed on 3 consecutive days (Day 1 [just after NIR light irradiation], Day 2, and Day 3; total amount, 90 μg/g). Open downwards arrow: 300 μg of Tra-IR700 i.v.; filled upwards arrow: 30 μg/g of 5-FU i.p. b Photoimmunochemotherapy resulted in marked antitumor effects compared to photoimmunotherapy (PIT) alone. A significant therapeutic effect was observed in mice treated with PIT in combination with 5-FU compared to in mice treated with PIT alone on Days 24 and 26 after the initial treatment. Data are presented as means ± SEM (at least n = 10 mice in each group, * P < 0.05 vs. treated with PIT alone, Mann–Whitney U test). c Histological observations of the negative control and treated NCI-N87 tumors (n = 3 mice, hematoxylin and eosin staining). Bleeding and massive granulation with inflammatory changes were observed, and only scant viable cells were present in the tumor nodules treated with PIT in combination with 5-FU. Scale bar, 100 μm