Establishing a xenograft mouse model of peritoneal dissemination of gastric cancer with organ invasion and fibrosis

Surgical specimens of human omentum were obtained from patients with no evidence of peritoneal inflammation and/or malignancy who underwent surgery in Kanazawa University Hospital between April and December 2013. Donors were not subjected to chemotherapy or radiation treatment prior to surgery. All patients provided written informed consent prior to participation in the study. The study was approved by the Research Ethics Committee of Kanazawa University (permission number 1747). HPMCs were isolated from surgical specimens of human omentum as previously described [18]. Briefly, small pieces of omentum were surgically resected under sterile conditions and were incubated in pre-warmed phosphate-buffered solution (PBS) containing 0.125% trypsin/EDTA (Gibco/Invitrogen, USA) for 30 min at 37 °C. The suspension was centrifuged at 1500 × g for 5 min. Collected cells were cultured in RPMI-1640 medium (Gibco/Invitrogen) supplemented with 20% heat-inactivated fetal bovine serum (FBS; Nichirei Bioscience Inc., Japan). The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 in air. For the following experiments, cells were used during the second or third passage after primary culture. HPMCs possibly contaminated with endothelial cells or fibroblasts at the time of harvest were not used. We used homogeneous HPMCs from a different donor for each experiment. We used the high-potential peritoneal dissemination cell line MKN45-P, which was established from the MKN45 gastric cancer cell line (Additional file 1: Figure S1) in our institution as described previously [19]. Briefly, female immunocompromised BALB/c-nu/nu mice (Charles River Laboratories Inc. Japan) were subcutaneously inoculated with MKN45 cells and the subcutaneous nodules were removed and injected into other mice intraperitoneally. The cancer cells from peritoneal nodules were injected into the abdominal cavity of other mice. The process was continued through to a seventh generation. The resulting cell line was named MKN45-P. Cells were maintained in RPMI-1640 medium supplemented with 10% FBS.

All animal experiments were performed according to Kanazawa University’s standard guidelines. Female immunocompromised BALB/c-nu/nu mice at 4–6 weeks of age were maintained in a sterile environment. MKN45-P cells were co-cultured with an equivalent number of normal HPMCs for 5 days, and a total of 5 × 10⁶ cells in 1000 μL of RPMI-1640 were then i.p. injected into nude mice on day 0 (co-cultured group). For the control group, 5 × 10⁶ MKN45-P cells alone were injected (control group). The scratch method of i.p. cell inoculation was employed. First, an abdominal incision was made under general diethyl-ether inhalation anesthesia. For the parietal peritoneum scratch model, the left ventral parietal peritoneum was scratched using a cotton swab (Fig. 1a, parietal group). For the visceral peritoneum scratch model, the intestinal tract and mesenterium was removed from the peritoneal cavity and scratched using a cotton swab (Fig. 1b, visceral group). The visceral were then returned into the peritoneal cavity and the abdominal wall was closed. Next, a total of 5 × 10⁶ cells of a MKN45-P and HPMCs co-culture was injected i.p. and animals were carefully monitored. After 14 days, mice were anesthetized with diethyl-ether, sacrificed, and the tumors and abdominal organs excised together. Tumor specimens were then collected for immunohistochemical examination.

Tumor specimens were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) and Azan stain for assessment of fibrosis, while the expression of α-smooth muscle actin (α-SMA; 1A4, mouse monoclonal IgG, diluted 1:100; Dako Cytomation, Denmark) and vimentin (V9, mouse monoclonal IgG, diluted 1:100; Santa Cruz Biotechnology, Inc.) was also assessed immunohistochemically. Deparaffinized sections were pretreated by autoclaving in 10% citric acid buffer (pH 8.0) at 120 °C for 15 min. Following treatment with protein block serum (Dako Cytomation, Kyoto, Japan) for 10 min and incubation with 2% skim milk for 30 min to block non-specific reactions, sections were incubated with primary antibody at 4 °C overnight. The Envision-polymer solution (horseradish peroxidase, HRP, Dako Cytomation) was then applied for 1 h. Signals were developed in 0.02% 3,3′-diaminobenzidinetetrahydrochloride solution containing 0.1% H₂O₂. Sections were then lightly counter stained with hematoxylin and examined using a fluorescence microscope (Olympus, Tokyo, Japan). The degree of fibrosis was calculated as a percentage of fibrosis within the whole section in all samples using a BZ-9000 BZII microscope (Keyence, Osaka, Japan).

Representative images depicting the macroscopic appearance of the tumors at Day 14 are shown in Fig. 2. All mice developed abdominal swelling with peritoneal tumors and bloody ascites. Tumors of both control and co-cultured groups underwent peritoneal dissemination, though this occurred on abdominal organ surfaces and organ invasion was not observed (Fig. 2a, b). Mice of the parietal group had tumors present on the left side of the peritoneal cavity at the scratch sites (Fig. 2c). However, dissemination nodules did not exhibit organ invasion (Fig. 2d). Dissemination nodules with adhesion and invasion to the intestinal tract, block formation, and distention and edema of the intestinal tract were observed in mice of the visceral group (Fig. 2e–g).

Control group tumors did not exhibit fibrosis or α-SMA and vimentin positive cells (Fig. 3a). Additionally, co-culture group tumors showed little fibrotic change (Fig. 3b). However, both the parietal and visceral peritoneum scratch model groups showed rich fibrosis and α-SMA positive cells (Fig. 3c, d). Furthermore, visceral group tumors invaded the intestinal tract by destroying the serosal membrane, as serosal edges stained positively for Azan and α-SMA (Fig. 3e). The scratch model group also exhibited cytoplasmic expression of vimentin. The fibrotic areas of tumors formed in conjunction with the scratch methods were significantly larger than those of control tumors (Fig. 4).