Lymph node metastasis of the rectal tumor.

<p>Lymph node metastasis of the rectal tumor.</p

Dual-color imaging of tumor–host interaction in nestin-driven green fluorescent protein (ND-GFP) nude mice.

<p>(<b>a</b>) CT26-RFP tumor growing in the rectal mucosa of ND-GFP nude mice (white arrow heads). Red line, the direction of the rectal tumor cross-section of (<b>b</b>). Scale bar, 2 mm. (<b>b</b>) Cross-section of the rectal tumor. Bright- light observation. Scale bar, 2 mm. (<b>c</b>) Fluorescence observation of (<b>b</b>). Scale bar, 2 mm. (<b>d</b>) Detail of the boxed region in (<b>c</b>). Host-derived ND-GFP-expressing blood vessels were visualized in the RFP-expressing CT26 rectal tumor in the ND-GFP nude mouse (white arrows). Scale bar, 200 µm.</p

Spontaneous hematogenous metastasis.

<p>(<b>a</b>) Macroscopic appearance of lung. Lung metastatic foci were detected with GFP fluorescence. Scale bar, 2 mm. (<b>b</b>) Macroscopic appearance of the liver. Fluorescence imaging detected GFP expression of CT26-GFP liver metastases (red arrows). Scale bar, 10 mm.</p

Disruption of the mucosal barrier of the rectum.

<p>(<b>a</b>) Normal appearance of mouse anus. Scale bar, 1 mm. (<b>b</b>) Retractor made from a drip infusion tube. (<b>c</b>) The anorectal lumen was dilated by inserting the retractor into the anorectum and instilled with an acetic acid solution. (<b>c1</b>) Before acetic acid preparation. (<b>c2</b>) The color of the rectal mucosa changed from reddish pink to whitish after treatment with acetic acid solution. m  =  rectal mucosa. Scale bar, 1 mm. (<b>d</b>) Histological examination just after acetic acid treatment showed that the epithelial cell layer of the rectal mucosa was traumatized. (<b>d1</b>) H&E section of normal anorectum. A  =  surface epithelium; B  =  mucosa; C =  muscularis mucosae; D  =  submucosa; E  =  muscularis externa. (<b>d2</b>) After acetic acid treatment. Note that only the upper part of the mucosa is disrupted. Top, × 40 magnification; bottom, × 100 magnification.</p

Intramucosal CT26-GFP tumor formation in the mouse rectum.

<p>(<b>a</b>) The rectum was imaged noninvasively 10 days after implantation. Left, brightfield observation; middle, CT26-GFP tumors growing on the rectal mucosa were clearly visible under fluorescence observation; right, simultaneous observation under bright-light and fluorescence imaging. Scale bar, 500 µm. (<b>b</b>) After laparotomy, the rectum was opened longitudinally from the anterior wall. Left, gross appearance of the abdominal cavity. Scale bar: 10 mm; middle, detail of the boxed region; right, simultaneous observation under bright-light and fluorescence. The location of tumor formation was limited on the posterior wall of the terminal rectum. Red line, the direction of rectal cross-section of (<b>c</b>). White line, the direction of tumor cross-section of (d). Scale bar, 2 mm. (<b>c</b>) Histological analysis confirmed that GFP-positive lesions were medullary-type adenocarcinomas with no glandular structures. Left, fluorescence detection of tumors in a frozen section; middle, GFP-positive tumors showed high cellularity and were confirmed as tumors growing in the mucosal layer of the rectum; right, merged image of H&E histological section and fluorescence detection. Note that cancer cells locate only in the mucosal layer of the rectum. (<b>d1-3</b>) CT26 medullary-type adenocarcinomas invading the submucosal layer beyond the limits of the muscularis mucosae (red arrow heads). T  =  tumor. Black arrow heads, muscularis mucosae. (<b>e</b>) Histological appearance of human medullary-type adenocarcinoma. Green arrow heads indicate tumor edge <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079453#pone.0079453-Winn1" target="_blank">[23]</a>.</p

Growth kinetics of CT26-GFP rectal tumors in mice.

<p>(<b>a</b>) All the mice eventually had only a single tumor formed on the rectal mucosa. Tumor size increased over time. (<b>b</b>) Prolapsed CT26 rectal tumor growing outside of the anus at 4 weeks after implantation (red arrow heads). White tube inserted into the rectum (white arrow) shows that anorectal passage is well preserved, and there is no obstruction. Scale bar, 10 mm</p

Hematogenous metastasis of the rectal tumor.

<p>Hematogenous metastasis of the rectal tumor.</p

Spontaneous lymph nodes metastasis.

<p>(<b>a</b>) Schematic drawing of the anatomy in (<b>b</b>). The caudal mesenteric lymph node at the origin of the caudal mesenteric artery. This lymph node is equivalent to the inferior mesenteric lymph node in humans. Scale bar, 2 mm. (<b>b1</b>) Caudal mesenteric lymph node under bright-light observation. (<b>b2</b>) GFP fluorescence of the caudal mesenteric lymph node in (<b>b1</b>), indicating that the lymph node contained a metastasis. Scale bar, 2 mm. (<b>c</b>) Two para-aortic lymph nodes (red and yellow arrows) were identified in another mouse. White arrow indicates metastasized caudal mesenteric lymph node. Scale bar, 2 mm. (<b>d</b>) Higher magnification observation of a para-aortic lymph node indicated by red arrow in (<b>c</b>). GFP fluorescence of a micro-metastasis was detected (white arrows). Scale bar, 1 mm.</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