Tumor-sealing Surgical Orthotopic Implantation of Human Colon Cancer in Nude Mice Induces Clinically-relevant Metastases Without Early Peritoneal Carcinomatosis

Materials and Methods

Cell culture. The HCT116 human colon cancer cell line has been well characterized in many studies and found to grow rapidly in nude mice (11-16). Thus, it was chosen for the present study. This cell line originated from a 48-year-old male who underwent an operation for ascending colon carcinoma at Dukes' D stage. Characteristics of this tumor included microsatellite instability, positivity for CpG island methylator phenotype panels 1 and 2, chromosomal instability pathway-negative, KRAS proto-oncogene, GTPase (KRAS^G13D), wild-type B-Raf proto-oncogene, serine/threonine kinase (BRAF), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)^H1047R wild-type phosphatase and tensin homolog (PTEN), and wild-type tumor protein 53 (TP53) (11). This cell line was originally obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum and 1% 2 mmol/l glutamine (Life Technologies, Inc., Grand Island, NY, USA) at 37°C in an incubator with 5% CO₂.

GFP transfection. HCT116 cells were transfected with GFP for more accurate detection by fluorescence imaging. Packaging cells 293 GP (Clontech, Mountain View, CA, USA) were co-transfected with a plasmid encoding vesicular stomatitis virus glycoprotein envelope protein and a retroviral vector encoding GFP and G418 resistance gene using FuGene (Invitrogen, Carlsbad, CA, USA). Viruses were collected 48 hours later. They were used to infect HCT116 cells. After 48 hours, infected HCT116 cells were selected by treatment with G418 for 5 days, resulting in stable expression of GFP (12, 15).

Animal care. Athymic nu/nu nude mice (AntiCancer Inc., San Diego, CA, USA) 5 to 6 weeks old were used in this study. These mice were bred and maintained in a high-efficiency particulate air-filtered environment at AntiCancer, Inc. with cages, food, and bedding sterilized by autoclave. Animal diets were obtained from Harlan Teklad (Madison, WI, USA). Ampicillin (5.0%, w/v; Sigma, St. Louis, MO, USA) was added to autoclaved drinking water. All surgical procedures and intravital imaging were performed with animals anesthetized by intramuscular injection of 0.02 ml of a mixture of 50% ketamine, 38% xylazine, and 12% acepromazine maleate. All animal studies were conducted under assurance number A3873-1 issued to AntiCancer, Inc. and the principles and procedures outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Animals (17).

Subcutaneous tumor implantation. HCT116 GFP-labeled cells (5×10⁶) were harvested from in vitro culture by trypsinization and washed twice with serum-free medium. These cells were injected into the subcutaneous layer on the right flank of nude mice within 30 min after harvesting. Subcutaneous tumors were allowed to grow for 2-4 weeks until large enough to supply adequate tumor size (about 1 cm) for orthotopic implantation.

Orthotopic tumor implantation. The subcutaneous tumor was resected aseptically, and necrotic tissues were cut away. Remaining healthy tumor tissues were then scissor-minced into fragments of about 1-mm in diameter in RPMI media. In the SSI group (8, 9, 12, 18-20) (Figure 1A), the cecum was delivered through a small 6 to 10-mm left paramedian vertical abdominal incision and a small segment of bowel and mesentery were exposed. Tumor fragments were fixed to the mesenteric border of the cecal wall using 8-0 nylon surgical sutures. The serosa of the site where the tumor fragment was to be implanted was partially torn in advance. In the TSM group (Figure 1B), after fixing the tumor fragment, the cecum was folded to cover the tumor fragment and the periphery was sutured with 8-0 nylon. A new set of surgical instruments was used after completing the SSI procedure to avoid cancer-cell contamination around the folded area. Upon completion, the cecum was returned to the abdomen and the wound was closed in two layers using 6.0 Ethibond non-absorbable sutures (Ethicon Inc., Somerville, NJ, USA).

Imaging to detect primary tumor growth, metastasis, and peritoneal carcinomatosis in vivo. In vivo images were acquired for all mice through a laparotomy to detect the presence or absence of peritoneal carcinomatosis, as well as primary tumor growth and metastasis, on the 20th day after orthotopic implantation (Figure 2). The mean survival time of mice carrying HCT116 cells has been reported to be 39 days [16]. Thus, we chose day 20 as half of the mean survival time. All abdominal organs were explored along the midline incision as described above. Two imaging devices were used to obtain fluorescence images: an OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan) containing an MT-20 light source (Olympus Biosystems, Planegg, Germany) with a DP70 CCD camera (Olympus Corp.) (21), and an iBox Small Animal Imaging System (UVP, Upland, CA, USA) equipped with a BioChemi HR500 CCD camera (UVP) and a BioLite automated multispectral light source (UVP) (18-22).

PC was graded based on the number of peritoneal seeding nodules: Grade 0, no seeding nodule was found; grade I, 1 to 15 nodules; grade II, 16 to 100 nodules; and grade III, more than 100 nodules. Tumor volume was estimated as (long diameter × short diameter²)/2.

Imaging to visualize distant metastases at necropsy. At animal death, a complete necropsy procedure was performed. Thoracic and abdominal organs were extracted. The presence of local tumor at the cecum was assessed, its long and short diameters measured, and all macroscopic and microscopic tumor deposits in the liver, lung, lymph nodes, and peritoneal carcinomatosis were recorded. Each organ, including primary tumors, livers, lungs, lymph nodes, and peritoneal seeding nodules of necropsied mice, was excised. Tissues were embedded in tissue-freezing medium (Triangle Biomedical Science, Durham, NC, USA), frozen in liquid nitrogen for 10 min, and placed at −80°C overnight. These tissue samples were then frozen in Tissue-Tek O.C.T. compound (Sakura Fintek, Torrance, CA, USA) and sectioned on a microtome. Frozen sections at 15-μm in thickness were prepared with a Leica CM1850 cryostat (23) for fluorescence microscopy. These sections were air-dried for 5-10 min and imaged with a confocal microscope (Fluoview FV1000; Olympus Corp.). A continuous-wave semiconductor laser at 473 nm for GFP excitation and a tunable Mai Tai HP femtosecond laser emitting at 700-1,020 nm (Newport-Spectra Physics, Irvine, CA, USA) were used for deep tissue imaging of GFP. Fluorescence images were obtained using Olympus 4×/0.13 and 20×/0.50 UPLFLN objectives (24).

Statistical analysis. Pearson's chi-square tests were used for categorical variables (PC, liver metastasis, and lung metastasis) and independent sample t-tests were used for continuous variables (tumor volume). Kaplan–Meier with the log-rank test was used for survival analysis. A two-tailed value of p<0.05 was considered statistically significant. All calculations were performed using SPSS version 24.0 (IBM SPSS Statistics, IBM Corporation, Armonk, NY, USA).