Supplementary information. Supplementary Materials and Methods

Table S1. Characteristics of Patients' Population (Test cohort, N=69). Table S2. Characteristics of Patients' Population (Validation Cohort-1, N=41). Table S3. Characteristics of Patients' Population (Validation Cohort-2, N=64). Table S4. Clinicopathologic features of TCGA cohort. Table S5. Characteristics of tissue microarray cohort (Validation cohort-3, N=173) Table S6. Differentially expressed miRNAs in MIF-High and MIF-Low PDAC cases and their association with survival (cox regression). Table S7. miR-301b candidate target gene associated with survival by Cox regression and Kaplan-Meier analyses. Table S8. qRT-PCR probes and primer sequences for plasmid construction. Table S9. Antibodies information

Supplementary figure S1. Increased cellular ROS levels in PDAC cells treated with gemcitabine.

Supplementary Table S1. Real-time PCR primer sequences of assessed genes.

Figure S1. Validation of miRNA microarray data by qRT-PCR in the test cohort (n=69). Figure S2. MIF regulates miR-301b expression in human pancreatic cancer Figure S3. NR3C2 is a potential target of miR-301b. Figure S4. Endogenous NR3C2 expression was negatively correlated with MIF and miR-301b in a panel of human pancreatic cancer cell lines. Figure S5. NR3C2 enhances sensitivity to gemcitabine in pancreatic cancer cell lines. Figure S6. Knockdown of NR3C2 increases proliferation, migration and invasion, and decreases sensitivity of pancreatic cancer cells to gemcitabine. Figure S7. A lower endogenous NR3C2 expression was associated with EMT phenotype. Figure S8. NR3C2 inhibits epithelial-to-mesenchymal transition (EMT). Figure S9. MIF-induced miR-301b expression was blocked by anti-miR-301b. Figure S10. MEK inhibitor AZD6244 didn't alter miR-301b or NR3C2 in MIF-overexpressing Panc-1 and Capan-2 cells. Figure S11. NR3C2 expression is decreased in tumors as compared to adjacent nontumor tissues in PDAC. Figure S12. A lower expression of NR3C2 is associated with poor survival in multiple independent cohorts of PDAC.

Congestive hepatopathy often leads to liver fibrosis and hepatocellular carcinoma. Imaging modalities provided clinical evidence that elevation of liver stiffness and tumor occurrence are mainly induced in the periphery of the liver in patients with congestive hepatopathy. However, clinical relevance of liver stiffness and liver fibrosis is unclear because liver congestion itself increases liver stiffness in congestive hepatopathy. It also unclear which factors configure such regional disparity of tumor development in patients with congestive hepatopathy. To answer these questions, we evaluated the macroscopic spatial distribution of liver fibrosis and tumors in the murine model of congestive hepatopathy. Chronic liver congestion was induced by partial ligation of the suprahepatic inferior vena cava. Distribution of liver congestion, fibrosis, and tumors in partial ligation of the suprahepatic inferior vena cava mice were assessed by histological findings, laser microdissection (LMD)-based qPCR and enhanced computed tomography. LMD-based RNA-sequencing was performed to identify causal factors that promote tumor development in congestive hepatopathy. Liver fibrosis was mainly induced in the periphery of the liver and co-localized with distribution of liver congestion. Liver tumors were also induced in the periphery of the liver where liver congestion and fibrosis occurred. LMD-based RNA-sequencing revealed the upregulation of extracellular matrix/collagen fibril-, wound healing-, angiogenesis-, morphogenesis-, and cell motility-related signaling pathways in periphery of liver compared with liver center. Our findings showed the experimental relevance of liver congestion, fibrosis, and tumor development in congestive hepatopathy, and may provide important locational information. Macroscopic regional disparity observed in this murine model should be considered to manage patients with congestive hepatopathy.

Supplementary figure S2. siRNA depletion of NF-κB/p65 decreased cellular ROS levels in MIAPaCa-2/GR cells or MIAPaCa-2 cells treated with gemcitabine.

