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To evaluate whether insertion of self-biodegradable stent into the pylorus to prevent delayed-gastric emptying after pylorus-preserving gastrectomy is feasible and safe through porcine experiment.Self-biodegradable dumbbell-shaped pyloric stents were designed from absorbable suture materials: poly(glycolide-co-caprolactone) (PGCL) or poly-p-dioxanone (PPDO). After gastrotomy on ten pigs, each stent was inserted: two shams, four PGCL stents, and four PPDO stents. Body weight (Bwt), body temperature (BT), complete blood cell (CBC) count, and plain X-ray were evaluated. On postoperative day (POD) 13, euthanasia was performed for histologic evaluation.Operation was successfully performed in all ten pigs. Without tagging suture, both stents migrated before POD 3. The migration was delayed up to POD 13, when the tagging sutures (-t) were applied between stent and stomach wall. Self-degradation of PGCL started from POD 3, and stents were completely excreted from the abdomen by POD 8. Although PPDO were also weakened as self-degradation progressed, its shape was maintained in gastrointestinal tract for 13 days. Unexpected sudden death occurred in the pig with PPDO-t2 on POD 10, which is more likely due to acute volvulus rather than stent-related complication. There was no significant difference between three groups in terms of Bwt, BT, CBC, and histology (sham vs. PGCL vs. PPDO, all p 0.05).The concept of biodegradable stents made of absorbent suture material seems feasible in porcine experiment. Among them, PGCL which has shown rapid absorption, appears to be a more suitable material for transient pyloric absorbable stent when considering safety aspect.

420 Background: TLR-7/8 agonist has been suggested to be an effective immunotherapeutic agent, possible systemic side effect has forbid its clinical usage. A new controlled-releasing compound of TLR-7/8 agonist was developed to overcome the limitations, and this study aimed to evaluate efficacy and safety of this new compound in xenograft mouse model using cell-lines derived from triple conditional (Tcon) mouse which develop gastric cancer spontaneously. Methods: A cell line was established by tumor tissue in Tcon mouse which was developed by activation of Kras and suppression of E-cadherin and P53. Tcon cell line was evaluated its growth rate, sensitivity to anticancer agents and in vivo tumorigenicity. Tcon cell line was subcutaneously injected to the flank of the syngeneic mice, and 5-FU and/or TLR-7/8 agonist were administered into 4 groups of mice (control group, 5-FU alone, TLR-7/8 agonist alone, and combination of 5-FU and TLR-7/8). 5-FU was administered i.p(intra-peritoneal) in once a week and TLR-7/8 agonist was injected i.t(intra-tumoral) 3 times a week. Tumor size and mouse weight were measured for drug efficacy and safety. Immune profile of the tumor was analyzed by fluorescence-activated cell sorting. Results: Tcon cell-line show a rapid growth with 24 hours of doubling time, and good in vivo tumorigenicity in syngeneic mice. Cell-line showed moderate sensitivity to 5-FU and paclitaxel but resistance to oxaliplatin. Tumors in 5-FU alone group showed slight decrease in size compared with that in control group, However, TLR-7/8 agonist alone and combination significantly inhibited tumor growth. Weight loss or any significant side effect was not observed in any group. In immune profiling, TLR-7/8 mono or combination group showed increased levels of infiltrating CD4, CD8 T cells, and recruited NK cell as well as significantly increase M1/M2 ratio of macrophages compared to 5-FU alone or control group. Conclusions: A new controlled releasing system of TLR-7/8 agonist with or without 5-FU showed a excellent tumor inhibition efficacy with low toxicity in gastric cancer xenograft model. The effect seemed to be through multiple immune activations including cell-mediated immune response, M1 macrophage polarization as well as innate NK cells.

Several carcinomas including gastric cancer have been reported to contain Epstein‐Barr virus (EBV) infection. EBV‐associated gastric cancer (EBVaGC) is classified as one of four molecular subtypes of gastric cancer by The Cancer Genome Atlas (TCGA) group with increased immune‐related signatures. Identification of EBV‐dependent pathways with significant biological roles is needed for EBVaGC. To compare the biological changes between AGS gastric epithelial cells and EBV‐infected AGS (AGS‐EBV) cells, proliferation assay, CCK‐8 assay, invasion assay, cell cycle analysis, RT‐PCR, Western blot and ELISA were performed. BI836845, a humanized insulin‐like growth factor (IGF) ligand‐neutralizing antibody, was used for IGF‐related signalling pathway inhibition. AGS‐EBV cells showed slower proliferating rate and higher sensitivity to BI836845 compared to AGS cells. Moreover, invasiveness of AGS‐EBV was increased than that of AGS, and BI836845 treatment significantly decreased the invasiveness of AGS‐EBV. Although no apoptosis was detected, entry into the S phase of the cell cycle was delayed in BI836845‐treated AGS‐EBV cells. In conclusion, AGS‐EBV cells seem to modulate their proliferation and invasion through the IGF signalling pathway. Inhibition of the IGF signalling pathway therefore could be a potential therapeutic strategy for EBVaGC.

