Patients and resource of samples
We performed immunostaining of paired primary lesions and peritoneal lesions from 28 GC patients with PM at our hospital from 2009 to 2016, and compared their immune environments. Tissue samples from primary lesions were obtained from biopsy specimens during upper gastrointestinal endoscopy, and peritoneal lesion were obtained from sampling during staging laparoscopy. All specimens were obtained before chemotherapy. Prior to this research, written informed consent was obtained from each patient. This study was approved by the Institutional Review Board of Kanazawa University Graduate School of Medical Science (study permission number 2789).
Cell lines and cell culture
YTN16 is a gastric cancer cell line transplantable into C57BL/6 mice. YTN16 cells were established from subcutaneous tumors by injection of primary cultured cells derived from a mouse gastric adenocarcinoma. Mouse gastric tumors were established in p53 heterozygous knockout C56BL/6 mice by addition of N-Methyl-N-nitrosourea (MNU) to the animals’ drinking water [23]. The resulting tumor cells were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich Japan, Tokyo, Japan) containing 1.0mL/L MITO (Coning Japan, Tokyo), 10mL/L L-Glutamine, 10mL/L Penicillin/Streptomycin and 10% fetal bovine serum (FBS), on plastic dishes coated with Type I collagen solution (0.5% Atelocollagen Acidic Solution IPC-50; Koken Co., Ltd., Japan) at 37°C in 5% CO2 atmosphere.
Mouse intestinal myofibroblast cell lines (LmcMFs) derived from mouse colonic mucosa were established by Takashi Ohama and Koichi Sato, Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University [24]. Cells were cultured in DMEM containing 10% FBS at 37°C in 5% CO2 atmosphere.
Co-culture
Indirect co-cultures were established as follows. YTN16 cells were seeded on a 6-well plate, while LmcMF cells were seeded in 1-μm pore-size Boyden chambers (BD Falcon, Franklin Lakes, NJ), both at a density of 1×105 cells per well or chamber in DMEM or high-glucose DMEM containing 10% FBS, respectively. After 24 h, the cells were washed twice with PBS, the chambers were placed into the wells of the plates, and the plates were incubated for 5 days in 2 mL of DMEM or high-glucose DMEM.
Mouse allograft model
The animal use proposal and experimental protocol (AP-183944) was reviewed and approved by the Animal Care and Use Committee of Kanazawa University. All animal experiments were performed in accordance with the standard guidelines of Kanazawa University. Female C57BL/6J mice (20 g, 6-8 weeks old) were purchased from Charles River Laboratories, Inc., Yokohama, Japan. The mice were housed with a 12h day-night cycle in a temperature-(21°C) and humidity-(50%) controlled room of the animal experimental institute. All mice were kept in individually ventilated cages, fed with sterile standard food and water ad libitum. Mice were randomly distributed into YTN16 inoculated group (n = 6) and YTN16 with LmcMF co-inoculated group (n = 6). To establishing peritoneal metastatic models, 1×107 of YTN16 cells alone in 1 mL of high-glucose DMEM were inoculated intraperitoneally under isoflurane anesthesia on day 0 as YTN16 inoculated group. YTN16 cells were co-cultured with an equivalent number of LmcMFs for 5 days, and a total of 1×107 cells in 1 mL of the same medium were then inoculated same manner as YTN16 inoculated group. In this study, total inoculated cell counts were aligned for comparing tumor weights because tumors consisted of both cancer cells and stroma cells including LmcMFs. After inoculation, on day 14, the mice were euthanized with isoflurane and cervical dislocation, and tumors were removed for weight calculation and immunohistochemical examination.
Immunohistochemistry
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 various antigens was assessed immunohistochemically. Deparaffinizing sections were pretreated by autoclaving in 10% citric acid buffer at 120 °C for 15 min. Following treatment with protein block serum (Dako Co., Kyoto, Japan) for 10 min and incubation with 2% skim milk for 30 min to block nonspecific reactions, sections were incubated with primary antibody at 4 °C overnight. Information on the antibodies used is listed in Table 1. After the sections were washed in PBS, immunoreactivity was visualized using EnVision reagent (Dako Co.), and the slides were developed with diaminobenzidine and counterstained with hematoxylin. All sections were 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).
Quantification of immunostaining parameters
Data were obtained by manually counting positively stained cells in five non-overlapping intratumoral fields. Due to the small size of specimens, clinical specimens were analyzed under x400 magnification to avoid counting the non-tumoral fields. Stained cells in mouse model tumors were accessed under ×100 magnification for CD4+ and CD8+ cells, and under x200 magnification for CD163+ cells. All immunostaining was interpreted by two independently (JK and TY).
Statistical analysis
Statistical analyses were conducted using SPSS statistical software, version 23 (IBM Corp., Armonk, NY, USA). Comparison of peritoneal tumor weight in each group was made using Student’s t-test. Differences among the cell count and fibrotic area data sets in each group were evaluated using the Mann-Whitney U-test. P values less than 0.05 indicated statistical significance.