Fluorescence-guided surgery in combination with UVC irradiation cures metastatic human pancreatic cancer in orthotopic mouse models

Abstract

The aim of this study is to determine if ultraviolet light (UVC) irradiation in combination with fluorescence-guided surgery (FGS) can eradicate metastatic human pancreatic cancer in orthotopic nude-mouse models. Two weeks after orthotopic implantation of human MiaPaCa-2 pancreatic cancer cells, expressing green fluorescent protein (GFP), in nude mice, bright-light surgery (BLS) was performed on all tumor-bearing mice (n = 24). After BLS, mice were randomized into 3 treatment groups; BLS-only (n = 8) or FGS (n = 8) or FGS-UVC (n = 8). The residual tumors were resected using a hand-held portable imaging system under fluorescence navigation in mice treated with FGS and FGS-UVC. The surgical resection bed was irradiated with 2700 J/m2 UVC (254 nm) in the mice treated with FGS-UVC. The average residual tumor area after FGS (n = 16) was significantly smaller than after BLS only (n = 24) (0.135±0.137 mm2 and 3.338±2.929 mm2, respectively; p = 0.007). The BLS treated mice had significantly reduced survival compared to FGS- and FGS-UVC-treated mice for both relapse-free survival (RFS) (p<0.001 and p<0.001, respectively) and overall survival (OS) (p<0.001 and p<0.001, respectively). FGS-UVC-treated mice had increased RFS and OS compared to FGS-only treated mice (p = 0.008 and p = 0.025, respectively); with RFS lasting at least 150 days indicating the animals were cured. The results of the present study suggest that UVC irradiation in combination with FGS has clinical potential to increase survival.

Figure 1. Schematic diagram of the experimental protocol.

Two weeks after orthotopic implantation of MiaPaCa-2-GFP pancreatic cancer, bright-light surgery (BLS) was performed on all tumor-bearing mice (n = 24). Postoperatively, the surgical resection bed was imaged with the OV100 at a magnification of 0.56x to detect residual tumor. Mice which underwent BLS were randomized into 3 treatment groups: BLS-only (n = 8), FGS (n = 8), or FGS-UVC (n = 8). Residual tumors in mice in the FGS and FGS-UVC groups were resected using the Dino-Lite imaging system under fluorescence navigation. After completion of FGS, the surgical resection bed was imaged with the OV100 at a magnification of 0.89x to detect micoscopic minimal residual cancer (MRC). The surgical resection bed in the mice in the FGS-UVC group was irradiated with 2700 J/m² UVC (emission peak, 254 nm) from the bottom of the chamber using a Benchtop 3UV transilluminator (UVP, LLC, Upland, CA).

Figure 2. Preoperative and postoperative images of the orthotopic pancreatic cancer model (A–F).

Upper panels are bright-field (BF), and lower panels show tumor fluorescence. The residual tumor after BLS was clearly detected with both the OV100 at a magnification of 0.56x (B) and the Dino-Lite at a magnification of 30x (E). The residual tumor after FGS was marginally detected with either the OV100 at a magnification of 0.56x (C) or the Dino-Lite at a magnification of 30x (F). The OV100 at a magnification of 0.89x clearly detected the minimal residual tumor after FGS (D). (G) The residual tumor area after FGS was significantly smaller than after BLS. All images were measured for residual tumor areas using ImageJ. **p<0.01.

Figure 3. Representative time course of tumor recurrence after FGS.

The recurrence was initially detected by non-invasive whole-body imaging using the OV100 at a magnification of 0.14x at week 11 after FGS (A; white arrowhead). The recurrent tumor progressed rapidly (B–D) and killed the mice by week 22 after FGS (D). Left axillary lymph-node metastasis (E; white arrowhead), large local recurrent tumor and many disseminating tumor nodules (F) were detected in the mice at time of death. In contrast, no recurrence was detected in the FGS-UVC group (G–J). Scale bars: 10 mm.

Figure 4. Relapse-free survival (RFS) (A) and overall survival (OS) (B) for tumor-bearing mice treated with BLS-only, FGS, or FGS-UVC.

Figure 5. Efficacy of UVC irradiation on non-colored BxPC-3, BxPC-3-Ab488 and BxPC-3-GFP in vitro.

(A) Representative images of non-colored BxPC-3, BxPC-3-Ab488 and BxPC-3-GFP in vitro. Cells were observed with the FV1000 confocal microscope (Olympus, Tokyo, Japan). Scale bars: 50 µm. (B) UVC was irradiated at various doses (0, 25, 50 and 100 J/m²). Compared to non-colored BxPC-3 cells, the number of BxPC-3-Ab488 and BxPC-3-GFP cells decreased significantly due to UVC irradiation with 25 and 50 J/m². The experimental data are expressed as the mean ± SD. *p<0.05, **p<0.01.