Fluorescence-Guided Surgery of Liver Metastasis in Orthotopic Nude-Mouse Models

<div><p>We report here the development of fluorescence-guided surgery of liver metastasis. HT29 human colon cancer cells expressing green fluorescent protein (GFP) were initially injected in the spleen of nude mice. Three weeks later, established liver metastases were harvested and implanted on the left lobe of the liver in other nude mice in order to make an orthotopic liver metastasis model. Fourteen mice with a single liver metastasis were randomized into bright-light surgery (BLS) or fluorescence-guided surgery (FGS) groups. Seven mice were treated with BLS, seven were treated with FGS. Three weeks after implantation, the left lobe of the liver with a single metastasis was exposed through a median abdominal incision. BLS was performed under white light. FGS was performed using a hand-held portable fluorescence imaging system (Dino-Lite). Post-surgical residual tumor fluorescence was visualized with the OV100 Small Animal Imaging System. Residual tumor fluorescence after BLS was clearly visualized at high magnification with the OV100. In contrast, residual tumor fluorescence after FGS was not detected even at high magnification with the OV100. These results demonstrate the feasibility of FGS for liver metastasis.</p></div

Pre-operative and post-operative images from the orthotopic liver metastasis model treated with FGS.

<p><b>(A)—(C)</b> Upper panels show bright field images, and lower panels are images of tumor fluorescence obtained with the OV100. Residual tumor fluorescence could not be detected even at high magnification <b>(C). (D,F,H)</b> Pre-FGS tumor fluorescence was clearly visualized with the Dino-Lite imaging system. <b>(E,G,I)</b> Dino-Lite imaging showed no evidence of tumor after FGS. <b>(J-K)</b> Dino-Lite settings. <b>(J)</b> After exposing the left lobe of the liver, the mouse was put under the Dino-Lite. <b>(K)</b> Connection between the Dino-Lite and computer. Tumor fluorescence was imaged on the monitor during FGS. Magnifications are indicated above the columns.</p

Evaluation of tumor fluorescence at day 28 after surgery.

<p><b>(A)</b> Upper panel shows the bright field image, and lower panel shows the GFP tumor fluorescence image obtained with the OV100 at a magnification of 0.56. Laparotomy was performed at the 28^th postoperative day. Bright field image shows tumor in the resection site in the liver (arrows). Strong GFP fluorescence from the tumor is seen in the lower panel. Arrows show recurrent tumor in the resection site. Arrowheads show operative scar on the liver. <b>(B)</b> The GFP tumor fluorescence area was significantly larger in the BLS group compared to the FGS group, where only autofluorescence was detected. Error bars show SD. *<i>P</i><0.05.</p

Pre-operative and post-operative images from the orthotopic liver metastasis model treated with BLS.

<p><b>(A)—(C)</b> Upper panels show bright field images, and lower panels are images of tumor fluorescence obtained with the OV100. At low magnification, residual tumor fluorescence was marginally detected. <b>(B)</b> However, at high magnification, residual tumor fluorescence was clearly visualized (arrows) <b>(C)</b>. Arrowheads show residual tumor fluorescence in <b>B</b> and <b>C</b>. <b>(D)</b> Resected specimen. Magnifications are indicated above the columns.</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) 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

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

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

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