AimCongestive hepatopathy often leads to liver fibrosis and hepatocellular carcinoma. Imaging modalities provided clinical evidence that elevation of liver stiffness and tumor occurrence are mainly induced in the periphery of the liver in patients with congestive hepatopathy. However, clinical relevance of liver stiffness and liver fibrosis is unclear because liver congestion itself increases liver stiffness in congestive hepatopathy. It also unclear which factors configure such regional disparity of tumor development in patients with congestive hepatopathy. To answer these questions, we evaluated the macroscopic spatial distribution of liver fibrosis and tumors in the murine model of congestive hepatopathy. MethodsChronic liver congestion was induced by partial ligation of the suprahepatic inferior vena cava. Distribution of liver congestion, fibrosis, and tumors in partial ligation of the suprahepatic inferior vena cava mice were assessed by histological findings, laser microdissection (LMD)-based qPCR and enhanced computed tomography. LMD-based RNA-sequencing was performed to identify causal factors that promote tumor development in congestive hepatopathy. ResultsLiver fibrosis was mainly induced in the periphery of the liver and co-localized with distribution of liver congestion. Liver tumors were also induced in the periphery of the liver where liver congestion and fibrosis occurred. LMD-based RNA-sequencing revealed the upregulation of extracellular matrix/collagen fibril-, wound healing-, angiogenesis-, morphogenesis-, and cell motility-related signaling pathways in periphery of liver compared with liver center. ConclusionsOur findings showed the experimental relevance of liver congestion, fibrosis, and tumor development in congestive hepatopathy, and may provide important locational information. Macroscopic regional disparity observed in this murine model should be considered to manage patients with congestive hepatopathy.

Supplementary figure S4. BSO showed similar effects as PEITC and GME rescued PEITC-induced apoptosis in MIAPaCa-2 cells.

Supplementary figure S3. PEITC-induced selective killing of PDAC cells with minimal toxicity to HPDE cells and nude mice.

Purpose To assess the efficacy of combination chemotherapy with nafamostat mesilate, gemcitabine and S-1 for unresectable pancreatic cancer patients. Materials and methods The study was conducted as a single-arm, single center, institutional review board-approved phase II trial. Patients received nafamosntat mesilate (4.8 mg/kg continuous transregional arterial infusion) with gemcitabine (1000 mg/m 2 transvenous) on days 1 and15, and with oral S-1 [(80 mg/day (BSA<1.25 m 2 ), 100 mg/day (1.25 ≤ BSA<1.5 m 2 ), or 120 mg/day (BSA ≥1.5 m 2 )] on days 1–14 or, days 1–7 and 15–21. This regimen was repeated at 28-day intervals. Results Forty-seven evaluable patients (Male/Female: 31/16, Age (median): 66 (range 35–78) yrs, Stage III/IV 10/37.) were candidates in this study. Two patients in stage III (20%) could undergo conversion surgery. Twenty-four patients (51%) underwent subsequent treatment (1 st line/ 2 nd line / 4 th line, 13/ 10/ 1, FOLFIRINOX: 12, GEM/nab-PTX: 18, TAS-118: 3, chemoradiation with S-1: 2, GEM/Erlotinib: 1, nal-IRI: 1, surgery: 2). Median PFS and OS were 9.7 (95% CI, 8.9–14.7 mo) and 14.2 months (99% CI, 13.3–23.9 mo), respectively. Median PFS in stage IV patients was 9.2 months (95% CI, 8.4–12.0 mo). Median OS in patients without subsequent treatment was 10.8 months (95% CI, 9.1–13.8 mo). Median OS in patients with subsequent treatment was 19.3 months (95% CI, 18.9–31.9 mo). Grade 4 treatment-related hematological toxicities were encountered in 7 patients. Two patients developed grade 3 allergic reaction after 6 cycles or later. No febrile neutropenia has been observed. Conclusion NAM/GEM/S-1 therapy is safe and could be promising option for unresectable pancreatic cancer, especially for stage IV cancer.

