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Color-coding cancer and stromal cells with genetic reporters in a patient-derived orthotopic xenograft (PDOX) model of pancreatic cancer enhances fluorescence-guided surgery

Precise fluorescence-guided surgery (FGS) for pancreatic cancer has the potential to greatly improve the outcome in this recalcitrant disease. In order to achieve this goal, we have used genetic reporters to color code cancer and stroma cells in a patient-derived orthotopic xenograft (PDOX) model. The telomerase-dependent green fluorescent protein (GFP) containing adenovirus OBP401 was used to label the cancer cells of the pancreatic cancer PDOX. The PDOX was previously grown in a red fluorescent protein (RFP) transgenic mouse that stably labeled the PDOX stroma cells bright red. The color-coded PDOX model enabled FGS to completely resect the pancreatic tumors including stroma. Dual-colored FGS significantly prevented local recurrence, which bright-light surgery (BLS) or single color could not. FGS, with color-coded cancer and stroma cells has important potential for improving the outcome of recalcitrant cancer.


Introduction
Accurate visualization of primary tumor margins and metastatic deposits at the time of surgery is critical to determine the success of a cancer operation. In order to achieve this goal, there is currently intense interest in labeling of tumors for fluorescence-guided surgery (FGS). In the clinic, sentinel lymph nodes were labeled by the near-infrared (NIR) fluorescing dye indocyanine. 1 However, indocyanine does not specifically label tumor cells. 5-Aminolevulinic acid, a precursor of hemoglobin, can selectively label malignant glioma. Porphyrin fluorescence in the brain tumor can then be visualized with a fluorescence neurosurgical microscope. 2 FGS resulted in a higher 6-month progression-free survival rates than did those who had bright-light surgery (BLS). 2 Folate conjugated to fluorescein isothiocyanate (FITC) targeted the folate receptor-α (FR-α), which is often overexpressed in ovarian cancers. FGS, with a real-time multispectral intraoperative fluorescence imaging system, enabled tumors less than 1 mm in diameter to be resected. However, FR-α may not generally overexpressed in cancer. 3 In other studies, monoclonal antibodies against cancer antigen 19-9 (CA19-9) or carcinoembryonic antigen (CEA), were conjugated to a green fluorophore and delivered intravenously into nude mice with orthotopic human pancreatic or colon tumors. 4 The fluorescent antibodies adhered to the CEA or CA19-9 antigens of CEA-or CA19-9expressing cancer cells, respectively, making the tumors become fluorescent and visible and resectable under fluorescence guidance, 4 which in subsequent studies resulted in reduced recurrence and increased survival compared to BLS. 3,[5][6][7][8][9][10][11][12] Kishimoto et al. 13 selectively labeled tumors with green fluorescent protein (GFP) using a telomerase-dependent adenovirus (OBP-401) that expresses the gfp gene only in cancer cells. The labeled tumors could then be resected under fluorescence guidance. OBP-401 GFP-labeled tumors that recurred after fluorescence-guided surgery maintained GFP expression. 14 Thus, imaging cancer recurrence and metastasis is also possible with OBP-401 GFP labeling, which is not possible with non-genetic probes such as fluorescent antibodies. 3 OBP-401 could selectively label soft-tissue sarcoma (STS) with GFP in an orthotopic nudemouse model. OBP-401-based FGS resulted in superior resection of STS compared to BLS. OBP-401 based-FGS improved disease free survival as well as preserved muscle function compared with BLS. 15 Glioblastoma multiforme (GBM) is one of the most invasive of cancers and is not totally resectable using standard BLS or current FGS. OBP-401 infection brightly and selectively labeled GBM with GFP for effective FGS in orthotopic nude-mouse models without recurrence. 16 It is also possible to selectively label the stroma of a tumor. Pancreatic-cancer patientderived orthotopic xenografts (PDOX) tumors were passaged orthotopically in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary patient tumors acquired RFP-expressing stroma. The RFP-expressing stroma included cancerassociated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). The RFP stroma persisted in the tumors after passage. 17 It was possible to image the brightly fluorescent tumors noninvasively longitudinally as they progressed after passage to nontransgenic nude mice. 18 In the present study, we demonstrate a novel approach to FGS, with cancer cells labeled with GFP by GFP-401 and stroma labeled with red fluorescence protein (RFP) by previous growth in RFP transgenic mice. Dual-color FGS was more effective than BLS or singlecolor FGS to prevent recurrence in a pancreatic cancer patient-derived orthotopic xenograft (PDOX) nude-mouse model.

