Established fibrous peritoneal metastasis in an immunocompetent mouse model similar to clinical immune microenvironment of gastric cancer

Peritoneal metastasis (PM) in gastric cancer (GC) is characterized by diffusely infiltrating and proliferating cancer cells accompanied by extensive stromal fibrosis in the peritoneal space. The prognosis of GC with PM is still poor regardless of the various current treatments. In order to elucidate the cause of difficulties in PM treatment, we compared the tumor immune microenvironment (TME) in primary and PM lesions in GC. In addition, a PM model with fibrous stroma was constructed using immunocompetent mice to determine whether its TME was similar to that in patients. Immuno-histochemical analyses of infiltrating immune cells were performed in paired primary and PM lesions from 28 patients with GC. A C57BL/6 J mouse model with PM was established using the mouse GC cell line YTN16 either with or without co-inoculation of mouse myofibroblast cell line LmcMF with α-SMA expression. The resected PM from each mouse model was analyzed the immunocompetent cells using immunohistochemistry. The number of CD8+ cells was significantly lower in PM lesions than in primary lesions (P < 0.01). Conversely, the number of CD163+ cells (M2 macrophages) was significantly higher in PM lesions than in primary lesions (P = 0.016). Azan staining revealed that YTN16 and LmcMF co-inoculated tumors were more fibrous than tumor with YTN16 alone (P < 0.05). Co-inoculated fibrous tumor also showed an invasive growth pattern and higher progression than tumor with YTN16 alone (P = 0.045). Additionally, YTN16 and LmcMF co-inoculated tumors showed lower infiltration of CD8+ cells and higher infiltration of M2 macrophages than tumors with YTN16 alone (P < 0.05, P < 0.05). These results indicate that LmcMF plays as cancer-associated fibroblasts (CAFs) by crosstalk with YTN16 and CAFs contribute tumor progression, invasion, fibrosis, and immune suppression. This model is the first immunocompetent mouse model similar to TME of human clinical PM with fibrosis. By using this model, new treatment strategies for PM, such as anti-CAFs therapies, may be developed.


Background
Gastric cancer (GC) is one of the most common malignancies worldwide, with an estimated 1,033,701 new cases and 782,685 associated deaths in 2018 according to the International Agency for Research on Cancer [1]. Peritoneal metastasis (PM) is the most common mode of metastasis in gastric cancer and a critical indicator of poor prognosis [2][3][4]. Although various approaches to the treatment of PM have been assessed, including systemic and/or intraperitoneal chemotherapy [5][6][7][8], hyperthermic intraperitoneal chemotherapy (HIPEC) [9,10], and aggressive surgery (peritonectomy) [11,12], satisfactory outcomes have not been achieved. PM is characterized by rapid infiltration and proliferation of cancer cells accompanied by extensive stromal fibrosis, causing potentially fatal disorders such as bowel obstruction, hydronephrosis, and jaundice [13]. Fibrosis interferes with drug delivery due to high intratumoral pressure [14]. These fibrous stroma are conducted by crosstalk between cancer cells and cancer-associated fibroblasts (CAFs) [15][16][17]. CAFs also induce endothelial mesenchymal transduction (EMT) in cancer cells, result to produce the invasion ability and chemo-resistance in cancer cells. Besides, cytokines and chemokines derived from CAFs interact tumor immune microenvironment (TME), which affect not only chemo-sensitivities but also efficacy of immune checkpoint inhibitors (ICIs).
Recently, ICIs have become a major tools of cancer therapy tool. ICI therapies have been shown to be extremely effective against numerous malignancies, such as melanoma, renal cell carcinoma, non-small cell lung carcinoma (NSCLC), and Hodgkin disease [18][19][20][21]. The anti-programmed death-1 (PD-1) antibody, nivolumab, showed an 11% of response rate and 40% of disease control rate for late line of metastatic GC patients in the ATTRAC TION2 trial [22]. Generally, the efficacy of anti-PD-1 antibody is believed to depend on the tumor microenvironment with PD-ligand 1 (PD-L1) expression in cancer cells and the presence of tumor-infiltrating lymphocytes (TILs), mediating adaptive immune resistance [23]. This tumor immune microenvironment (TME) phenotype was found in 10-11% of Japanese GC patients [24,25], which is consistent with data from the ATTRACTION2 trial [22]. However, the antitumor response to nivolumab was independent of tumor PD-L1 status in this trial. These results suggest that other components of TME, such as cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs), might affect the immune system in PM. To obtain a favorable outcome using ICIs, tailoring combined therapy with modification of the TME will need to be considered. In the present study, we compared the TME in primary and peritoneal lesions to elucidate the cause of difficulties in treatment of metastatic GC. Additionally, a PM model with fibrous stroma features similar to those of human clinical samples was established using immunocompetent mice. The model was used to confirm whether the TME was similar to clinical PM for the development of new treatment strategies.