症例は48歳女性．生理不順を主訴に当院婦人科受診，肝腫瘍を指摘された．入院時身体所見は腹部に腫瘤を触知するも圧痛なし．HBs-Ag陰性，HCV-Ab陰性で，腫瘍マーカーはCA19-9のみ軽度上昇していた．腹部CT検査では肝外側区域に境界明瞭な14cm大の単発性腫瘍を認めた．内部は多血性で，不均一に造影され，複数の発達した肝静脈にドレナージされていた．消化管には明らかな悪性所見は認めなかった．以上より，肝血管筋脂肪腫（angiomyolipoma：AML）を最も疑ったが，悪性腫瘍が否定できないことと破裂の可能性を考慮し肝左葉切除術を施行した．術後の病理組織学所見ならびに免疫組織化学所見よりperivascular epithelioid cell tumor（PEComa）と診断した．肝PEComaは稀な腫瘍であり，その悪性度評価，治療法には現状では一定の見解はない．文献的考察を加えて報告する．

Objective Gallbladder carcinoma (GBC) is one of the digestive cancers with poor prognosis, for which surgical resection is the only potentially curative therapy. Prognostic value of macroscopic inflammatory status of the resected gallbladder in patient with GBC has not been fully investigated. We retrospectively investigated the relation between macroscopic inflammatory status and disease-free as well as overall survival after radical resection for GBC. Method The subjects were 44 patients who underwent radical resection for GBC between January 2004 and April 2011 at Jikei University Hospital. We retrospectively investigated the relationship between clinicopathologic variables, including macroscopic inflammatory status and disease-free as well as overall survival. Results In univariate analysis, disease-free survival was poor in patients with Tumor-Nodes-Metastasis (TNM) stage ≥III (P < 0.0001) and positive vascular invasion (P = 0.0001). Patients with macroscopic chronic inflammation tended to have poor disease-free survival than those with normal type (P = 0.0930). Overall survival was poor in patients with TNM stage ≥III (P < 0.0001), presence of intraoperative blood transfusion (P = 0.0125), positive vascular invasion (P = 0.0055), and macroscopic chronic inflammation (P = 0.0281). In multivariate analysis, TNM stage ≥III (P < 0.0114) and macroscopic chronic inflammation (P = 0.0350) were independent predictors of disease-free survival. For overall survival, TNM stage ≥III (P = 0.0054) and macroscopic chronic inflammation (P = 0.0124) were the independent predictors. Moreover, macroscopic chronic inflammation correlated with the presence of gallstones. Conclusion The macroscopic Inflammation status of resected gallbladder cancer correlates with oncologic outcome in patients with GBC treated by radical resection.

Abstract Background Prevention and treatment of postoperative pancreatic fistulas is a crucial problem associated with pancreaticoduodenectomies and distal pancreatectomies. The objective of this study was to determine the outcomes of the “crushing and ligation” technique for liver dissection during pancreaticoduodenectomy and distal pancreatectomy procedures. Methods We evaluated the outcomes of 44 patients who underwent pancreatic dissections using the crushing and ligation technique and 44 patients who underwent the ultrasonically activated scalpel technique. Operative duration, blood loss, length of hospital stay, incidence of postoperative pancreatic fistulas (grade BC), and postoperative drainage amylase fluid level were compared between groups. Results Postoperative pancreatic fistulas (grade BC) occurred in 2 (8.0%) and 13 (44.8%) patients in the pancreaticoduodenectomy-crushing and ligation and pancreaticoduodenectomy-ultrasound groups, respectively (p = 0.0026). The pancreaticoduodenectomy-crushing and ligation group had significantly lower maximum amylase levels in drainage fluid (p = 0.0387) and significantly longer operative durations (p = 0.0422), but a shorter postoperative length of hospital stay (p = 0.0159), than the pancreaticoduodenectomy-ultrasound group. Postoperative pancreatic fistulas (grade BC) occurred in 2 (10.5%) patients in the distal pancreatectomy-crushing and ligation group, which was significantly lower than in the distal pancreatectomy-ultrasound group (7 patients, 46.7%). Conclusion Crushing and ligation may lead to better outcomes and fewer complications than the conventional ultrasonically activated scalpel technique.