GFP-expressing telomerase-specific adenovirus
Adenovirus OBP-401 contains a promoter element of the human telomerase reverse transcriptase (hTERT) gene which drives the expression of E1A and E1B genes linked to an internal ribosome entry site. OBP-401 also contains the GFP gene which is driven by the CMV promoter. The virus only replicates in cancer cells, labeling them with the GFP gene. 13

Mice
Athymic nude nu/nu mice or transgenic RFP nu/nu mice 19 (AntiCancer, Inc., San Diego, CA, USA) (5 weeks old), were kept in a barrier facility under HEPA filtration. Mice were fed with autoclaved laboratory rodent diet (Tecklad LM-485, Western Research Products). All animal studies were conducted in accordance with the principals and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals under Assurance no. A3873-01.

Establishment of patient pancreatic cancer
Pancreatic cancer tumor tissue from a single patient was originally obtained at surgery at the MD Anderson Cancer Center and cut into 3-mm 3 fragments and transplanted subcutaneously in NOD/SCID mice. 20,21

Patient-derived orthotopic xenograft (PDOX®)
The fluorescent PDOX (fPDOX) model was passaged, using surgical orthotopic implantation (SOI) 22 of tumor previously grown in the NOD/SCID mice, in transgenic RFP Yano et al. Page 3 Cancer Gene Ther. Author manuscript; available in PMC 2016 January 01. nude mice. 17,18,19 A small 6-to-10 mm transverse incision was made on the left flank of the RFP nude mouse through the skin and peritoneum. The tail of the pancreas was exposed through this incision, and a single 1-mm 3 tumor fragment was sutured to the tail of the pancreas using 8-0 nylon surgical sutures (Ethilon; Ethicon, Inc., NJ). On completion, the tail of the pancreas was returned to the abdomen and the incision was closed in one layer using 6-0 nylon surgical sutures (Ethilon). 22-26

OBP-401 labeling of the RFP-expressing PDOX
The PDOX tumor was exposed in the peritoneal cavity during laparatomy. OBP-401 (1 × 10 8 PFU/tumor) was then injected into the exposed tumor.

Fluorescence-guided surgery
Mice were given a ketamine mixture (10 μl ketamine HCl, 7.6 μl xylazine, 2.4 μl acepromazine maleate, and 10 μl PBS) (i.m.) before fluorescence-guided surgery. .The Dino-Lite mobile imaging system was used for imaging in live mice. The portable system has seven filtered (wavelength) blue LEDs for excitation lighting; a 510 nm emission filter; a white LED switched by software, and a 1.3 megapixel sensor. This digital camera can magnify from ×30 up to ×200 and can readily take both pictures and videos of magnified green, fluorescent objects. The camera's dimensions are 10.5 × 3.2 cm and the weight is only 105 g. This all-in-one compact digital camera makes the Dino-Lite imaging system easily transportable and thereby suitable for FGS. 30 All animal procedures were performed under anesthesia using s.c. administration of a ketamine mixture (see above). OBP-401 GFP-labeled cancer cells and/or RFP stromal cells in the PDOX were imaged with the Dino-Lite hand-held fluorescence scope to determine the resection area and margin. A 20 mm incision was made at the lateral midline of the abdomen. The pancreatic tumor was then exposed. Either under bright light or fluorescence tumor was resected using a scalp and foreceps. Fluorescence imaging with the Dino-Lite was performed to visualize residual GFP cancer cells or RFP stroma. The resected area of the pancreas was closed with a 6-0 suture. After surgery, the abdominal wall was closed with 6-0 suture.

Statistical analysis
PASW Statistics 18.0 (SPSS, Inc) was used for all statistical analyses. Data are shown as mean ± SD. For comparison between two groups, significant differences were determined using Student's t-test. P values of < 0.05 were considered significant.