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-Nnitrosourea (MNU) to the animals' drinking water [26]. The resulting tumor cells were cultured in high-glucose Dulbecco's modified Eagle medium (DMEM, Sigma-Aldrich Japan, Tokyo, Japan) containing 1.0 mL/L MITO (Coning Japan, Tokyo), 10 mL/L L-Glutamine, 10 mL/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% CO 2 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 [27]. Cells were cultured in DMEM containing 10% FBS at 37°C in 5% CO 2 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 × 10 5 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/6 J mice (20 g, 6-8 weeks old) were purchased from Charles River Laboratories, Inc., Yokohama, Japan. The mice were housed with a 12 h 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 × 10 7 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 × 10 7 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). We used Olympus PLFLN 100X as objective lenses, DP70-SET as microscope digital

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 × 400 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 × 200 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 was made using Student's t-test. Differences among the cell count and fibrotic area data sets were evaluated using the Mann-Whitney U-test. P values less than 0.05 indicated statistical significance.

Patient characteristics
Patient characteristics are shown in Table 2. The median age of the study population was 66 years (range, 27-83 years). Overall, 17 men and 11 women were enrolled in the study. Twenty-five of the twenty-eight patients (89%) had diffuse type GC (Bormann 3 or 4), while the other three patients showed intestinal type GC based on the Lauren classification. According to the Japanese Classification of Gastric Carcinoma 15th edition, the P statuses were P1a in 11 cases, P1b in 4 cases, and P1c in 13 cases.

Immunostaining of GC primary and peritoneal lesions
We performed immunostaining of biopsy specimens of primary and peritoneal lesions to analyze the microenvironment. We used antibodies against CD4 and CD8 as markers for lymphocytes and the CD163 antibody as a marker for M2 macrophages. Representative microscopic views of the peritoneal lesions are shown in Fig. 1a. There was no significant difference in the number of CD4 + cells between primary and peritoneal lesions (8.9 ± 5.5 vs. 7.4 ± 7.7). The number of CD8 + cells observed in the peritoneal lesions was significantly lower than in the primary lesions (28.3 ± 12.7 vs. 8.7 ± 7.2, P = 0.01). CD163 positive cells in the peritoneal lesion were significantly higher than in the primary lesion (18.4 ± 2.5 vs. 32.1 ± 5.0, P = 0.016) (Fig. 1b).

Establishment of fibrous allograft model
All mice were successfully established peritoneal tumors in each group and were analyzed using their datasets of day 14. YTN16 cells were co-cultured with LmcMF and co-inoculated intraperitoneally into mice. As shown in Fig. 2a and b, the tumor weight of peritoneal tumors from mice co-inoculated with YTN16 and LmcMF was significantly higher than that of tumors from mice inoculated with YTN16 alone (0.57 ± 0.33 g vs. 1.25 ± 0.25 g, P = 0.045). Microscopic findings of peritoneal tumors in co-inoculated mice showed invasive progression into spleen, but tumors in mice inoculated with YTN16 alone showed expansive progression, not invasive (Fig. 2c). α-SMA expressing fibroblasts were found in co-inoculated peritoneal tumors as CAFs (Fig. 2d, Additional file 1). The degree of fibrosis in the tissues was compared by Azan staining (Fig. 3a, Additional file 2). Areas that stained blue were regarded as fibrosis, and the percentage of stained tissue was compared. In the YTN16 control group, some fibrosis was observed, but in the coinoculated group (YTN16 and LmcMF), the fibrotic area was significantly increased in peritoneal tumors (1.4%; range 0.13-3.38 vs. 19%; range 12.8-25.3, P < 0.05) (Fig.  3b). Co-inoculated LmcMF cells that were pretreated with PKH staining were confirmed as a component of fibrous tumors (data not shown).