Casein kinase (CK) 2 activation has been implicated in the proliferation of various tumor types and resistance to chemotherapy. We investigated the mechanistic basis for the association between CK2 activation and paclitaxel resistance in a gastric cancer (GC). CK2 expression was evaluated in 59 advanced GC patients treated with paclitaxel as the second-line therapy. The efficacy of a CK2 inhibitor, CX-4945, and paclitaxel was evaluated in GC cell lines and a xenograft model. Patients with high CK2 expression (29/59, 39%) showed lower disease control rates (47.7% vs. 72.3%, p = 0.017) and shorter progression-free survival (2.8 vs. 4.8 months, p = 0.009) than patients with low CK2 expression. CK2 protein expression was associated with sensitivity to paclitaxel in 49 GC cell lines. Combination therapy with CX-4945 and paclitaxel exerted synergistic antiproliferative effects and inhibited the downregulation of phosphatidylinositol 3-kinase/AKT signaling in SNU-1 cells. In the SNU-1 xenograft model, the combination treatment was significantly superior to either single agent, suppressing tumor growth without notable toxicities. These results demonstrated that CK2 activation was related to paclitaxel resistance and that CX-4945 in combination with paclitaxel could be used as a potential treatment for paclitaxel resistance in GC.

Platinum-based antineoplastic drugs are chemotherapeutic agents to usually treat gastric cancer (GC) include cisplatin. However, the majority of cancer patients will eventually relapse with cisplatin-resistant disease. Especially, increased DNA repair is drug targetable mechanism and useful in the treatment strategy of cisplatin-resistant cancer. Casein kinase 2 (CK2) has critical role of multiple cellular processes with DNA repair. For this reason, research for CK2 expression correlated with DNA repair mechanism is important in gastric cancer. Combination of cisplatin and CK2 inhibitor (CX-4945, also known as Silmitasertib, Senhwa Biosciences, USA) may improve cisplatin-induced DNA damage for GC treatment. In this study, we screened sensitivity of cisplatin and CX-4945 in 49 GC cell lines by MTT assay. For molecular profiling, we analyzed variants and gene expression using whole exome sequencing and RNA sequencing. RNA and protein expression of CK2 subunits (α/α’) using real-time RT-PCR and Western blot. Also, activity of CK2α was measured by ELISA. Combination treatment performed different schedules including concurrent and sequential. Synergistic effect was analyzed by Bliss Independence model. As CK2 profiling, CK2α’ mRNA expression was a tendency to correlated with CX-4945 sensitivity(p=0.0504). Moreover, CK2α’ protein expression was a correlated with CX-4945 sensitivity(p=0.0252). Other molecular profiling did not reveal any clear correlations. Twenty one (Group1: cisplatin extremely resistant and CK2 high, Group2: cisplatin intermediate resistant regardless of CK2 expression) cell lines were performed combination treatment. Both YCC-21 and YCC-28 cell lines show synergistic effect (+20% and +22%, respectively) in concurrent schedule. Only MKN-74 cell line show synergistic effect (+11%) in Pre-addition. The 4 cell lines (YCC-18, YCC-38, SNU-1 and -216) were show synergistic effect (+10%, +37%, +11% and +19%, respectively) in Post-addition. In conclusion, early inhibition of CK2 shows synergistic effect in group 1, because CK2 is high expression. In group 2, late inhibition of CK2 demonstrated synergistic effect. It means CK2-related DNA repair is up-regulating for repair to cisplatin-induced DNA damage. Personalized the treatment schedule for inhibit CK2-induced DNA repair is a new strategy to restore cisplatin resistant in GC. Citation Format: Hyun Myong Kim, Inhye Jeong, Kyu Hyun Park, Tae Soo Kim, Woo Sun Kwon, Hei-Cheul Jeung, Minkyu Jung, Sun Young Rha. Overcoming cisplatin resistance through the combination treatment with CK2 inhibitor, CX-4945, in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1216. doi:10.1158/1538-7445.AM2017-1216

