Tumor-selective, adenoviral-mediated GFP genetic labeling of human cancer in the live mouse reports future recurrence after resection

Abstract

We have previously developed a telomerase-specific replicating adenovirus expressing GFP (OBP-401), which can selectively label tumors in vivo with GFP. Intraperitoneal (i.p.) injection of OBP-401 specifically labeled peritoneal tumors with GFP, enabling fluorescence visualization of the disseminated disease and real-time fluorescence surgical navigation. However, the technical problems with removing all cancer cells still remain, even with fluorescence-guided surgery. In this study, we report imaging of tumor recurrence after fluorescence-guided surgery of tumors labeled in vivo with the telomerase-dependent, GFP-containing adenovirus OBP-401.. Recurrent tumor nodules brightly expressed GFP, indicating that initial OBP-401-GFP labeling of peritoneal disease was genetically stable, such that proliferating residual cancer cells still express GFP. In situ tumor labeling with a genetic reporter has important advantages over antibody and other non-genetic labeling of tumors, since residual disease remains labeled during recurrence and can be further resected under fluorescence guidance.

Figure 1

In situ genetic labeling of disseminated peritoneal carcinomatosis. Red fluorescence indicates HCT-116-RFP-expressing disseminated nodules (left). Peritoneal disseminated HCT116-RFP cells were labeled by GFP after i.p. injection of OBP-401 (right). Fluorescence imaging revealed co-localization of red and green fluorescence.

Figure 2

Genetic labeling of microscopic tumors. Cells collected by peritoneal lavage from the abdominal cavity of mice 5 d after OBP-401 treatment were plated and cultured with RPMI 1640 medium supplemented with 10% FBS. (A) Plating cells in the peritoneal lavage fluid (5 d after viral administration). Most RFP-expressing cancer cells expressed GFP fluorescence induced by OBP-401 as well, x200 magnification. White arrows, cells unlabeled with GFP. (B) Eight days after plating (i.e., 13 d after viral administration). Cancer cell colonies expressing RFP were observed in the culture dish under fluorescence microscopy. The cancer cells also expressed GFP induced by OBP-401. x40 magnification. Boxes highlight colonies indicated by white circles. Original magnification x100.

Figure 3

Fluorescence-guided resection of tumors labeled with GFP in situ. (A) Peritoneal disseminated nodules were labeled by GFP expression 5 d after OBP-401 virus administration. (B) Laparotomy was performed. (C) Disseminated nodules labeled with GFP were removed under GFP-guided surgical navigation. (D) Disseminated nodules removed under GFP-guided navigation. Top, bright field observation; Bottom, fluorescent detection. (E) Section of disseminated nodules. Top, H&E section; Bottom, frozen section with fluorescence detection.

Figure 4

In vivo detection of recurrent tumors after fluorescence-guided surgery. (A) Brightfield observation several weeks after fluorescence-guided surgery of OBP-401 GFP-labeled tumors. Disseminated disease re-emerged. (B) Fluorescence observation of field observed by brightfield in (A). (C) Merge of (A and B). The red box outlines a region of (D) below. (D) Detail of the boxed region of (C). Black line indicates the direction of cross-sections. (E) Histologic sections stained with H&E showing that GFP-labeled lesions are recurrent tumor tissues (arrow heads). x40 magnification. (F) Detail of the boxed region of (E). x200 magnification.