Objective: Postoperative pancreatic fistula (POPF) following pancreaticoduodenectomy is the most serious complication of these surgical procedures; therefore, we examined the effectiveness of fasting, and administration of octreotide acetate and ulinastatin as a method of prevention. Summary of Background Data: Although various drug therapies and surgical techniques have been used for the treatment of POPF, no decisive treatment for POPF exists. Methods: The clinical course of 30 patients who developed POPF was retrospectively evaluated and compared among no dietary intake (n = 18), octreotide acetate (n = 8), and ulinastatin (n = 8) using an overlapping design. Patients were allocated to either the dietary intake or fasting (no dietary intake) group, and those in the no dietary intake group were further divided into the octreotide acetate or ulinastatin group. Results: Length of hospitalization was longer for the no dietary intake group than for the dietary intake group (P = 0.002). When considering only grade B or C POPF cases, the no dietary intake group had a longer length of hospitalization and a higher white blood cell count on day 7 after the diagnosis of POPF than the dietary intake group (P , 0.05). The white blood cell count was also higher in the octreotide acetate group than in the ulinastatin group (P = 0.021). The length of hospitalization was shorter in the ulinastatin group than in the octreotide acetate group (P = 0.025). Conclusions: The use of no dietary intake, octreotide acetate, and ulinastatin does not seem to contribute to the clinical course of patients with POPF after pancreatoduodenectomy.

Nuclear factor kappa B (NF-κB) is a transcriptional factor that can be activated by radiotherapy and chemotherapy. The synthetic protease inhibitor nafamostat mesilate (NM) inhibits NF-κB activity and exerts antitumor actions in various types of cancer. In the present study, we hypothesized that NM might enhance the antitumor action of radiotherapy on gallbladder cancer (GBC) cells by inhibiting radiation-induced NF-κB activity. Thus, we investigated the correlation between radiotherapy and NF-κB activity in GBC cells. We assessed the in vitro effects of radiotherapy with or without NM on NF-κB activity, apoptosis of GBC cells (NOZ and OCUG-1), induction of apoptotic cascade, cell cycle progression, and viability of GBC cells using four treatment groups: 1) radiation (5 Gy) alone; 2) NM (80 μg/mL and 40 μg/mL, respectively) alone; 3) combination (radiation and NM); and 4) vehicle (control). The same experiments were performed in vivo using a xenograft GBC mouse model. In vitro , NM inhibited radiation-induced NF-κB activity. Combination treatment significantly attenuated cell viability and increased cell apoptosis and G2/M phase cell cycle arrest compared with those in the other groups for NOZ and OCUG-1 cells. Moreover, combination treatment upregulated the expression of apoptotic proteins compared with that after the other treatments. In vivo , NM improved the antitumor action of radiation and increased the population of Ki-67-positive cells. Overall, NM enhanced the antitumor action of radiotherapy on GBC cells by suppressing radiation-induced NF-κB activity. Thus, the combination of radiotherapy and NM may be useful for the treatment of locally advanced unresectable GBC.

BACKGROUND AND AIMS:Chronic liver congestion reflecting right-sided heart failure (RHF), Budd-Chiari syndrome, or Fontan-associated liver disease (FALD) is involved in liver fibrosis and HCC. However, molecular mechanisms of fibrosis and HCC in chronic liver congestion remain poorly understood.APPROACH AND RESULTS:Here, we first demonstrated that chronic liver congestion promoted HCC and metastatic liver tumor growth using murine model of chronic liver congestion by partial inferior vena cava ligation (pIVCL). As the initial step triggering HCC promotion and fibrosis, gut-derived lipopolysaccharide (LPS) appeared to induce LSECs capillarization in mice and in vitro. LSEC capillarization was also confirmed in patients with FALD. Mitogenic factor, sphingosine-1-phosphate (S1P), was increased in congestive liver and expression of sphingosine kinase 1, a major synthetase of S1P, was increased in capillarized LSECs after pIVCL. Inhibition of S1P receptor (S1PR) 1 (Ex26) and S1PR2 (JTE013) mitigated HCC development and liver fibrosis, respectively. Antimicrobial treatment lowered portal blood LPS concentration, LSEC capillarization, and liver S1P concentration accompanied by reduction of HCC development and fibrosis in the congestive liver.CONCLUSIONS:In conclusion, chronic liver congestion promotes HCC development and liver fibrosis by S1P production from LPS-induced capillarized LSECs. Careful treatment of both RHF and liver cancer might be necessary for patients with RHF with primary or metastatic liver cancer.

Prognostic factors after treatment for intrahepatic recurrent hepatocellular carcinoma (RHCC) after hepatic resection (Hx) are controversial. The current study aimed to examine the impact of treatment modality on the prognosis of intrahepatic RHCC following Hx.