Fluorescence-guided Surgery with Splenic Preservation Prevents Tumor Recurrence in an Orthotopic Nude-mouse Model of Human Pancreatic Cancer

Materials and Methods

Experimental design. Figure 1 shows the overall experimental design of this study. MiaPaCa2 green fluorescence protein (GFP)-labeled cells (2×10⁶) were injected subcutaneously into the right and left flanks of nude mice (Anticancer, Inc., San Diego, CA, USA). Subcutaneously-injected tumors grew to approximately 20 mm by 6 weeks after implantation. The tumors were then harvested for subsequent orthotopic implantation. A small fragment (3 mm³) was orthotopically implanted on the tail of the pancreas of nude mice. Fluorescence-guided tumor resection was performed on day 21 after orthotopic tumor implantation. At this time, the mice were randomly divided into two groups according to splenectomy or not. Tumor recurrence was detected with laparotomy on day 21 after FGS.

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Figure 1.

Schematic illustration for the experiment. MiaPaCa2-green fluorescent protein (GFP) cells (2×10⁶) were injected subcutaneously into the right and left flanks of nude mice. The subcutaneously implanted tumors grew to 20 mm by 6 weeks after implantation. The tumors were harvested for subsequent orthotopic implantation. A small fragment (3 mm³) was orthotopically implanted on the tail of the pancreas of the nude mice. Fluorescence-guided tumor resection was performed on day 21 after orthotopic tumor implantation. At this time, the mice were randomly divided into two groups according to splenectomy or not. Tumor recurrence was detected with laparotomy on day 21 after fluorescence-guided surgery (FGS).

Establishment of GFP-labeled cancer cell line. The MiaPaCa2 human pancreatic cell line was stably transfected with GFP as previously described (9, 10).

Cell culture. The MiaPaCa2-GFP human pancreatic cancer cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA). The cells were incubated at 37°C in a humidified incubator with 5% CO₂ in air. The cells were collected after trypsinization and stained with trypan blue (Sigma-Aldrich, St. Louis, MO, USA). Only viable cells which excluded trypan blue were counted with a hemocytometer (Hausser Scientific, Horsham, PA, USA).

Mice. Athymic nu/nu nude mice, 4-6 weeks old, were used for subcutaneous cancer cell injection models and orthotopic tumor implant models. Mice were kept in a barrier facility under HEPA filtration and fed with autoclaved laboratory rodent diet. All mouse surgical procedures were performed with the animals anesthetized by intramuscular injection of a 0.02 ml solution of 50% ketamine, 38% xylazine, and 12% acepromazine maleate. The animals were sacrificed on the 21st day after surgery for investigating tumor recurrence. Intravenous injection of the ketamine mixture solution was used for euthanasia. To ensure death following injection, cervical dislocation was performed. All animal studies were conducted with an AntiCancer Institutional Animal Care and Use Committee protocol specifically approved for this study and in accordance with the principles and procedures outlined in the National Institute of Health Guide for the Care and Use of Animals under Assurance Number A3873-1.

Subcutaneous cancer cell injection. MiaPaCa2-GFP cells were harvested by trypsinization and washed twice with phosphate-buffered saline (PBS; Sigma Aldrich). The cells (2×10⁶) were injected subcutaneously into the right and left flanks of nude mice within 30 min of harvesting. Subcutaneously-injected tumors grew by about 20 mm at 6 weeks after implantation. The tumors were harvested for subsequent orthotopic implantation.

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Figure 2.

Fluorescence-guided surgery (FGS) was performed with a Dino-Lite system. Orthotopically-implanted tumors were identified by bright light (BL) and fluorescence (FL) before surgery (A). After surgery (B), the operative field showed no residual tumor.

Orthotopic tumor implantation. The surgical orthotopic implantation (SOI) of tumor fragment was performed as previously described (11-13). A small 6-10 mm transverse incision was made on the left flank of the mouse through the skin and peritoneum. The pancreatic tail and spleen were exposed through this incision, and a single 3 mm tumor fragment retrieved from subcutaneous tumor of a nude mouse was sutured to the tail of the pancreas using 7-0 nylon surgical sutures (Dermalon™, Covidien; Medtronic Inc., MN, USA). The pancreatic tail and spleen were returned to the abdomen, and the incision was closed in one layer using 6-0 nylon surgical sutures (Dermalon™).

Fluorescence-guided surgery. In order to achieve complete tumor resection, 3 weeks after SOI of MiaPaCa2-GFP to the pancreas, FGS was performed using a Dino-Lite imaging system (AM4113TGFBW Dino-Lite Premier; AnMo Electronics Corporation, Taiwan, ROC) (14-16). When tumor resection was performed, the mice were randomly divided into two groups according to splenectomy or not. In the control group, a 10 mm transverse incision was made on the left flank, which was the same area during SOI of the mouse. The tail of the pancreas and spleen were exposed through this incision. The tumor was completely removed. The surgical resection field was carefully imaged with the Dino-Lite imaging system and the OV100 Small Animal Imaging System (Olympus, Tokyo, Japan) to detect microscopic residual tumor (16). The pancreatic tail and spleen were returned to the abdomen, and the incision was closed in one layer using 6-0 nylon surgical sutures (Dermalon™). In the splenectomy group, after tumor resection, the splenic artery and vein in the splenic hilum and short gastric vessels communicating with the splenic upper pole were securely ligated with 7-0 nylon suture (Dermalon™), and the spleen was removed from the pancreas tail. Resected tumor length and width were measured with calipers and tumor volume was calculated by the following formula: tumor volume=(length x width²)/2.

Analysis of the tumor recurrence pattern. Tumor recurrence patterns were analyzed by laparotomy on day 21 after FGS. After laparotomy, the peritoneal cavity was carefully assessed for the evidence of peritoneal, hepatic, or other site metastases. Histology. The fresh resected tumors were fixed in 10% formalin, and then embedded in paraffin. The tissue sections, at a thickness of 4-μm, were deparaffinized in xylene and rehydrated in an ethanol series. Hematoxylin and eosin staining was performed according to standard protocols for histological confirmation of tumor.

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Figure 3.

Complete tumor resection after fluorescence-guided surgery (FGS) was confirmed with the OV 100 imaging system. In the control group (A, B), the operative field was observed with the OV 100 imaging system before (A) and after (B) FGS. In the splenectomy-treated group (C, D), FGS and splenectomy were simultaneously performed. The operative field was observed before (C) and after (D) FGS and splenectomy. There was no residual tumor on the pancreas in any mouse in the control and splenectomy group. BL: Bright light; FL: fluorescence. Dotted circles indicate the resected tumor visualized both in bright light and fluorescence.

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Figure 4.

Tumor recurrence detection in control and splenectomy-treated groups. In the control group (A), no recurrence was found in any mouse. Multiple peritoneal recurrences (yellow arrows) were observed in two mice in the splenectomy-treated group (B). Seeded nodules were confirmed as recurred pancreatic cancer by histological analysis (insets).

Statistical analysis. All statistical analyses were performed with SPSS 20.0 software (IBM, Armonk, NY, USA). Categorical variables were compared using chi-squared or Fisher exact tests. Continuous variables are presented as the mean±standard deviation (SD) and the significance of differences was determined using Student's t-test. A p-value of 0.05 or less indicated statistical significance.