FGS of OBP-401-GFP
Time-lapse imaging showed that pancreatic cancer cells in the PDOX model were labeled by OB-401 three days after OBP-401 infection. GFP fluorescence remained sufficiently strong to use FGS 5 days after OBP-401 infection (Fig. 1A). The margin between the tumor and pancreas was very unclear under bright-light (Fig. 1A). In contrast, OBP-401 made the tumor margin much clearer than under bright-light (Fig. 1A). Using the Dino-Lite fluorescence digital camera system, the margin between tumor and normal was visualized sufficiently clearly for FGS of the pancreatic tumor (Fig. 1B, 1C). Whole-tumor and crosssection imaging demonstrated that OBP-401 labeled the whole tumor (Fig. 1D).

FGS detects and resects residual cancer cells in OBP-401-GFP-labeled PDOX after BLS
PDOX tumors were initially resected by BLS 5 days after injection of OBP-401 ( Fig. 2A). After BLS, confocal laser microscopy detected residual GFP-expressing cancer (Fig. 2B) at the single cell level. The residual cancer cells were then resected by FGS (Fig. 2C).

BLS of PDOX labeled with RFP stroma
Pancreatic cancer PDOX, previously grown in RFP transgenic nude mice in order to acquire RFP stroma cells, were orthotopically transplanted to non-colored nude mice. 17,18,31 The RFP-expressing stroma cells were readily imaged in the PDOX (Fig. 3A). Residual RFPexpressing stromal cells were readily visualized after BLS (Fig. 3A, 3B).

OBP-401-GFP labeling of PDOX with RFP stromal cells
Pancreatic cancer PDOX with RFP-expressing stroma were labeled with GFP in their cancer cells by administration of OBP-401 ( Fig 4A). The pancreatic cancer PDOX with RFPexpressing stroma and GFP-labeled cancer cells was incompletely resected by BLS (Fig.  4B). Fluorescence imaging demonstrated that there were residual GFP-expressing cancer cells and RFP-expressing stroma cells after BLS (Fig. 4B).

FGS completely resects pancreatic cancer PDOX with GFP-expressing cancer cells and RFP-expressing stroma cells
GFP and RFP imaging showed that cancer and stromal cells in the color-coded labeled PDOX invaded beyond the tumor capsule (Fig. 4C, 4D). However, FGS enabled complete resection of the PDOX with GFP-expressing cancer cells and RFP-expressing stroma cells (Fig. 4B, Fig. 5, Supplementary Movie 1).

Color-coded FGS enables recurrence-free surgery
Two months after BLS, eight of ten mice had local recurrences ( Table 1). Five of twelve mice which received FGS of RFP stroma-labeled PDOX had local recurrences ( Table 1). Four of eleven mice which received FGS of GFP-labeled PDOX had local recurrences (Table 1). In contrast, there was no local recurrence in nine mice which received FGS of the color-coded PDOX with GFP-labeled cancer cells and RFP stroma cells (Fig. 6, Table 1). These data demonstrated that dual-color FGS resulted in complete resection and prevented local recurrence of the pancreatic cancer PDOX, which BLS or single-color FGS failed to do.
OBP-401 selectively labeled cancer cells in the pancreatic cancer PDOX with RFPexpressing stroma. The double-labeled PDOX enabled precise visualization of color-coded cancer and stromal cells after BLS and improved the efficacy of FGS.
Tumor-specific antibodies conjugated with fluorophores can only access the tumor from the outside and cannot label all the cancer cells in the central part of the PDOX. The present study demonstrated that OBP-401 infection labels cancer cells with GFP within the tumor and this signal, along with the RFP-expressing stromal cells, remained sufficiently strong to perform FGS 5 days after infection.
Future experiments will develop reporter gene vectors to target the stromal cells in order to apply color-coded cancer cell-stromal cell labeling for FGS in the clinic.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgements
This study was supported in part by the National Cancer Institute grant CA142669. This study was also supported in part by grants-in-Aid from the Ministry of Education, Science and Culture, Japan, and grants from the Ministry of Health and Welfare, Japan.