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

<p>*compared to BLS.</p><p>**compared to FGS.</p

Fluorescence-Guided Surgery in Combination with UVC Irradiation Cures Metastatic Human Pancreatic Cancer in Orthotopic Mouse Models

<div><p>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; <i>p</i> = 0.007). The BLS treated mice had significantly reduced survival compared to FGS- and FGS-UVC-treated mice for both relapse-free survival (RFS) (<i>p</i><0.001 and <i>p</i><0.001, respectively) and overall survival (OS) (<i>p</i><0.001 and <i>p</i><0.001, respectively). FGS-UVC-treated mice had increased RFS and OS compared to FGS-only treated mice (<i>p</i> = 0.008 and <i>p</i> = 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.</p></div

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

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

Recurrence rate of PDOX in each treatment group.

<p>* p = 0.041, compared to FGS only.</p><p>Recurrence rate of PDOX in each treatment group.</p

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

<p>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. **<i>p</i><0.01.</p

Representative gross and histological images of excised tumors in each treatment group.

<p>Left panels of (A) and (B) indicate bright field (BF) images and right panels indicate fluorescence images for DyLight 650 (650). Histopathological response to GEM treatment was defined according to Evans’s grading scheme. The tumors without GEM treatment (FGS only) were comprised of viable cancer cells that formed glandular structures and judged as Grade I (C). In the tumors with GEM treatment, over 50% of cancer cells were dead and replaced by necrotic tissue or stromal cells (D). Treatment efficacy of GEM on the pancreatic cancer PDOX was judged as grade IIb - III (D). Fluorescence decreased in some areas of the tumor treated with GEM, but was sufficient for FGS (B). Scale bars: 5 mm (A and B), 250 µm (C and D).</p

Antibody labelling of the pancreatic cancer patient derived orthotopic xenograft.

<p>The patient’s pancreatic cancer was diagnosed as moderately differentiated adenocarcinoma with H&E staining (A). The tumor was strongly stained with anti-CA19-9 antibody (B), whereas the signal was very weak with anti-CEA antibody (C). Scale bars: 100 µm. (D and E) Whole body images of a subcutaneous tumor in nude mice labeled with anti-CA19-9- or anti-CEA-conjugated DyLight 650. Fifty µg anti-CA19-9 DyLight 650 or anti-CEA DyLight 650 was injected in the tail vain of the mice with subcutaneous tumors. Twenty-four hours later, whole body images were taken with the OV100 (Olympus). Yellow arrowheads indicate subcutaneous tumors. The subcutaneous tumors were brightly labeled with anti-CA19-9 DyLight 650 (D), whereas, the fluorescence signal from the tumor labeled with anti-CEA DyLight 650 was very weak (E). Scale bars: 10 mm.</p

Representative time course of tumor recurrence after FGS.

<p>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.</p

Experimental schema and FGS imaging system.

<p>(A) Schema of the experimental design. After confirmation of tumor growth, the PDOXs were randomized to 4 groups: BLS only; BLS+NAC; FGS only; or FGS+NAC. Each treatment arm involved 8 tumor-bearing mice. The mice randomized to the NAC groups were treated with GEM (80 mg/kg) on day 8, 15 and 22. All animals underwent surgery on day 29. BLS was performed under standard bright-field using the MVX10 microscope. Fifty µg of anti-CA19-9 antibody conjugated with DyLight 650 was injected in the tail vain of mice with tumors in the FGS group 24 hours before surgery. FGS was performed using the MINI MAGLITE LED PRO flash light (MAG INSTRUMENT, Ontario, CA, USA) with excitation filter ET640/30X (Chroma Technology Corporation, Bellows Falls, VT, USA) and a Canon EOS 60D digital camera with an EF–S18–55 IS lens (Canon, Tokyo, Japan) and emission filter HQ700/75M-HCAR (Chroma Technology Corporation) under fluorescence navigation (B). Twelve weeks after surgery, animals underwent laparotomy, and the tumors were imaged, weighed and harvested for analysis. Scale bars: 2 cm (filters) and 5 cm (flash light).</p

Representative images during FGS with or without NAC.

<p>Upper panels indicate bright field (BF) images and lower panels indicate fluorescence images for DyLight 650 (650). The fluorescence in the tumors treated with GEM (B) decreased compared to untreated tumors (A), but were still clearly detected. Scale bars: 10 mm.</p