The luciferase-expressing human GC cell line NCI-N87-Luc was obtained from Shanghai Biomodel Organisms Center Co., Ltd. (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (100 mg/ml streptomycin and 100 U/ml of penicillin) (all from (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified incubator with 5% CO₂.

A total of 100 female BALB/c nude mice (4–5 weeks old, ~20 g) were provided by the Shanghai Laboratory Animal Center (Chinese Academy of Sciences, Shanghai, China). The mice were fed in a specific pathogen-free environment at room temperature, with free access to food and water and 12:12 h light/dark cycle. The animal experiment designed in the present study was approved by the ethical committee of Shanghai Jiao Tong University School of Medicine (Shanghai, China).

The orthotopic injection technique was performed using a previously reported method (13). The mouse abdomen was opened via a midline incision under aseptic conditions, and the stomach was exteriorized. A volume of 40 µl DMEM containing 5×10⁶ NCI-N87-Luc cells was injected into the subserous layer of the middle of the stomach using a 100 µl syringe and 30G needle. A cotton swab was pressed against the injection site for ≥20 sec to prevent tumor cell leakage into the peritoneal cavity. Subsequently, the stomach was returned to the peritoneal cavity, and peritoneum and skin sutured sequentially. The feasibility of the injection method was confirmed using trypan blue as an indicator, which demonstrated an accurate location of the injected dye in the subserosa of the gastric wall.

Female BALB/c nude mice (4–5 weeks old) were used for animal studies. At 4, 6, 8 and 10 weeks after tumor cell injection, mice (n=5) were intraperitoneally injected with 200 µl D-luciferin solution (150 mg/kg body weight, J&K Scientific Ltd, Shanghai, China). Subsequently, mice were anesthetized with 2% isoflurane, and at 8 min after D-luciferin injection, placed in the Xenogen IVIS 200 (PerkinElmer, Inc., Waltham, MA, USA) chamber with right lateral recumbency for bioluminescence imaging of the orthotopic NCI-N87-Luc tumor according to the manufacture's protocols. Body weights of mice were recorded weekly throughout the test. To detect metastasis in LN and other major organs at different time points, mice (n=5) were sacrificed by cervical vertebra dislocation between week 3 and 10. LNs at four anatomical positions in the abdomen (14), including gastric LN (GLN), pancreaticoduodenal LN (PLN), mesenteric LN (MLN) and lumbar LN (LuLN), and other major organs, including the heart, liver, spleen, lung and kidney, were excised for ex vivo bioluminescence imaging. A metastasis heat map was generated using GraphPad Prism 7 software (GraphPad Software, Inc., La Jolla, CA, USA).

Excised LN and organs were fixed in 4% paraformaldehyde at room temperature overnight, embedded in paraffin and cut into 4 µm sections. Subsequently the sections were stained with hematoxylin for 3 min and eosin for 2 min at room temperature. The sections were observed and photographed under upright metallurgical microscope with digital image capturing system (DP50; magnification, ×40 or ×200; Olympus Cooperation, Tokyo, Japan).

Data analysis was conducted with the GraphPad Prism 7.0 software (GraphPad Software, Inc., La Jolla, CA, USA). The quantified time-dependent bioluminescence signal of the tumors and also the mouse body weight are presented as mean ± standard deviation.

Trypan blue dye was used to confirm the reliability of the injection technique (Fig. 1A). Following the NCI-N87-Luc cell injection, a small white plaque was observed under the serosa of the gastric body (Fig. 1B). The cells formed an orthotopic tumor, which was detected via sensitive bioluminescence imaging (Fig. 1C). Tumors on the gastric wall were observed following opening the abdomen (Fig. 1D) or directly from the resected stomach (Fig. 1E). Ex vivo bioluminescence imaging highlighted the tumor position in the stomach (Fig. 1F). Histopathological assays further confirmed tumor formation and anatomical location of subserosa (Fig. 1G). All mice receiving this treatment grew tumors on the stomach, establishing a 100% success rate of the model.

The growth of stomach tumors was monitored via bioluminescence imaging (Fig. 2A). Quantification of bioluminescence intensity demonstrated the time-dependent tumor growth until week 10 after tumor cell injections (Fig. 2B). During the same time-period, the body weight of the mice increased in the first 7 weeks. However, subsequent to week 7 the body weight of the mice declined (Fig. 2C), suggesting the involvement of harmful gastric tumors in the deterioration of the mice's health.

Firstly, the four anatomical positions of LNs (GLN, PLN, MLN, and LuLN) were identified in the abdomen (Fig. 3A-C). H&E staining further confirmed the presence of LNs, and not adipose tissues or omentum, at these positions (Fig. 3D).

From week 3, LNs of mice (n=5) were resected for ex vivo bioluminescence imaging to investigate the metastasis patterns (Fig. 4A). The overall LN metastasis patterns from weeks 3–10 were summarized in a heat map (Fig. 4B). Histopathological examination with bioluminescence imaging further confirmed the presence of metastatic tumor cells in the LNs (Fig. 4C). This demonstrated that no metastasis occurred in the LN at week 3 and 4. At week 5, GLN of one mouse was positive for metastasis, based on the bioluminescence signal. At week 6, metastasis was observed at PLN, MLN and LuLN in one mouse, indicating distant spreading. From weeks 7–9, metastasis occurred in a greater number of mice and spread into LNs at various anatomical positions. At week 10, all tested mice exhibited positive bioluminescence signals in LNs. It is also notable that the metastasis frequency for GLN, MLN and LuLN decreased from 40% at week 7 to 20% at week 8. This phenomenon may be ascribed to the individual differences of the mice at each week, which may be avoided if a greater number of animals (n>5) were examined at each time point. Additionally, it can be concluded, though this observation, that the metastasis frequency for GLN, MLN and LuLN varied in the range of 20–40% in this duration.

Organ metastasis was observed at week 5 (the same time as LN metastasis). One mouse exhibited metastasis to the liver and one to the spleen (Fig. 5); however, the bioluminescence signals detected were weak, indicating the metastasis of only a small number of tumor cells. From weeks 6–10, the spleen and liver remained the primary metastatic organs. Lung metastasis was only observed in one mouse at week 7. It also demonstrated a liver metastasis frequency of 20% at week 5 and 6, which reduced to 0% at week 7 and 8. It is notable that the observations at each week were from different mice. If a greater number of animals (n>5) were included for each week, the influence of animal individual differences on the metastasis frequency may be avoided. These observations also indicated that the liver metastasis were not frequent in the first 8 weeks in the present model.