Analysis of immune microenvironment of mouse fibrous tumor
There was no significant difference in the median CD4 + cell counts between tumors from co-inoculation with YTN16 and LmcMF tumor (fibrous tumors) and those from inoculation with YTN16 alone (data not shown). Significantly fewer CD8 + cells were found in fibrous tumors than in non-fibrous tumors (median: 64 cells; range 40-68 vs. 9 cells; range 3-15, P < 0.05) (Fig. 4,  Additional file 3). On the other hand, we found a higher rate of CD163 + cell infiltration in fibrous tumors than in non-fibrous tumors (median: 26 cells; range 19-39 vs. 49 cells; range 35-73, P < 0.05) (Fig. 5, Additional file 4).

Discussion
In this clinical analysis, the TME in peritoneal lesions was quite different from the TME in primary lesions. There were significantly fewer CD8 + cells corresponding to CTL in peritoneal lesions than in primary lesions. The presence of CD8 + cells in the tumor infiltrate prior to the onset of chemotherapy could predict pathological complete response [28,29]. These results indicate that it is difficult to treat with not only ICIs but also chemotherapy in TME of PM. Stromal fibroblasts so called CAFs suppress CD8 + cells function via PDL2 and FASligand, and enhance secretion of interleukin (IL)-6 via crosstalk with cancer cells resulting decreasing accumulation of CD8 + cells [30,31]. Accordingly, it is important to suppress the function of CAFs for recruitment of CD8 + cells.
There was no significant difference in the number of CD4 + cells between primary and peritoneal lesions. CD4 + cells include helper T cells, monocytes, and macrophages, which have various functions in tumor progression. CD4 infiltration status is not statistically associated with prognosis [32], but may affect the efficacy of ICIs.
CD163 + cells corresponding to M2 macrophages were found to be significantly higher in PM than in primary lesions. Although macrophages are thought to function as both antitumor agents (M1 macrophages) and protumor agents (M2 macrophages) [33], protumoral tumorassociated macrophages (TAMs) are believed to exhibit characteristics similar to M2 macrophages [34]. Monocytes migrate into peritoneal lesions using the C-C chemokine ligand 2 (CCL2) derived from CAFs and differentiate into the M2 phenotype by macrophage colony-stimulating factor (M-CSF) and IL-6 secreted from CAFs and cancer cells [35]. These M2 macrophages contribute to tumor angiogenesis, immune suppression, and metastasis [36,37]. The high density of M2 macrophages in PM may contribute to the low efficacies of ICIs treatments for metastatic GC, including PM.
Low-dose paclitaxel changed macrophage phenotype from M2 to M1 through the toll-like receptor (TLR)-4, resulting in tumor growth inhibition [38,39]. This theory is consistent with taxan-containing therapies showing superior results than other regimens for treating gastric cancer [40,41]. Half of the patients with unresectable GC have PM at the time of second-line chemotherapy induction [2,41]. Because ICI was used after treatment failure of taxan-containing therapies, the phenotype of macrophages may have changed from M1 to M2 in the PM TME. Therefore, another strategy for reprogramming macrophages from the M2 to M1 phenotype is needed.
We established a fibrous peritoneal tumor model by co-inoculating the mouse gastric cancer cell line YTN16 and the mouse myofibroblast cell line LmcMF into immune-competent C57BL6/J mice. LmcMF cells played as CAFs by crosstalk with YTN16 resulted in creating much more extracellular matrix represented by collagen fiber than tumors inoculated with YTN16 alone. Tissue fibrosis may interfere with drug delivery and immune cells infiltration due to high intratumor pressure [14]. In the YTN16 and LmcMF co-inoculated tumor model, we observed less infiltration of CD8 + cells, but more infiltration of M2 macrophages. In addition, the tumors were hyperprogressive and more invasive compared to YTN16 inoculated tumors. These results indicate that CAFs interfere accumulation of CD8 + cells and induce recruitment and M2 polarization of macrophages by secreting various cytokines and/or chemokines. Thus, the importance of CAFs in PM was clarified by demonstrating difference of TME in both mouse model with or without LmcMF cells.
This study has some limitations. First, the number of clinical samples was small, due to difficulty of obtaining non-treated paired specimens from GC patients with PM. Second, TME of biopsy specimens from the surface of the primary tumors may differ from serosal site specimens. Third, TME differences between solid peritoneal tumors were investigated for only one cell line. However, to our knowledge, this is the first report of the establishment of a fibrous peritoneal model in immunocompetent mice.

Conclusion
In this study, we have clarified the TME of PM in GC and successfully established a mouse model that closely resembles human clinical findings. By using this model, it could be possible to develop new treatment strategies through anti-CAFs therapy.