In vitro culture system is a long-standing useful tool in the study of cell biology for developing new therapeutic modalities and discovering new anti-cancer drugs. Therefore establishment and characterization of cell lines are very important in vitro study. However, establishment of GC patients derived cell lines are very challenging, especially with diffuse type which has different biology and worse prognosis from intestinal type. Yonsei Cancer Center (YCC) has been consistently worked to build human cancer cell lines, and resulting in over 30 novel cancer cell lines from metastatic gastric cancer patients. Most of YCC GC cell lines were established by primary culture of peritoneal fluid from metastatic gastric cancer patients. Interestingly, YCC-16 cells were isolated from the peripheral blood of gastric cancer patients. 20 cells were established from diffuse type GC, and 21 cells were from signet ring cell type. In this study, we evaluated 29 YCC GC cell lines and 23 GC cell lines from other sources (ATCC, KCLB, and JCRB). For molecular characterization of 52 GC cell lines, we analyzed copy number variant (CNV), single number variant (SNV) and gene expression using whole-exome sequencing (WES) and RNA sequencing. The 7 cell lines (YCC-17, 18, 19, 20, 29, 34, 36) were compared with their germline variants of paired PBMC. Also, these cell lines were investigated expression of proteins related to cancer progression and the sensitivity of chemotherapies and diverse molecular targeted drugs/antibodies. We evaluated the tumorigenesis with soft-agar assay and invasiveness by transwell assay. In addition to previous novel EBV infected cell line (YCCEL1/YCC-10, J Gen Virol. 2013), we observed amplification and overexpression of receptor tyrosine kinase (RTK) including HER2 (YCC-19, 32, 33, 38, 42), EGFR (YCC-11, 21), Met (YCC-31, 42), and FGFR2 (YCC-28, 30); confirming that protein was overexpression by Western blot in 20/52 (38.5%). Interestingly, amplification of RTKs was detected mutually exclusive pattern with other RTK amplification. Furthermore, RTK amplified cell lines were shown to be sensitive to target specific small molecules such as BGJ398. Also, we identified cell line specific genetic variations including ARID1A which might be the potential new targets in diffuse type GC cell lines. Then we could subgroup the cell lines based on 4 subtypes of TCGA. In conclusion, our GC cell line bank with genomic and biological characteristics based on the clinical information, would be useful in subgroup specific target selection, drug screening and mechanism evaluation for improving GC treatment. Citation Format: Tae Soo Kim, Kyu Hyun Park, Woo Sun Kwon, Won Suk Lee, In Hye Jeong, Sun Kyoung Kang, Hyun Myong Kim, Sun Young Rha, Hyun Cheol Chung. Novel gastric cancer cell lines established from diffuse type gastric cancer patients for potential subgroup-specific therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 139.

Peritoneal metastasis is the most frequent pattern of recurrence in patients with gastric cancer (GC). Patient derived xenograft (PDX) models using peritoneal fluid have been required to understand its biology and for the drug screening. So, we established the PDX model by peritoneal fluid in stage IV GC patients. First, we confirmed the preparation condition of peritoneal fluid cells for PDX model as approximately 1×107 cells in PBS. Then, we observed the engraftment rate of PDX which was 15.8% (3 of 19 patients, P2, P10 and P11). The time to 100 mm3 tumor size at F1 of 3 models was 47 ± 1, 26.7 ± 0.6 and 68 days respectively. Then, we checked the change of serial engraftment rate until F3. The more passage was progressed, the higher engraft rate was, 75% (3 of 4 mice) to 90% (9 of 10 mice) at P2, 60% (3 of 5 mice) to 85% (17 of 20 mice) at P10 and 33.3% (1 of 3 mice) to 86.7% (13 of 15 mice) at P11. In addition, time to 100mm3 tumor size of engrafted PDXs was faster at F3 than F1 in all 3 cases (41.1% in P2, 46.4% in P10 and 47.5% in P11). Also, tumor doubling time showed decreased in all engrafted PDXs according to passage (70.8% in P2, 11.5% in P10 and 71.5% in P12). Then, we checked the re-engraftment rate after thawing the frozen F3 tissue section of P2. F4 PDXs were re-engrafted 100% (3/ 3 mice) after thawing, and time to 100mm3 tumor size and tumor doubling times were not different between F3 and F4. It means that freezing and thawing of tumor fragment did not affect to engraft. After that, we compared the characterization of F0 peritoneal fluid of patient, F1∼F3 PDX tumor and the cell line established from F0. The morphologies of them were comparable by H&E staining. Also, molecule expressions ofF0 to F3 and cell lines were analogous. The genetic alterations according to passages in F0 to F3 are on-going. As a result, we observed PDX from peritoneal fluid of advanced gastric cancer is feasible but various according to each patient. The PDX model by peritoneal fluid of GC patients might be helpful the biological understanding and drug development for personalized therapies. Citation Format: Jeong Min Kim, Won Suk Lee, Woo Sun Kwon, Han Na Park, Hye Rin Lee, Hyun Myong Kim, Tae Soo Kim, Hyunki Kim, Jae Kyung Roh, Hyun Cheol Chung, Sun Young Rha. Patient-derived xenograft model using peritoneal fluid of gastric cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1454. doi:10.1158/1538-7445.AM2015-1454