Virally-directed fluorescent imaging (VFI) can facilitate endoscopic staging

Abstract

Background: Replication-competent, tumor specific herpes simplex virus NV1066 expresses green fluorescent protein (GFP) in infected cancer cells. We sought to determine the feasibility of GFP-guided imaging technology in the intraoperative detection of small tumor nodules.

Methods: Human cancer cell lines were infected with NV1066 at multiplicities of infection of 0.01, 0.1 and 1. Cancer cell specific infectivity, vector spread and GFP signal intensity were measured by flow cytometry and time-lapse digital imaging (in vitro); and by use of a stereomicroscope and endoscope equipped with a fluorescent filter (in vivo).

Results: NV1066 infected all cancer cell lines and expressed GFP at all MOIs. GFP signal was significantly higher than the autofluorescence of normal cells. One single dose of NV1066 spread within and across body cavities and selectively infected tumor nodules sparing normal tissue. Tumor nodules undetectable by conventional thoracoscopy and laparoscopy were identified by GFP fluorescence.

Conclusion: Virally-directed fluorescent imaging (VFI) is a real-time novel molecular imaging technology that has the potential to enhance the intraoperative detection of endoluminal or endocavitary tumor nodules.

Figure 1

NV1066 infects and express GFP in cancer cells. NV1066 infected cancer cells express GFP as identified by fluorescent microscopy. Shown in Figure A are the GFP expressing cancer cells 24 hours following infection at an MOI of 1. Shown in Figure B is the overlay of GFP expression with HSV immunohistochemistry to confirm GFP expression is due to HSV infection (GFP, green color; HSV antibody staining, brown color; nuclear Hoechst staining, blue color). VAMT malignant mesothelioma cells and HCT-8 colon cancer cells were infected with NV1066 at an MOI of 0.01 (open triangles) or 0.1 (filled circles) or 1 (filled squares). Cells were harvested at day 1, 2, 3, 4, 5 and 6 and analyzed by flow cytometry for GFP expression. GFP expressing cells were represented as percentage of control of uninfected cells in Figures C (VAMT) and D (HCT-8). Lactate dehydrogenase cytotoxicity assay was performed to assess viable cells on day 3, 4, 5, 6 and 7. Results for the treated group were expressed as cell survival compared to untreated control cells grown under identical conditions in Figures E (VAMT) and F (HCT-8). GFP: green fluorescent protein, MOI: multiplicity of infection; HSV: herpes simplex virus

Figure 2

Thoracoscopic identification of pleural carcinomatosis by GFP expression. Using fluorescent thoracoscopy, the pleural cavities of mice with macroscopic tumor deposits were examined in bright-field (A & E, parietal pleura; G, parietal and visceral pleura) and GFP (B, D, F and H) modes. Diaphragmatic pleural deposits (C) can be readily identified by the fluorescent laparoscopy (D). Following intrapleural administration, NV1066-induced GFP expression localized to tumor deposits, sparing normal tissues as identified by fluorescent thoracoscopy (J and L, bright-field: I and K). GFP: green fluorescent protein

Figure 3

Laparoscopic identification of peritoneal carcinomatosis by GFP expression. Intraperitoneal administration of NV1066 facilitates visualization of intraabdominal macroscopic and microscopic carcinomatosis. Peritoneal macroscopic carcinomatosis is visualized by fluorescent laparoscopy (A and C, intraperitoneal deposits; B, subdiaphragmatic deposits). Macroscopically undetectable peritoneal nodules can be accurately visualized by fluorescent laparoscopy (D, overlay of bright-field and GFP). Similarly, disseminated tumor nodules can be clearly identified by GFP expression (G, bright-field; H, GFP mode). Microscopic nodal deposits that cannot be visualized by bright-field (I) can be readily identified by GFP expression (J). Fluorescent upper gastrointestinal endoscopy identified cancerous lesions (<1 mm.) (K and L). GFP: green fluorescent protein

Figure 4

Histopathological confirmation that NV1066 selectively infects and expresses GFP in tumor deposits, sparing normal tissue: Tissue specimens were selected by GFP expression under fluorescent microscopy. Following sectioning, specimens were examined under fluorescent microscopy (A), and then H & E stained (B) for identification of tumor cells. All sections that expressed GFP had tumor cell infiltrates. H & E: Hematoxylin and Eosin staining, GFP: green fluorescent protein