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The incidence of non-alcoholic steatohepatitis (NASH) is dramatically increasing, but the effect of NASH on colon cancer liver metastasis (CLM) is controversial. The aim of this study was to investigate the impact and mechanism of action of NASH on CLM using a western diet (WD)-fed mouse model.Six-week-old male C57BL/6 J mice were used. They were divided into the WD group and control group with normal diet. MC38 colon cancer cells were injected into the spleen at 2, 6, 8 and 16 weeks, and mice were killed at 2 weeks after injection to evaluate hepatic steatosis, fibrosis, metastasis and mRNA/protein expression in the liver.Only mice fed a WD for 16 weeks showed hepatic fibrosis. These mice showed significantly higher alanine aminotransferase and total cholesterol levels compared with the control group (p 0.05). The WD group showed significantly lower tumor number and smaller tumor diameter (p 0.05). In the WD group, expression of SAA1, IL6, STAT3 and MMP9 mRNA in the liver was significantly lower than in the control group (p 0.05). Serum amyloid A1 protein expression was also lower in the WD group.The WD-fed NASH mouse model showed an inhibitory effect on CLM. Suppressed interleukin-6/signal transducer and activator of transcription 3 signaling and serum amyloid A/matrix metalloproteinase 9 expression may affect this phenomenon.

Background : Several reports have shown a high mortality rate in patients with liver cirrhosis (LC) who undergo pancreaticoduodenectomy, however, there are few reports on its long-term prognosis. Methods : Twelve patients with LC who had undergone pancreatic resection were enrolled. To compare clinicopathological variables, 159 non-LC patients who had undergone resection for pancreatic cancer were enrolled. Results : Pancreaticoduodenectomy (PD) was performed in 5 LC patients and distal pancreatectomy (DP) was performed in 7 LC patients. Patients in the LC group had more co-morbidities, lower platelet counts and higher Fib4 index than the non-LC group. The postoperative complication rate was higher in the LC group (83.3% vs 47.8%). While the postoperative hospital stay and 30-day mortality rate were not different, the 90-day mortality rate was higher in the LC group (25.0% vs 2.5% ; p < 0.01). Comparison by operative procedure showed no significant differences of postoperative outcomes in DP cases. However, in PD cases, postoperative complications were more frequent (100% vs 42.3%) and 90-day mortality was higher (40.0% vs 2.9% ; p < 0.01) in the LC group. Conclusions : PD resulted in higher postoperative morbidity and mortality rates in patients with LC compared with non-LC patients. DP could be tolerated in the LC patients.

Background : Several reports have shown the high mortality rate of pancreatic resection in patients with hemodialysis (HD), however, its long-term outcome remains unclear. In this study, we examined cases of pancreatic resection in patients with HD and conducted a literature review. Methods : Four patients with HD who underwent pancreatic resection from 2004 to 2019 were enrolled. To compare the clinicopathological variables of HD and non-HD patients, 161 non-HD patients who had undergone surgical resection for pancreatic cancer were enrolled. Results : Among four cases of pancreatic resection with HD, three cases were malignant diseases. All patients with HD had some co-morbidities (100% in HD group, 45.3% in the non-HD group) and postoperative complications (100% in the HD group, vs 46.6% in the non-HD group). Although one patient had severe postoperative complications and length of postoperative hospital stay was longer, the 30- and 90-day mortality rates were both 0% in patients with HD. However, three cases in the HD group (75%) died approximately 6 months after surgery, including one cancer-related death. Conclusions : Pancreatic surgery in patients with HD should be carefully indicated, especially pancreaticoduodenectomy or total pancreatectomy, because of the poor prognosis induced by non-cancer-related causes of death.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Poor regeneration after hepatectomy in NAFLD is well recognized, but the mechanism is unclear. Endoplasmic reticulum (ER) stress plays an important role in the development of NAFLD. Here, we show that an impaired ER stress response contributes to poor liver regeneration in partially hepatectomized mice.Non-alcoholic fatty liver (NAFL) or non-alcoholic steatohepatitis (NASH) was induced in mice using our patented feed and 70% partial hepatectomy (PH) was performed. Mice were sacrificed 0, 4, 8, 24, or 48 hours, or 7 days after PH, and liver regeneration and the mRNA expression of ER stress markers were assessed.Non-alcoholic fatty liver disease activity score was calculated as 4-6 points for NAFL and 7 points for NASH. NASH was characterized by inflammation and high ER stress marker expression before PH. After PH, NASH mice showed poorer liver regeneration than controls. High expression of proinflammatory cytokine genes was present in NASH mice 4 hours after PH. Xbp1-s mRNA expression was high in control and NAFL mice after PH, but no higher in NASH mice.Dysfunction of the ER stress response might be a cause of poor liver regeneration in NASH.

Tumor-infiltrating lymphocytes (TILs) are a prognostic factor or an indicator of chemotherapy response for various malignancies. The aim of this study was to investigate the prognostic impact of TILs in resected intrahepatic cholangiocarcinoma (IHCC). We also investigated the usefulness of the apparent diffusion coefficient (ADC) in diffusion-weighted magnetic resonance imaging (DW-MRI) to predict TILs. We enrolled 23 patients with IHCC who underwent initial hepatic resection in Tokushima University Hospital from 2006 to 2017. We evaluated stromal TILs in the tumor marginal area and central area in surgical specimens. Patients were divided into low vs high stromal TILs groups. We analyzed the patients’ clinicopathological factors, including prognosis, according to the degree of stromal TILs. We also analyzed the correlation between stromal TILs and the minimum ADC value. Stromal TILs in the marginal area reflected overall survival more accurately than that in the central area. Additionally, marginal low TILs was significantly associated with lymph node metastasis and portal vein invasion. Both overall- and disease-free survival rates in the marginal low TILs group were significantly worse than those in the marginal high TILs group (P

Background/aim The aim of this study was to investigate frailty as a prognostic factor in patients with colorectal liver metastasis undergoing hepatectomy. Patients and methods Eighty-seven patients who underwent hepatectomy at our institution were enrolled. Frailty was defined as a score of ≥4 on a clinical frailty scale. Patients were divided into frailty (n=29) and non-frailty (n=58) groups. Results Overall and cancer-specific survival rates were significantly worse in the frailty group compared with the non-frailty group, and multivariate analysis revealed frailty as an independent prognostic factor. Disease-free survival tended to be worse in the frailty group. Fifty-eight patients relapsed after the first hepatectomy. Twenty-one of 58 recurrent patients were allocated to the frailty group. After recurrence, chemotherapy was significantly more frequently performed in the non-frailty group compared with the frailty group. Conclusion Frailty can predict the prognosis of patients with colorectal liver metastasis undergoing hepatectomy.

Background The aim of this study was to clarify the effectiveness of a new three-dimensional (3D) culture system for hepatocyte-like cells (HLCs) generated from human adipose-derived mesenchymal stem cells (ADSCs). Methods Human ADSCs (2 × 104 ) with or without 0.1 mg/mL human recombinant peptide μ-piece per well were seeded in a 96-well U-bottom plate and then our three-step differentiation protocol was applied for 21 days. At each step, cell morphology and gene expression were investigated. Mature hepatocyte functions were evaluated after HLC differentiation. These parameters were compared between 2D- and 3D-cultured HLCs, and, DNA microarray analysis was also performed. Finally, HLCs were transplanted in to CCl4 induced acute liver failure model mice. Results Two-dimensional-cultured HLCs at day 21 did not have a spindle shape and had formed spheroids after day 6, which gradually increased in size for 3D-cultured HLCs. Definitive endoderm, hepatoblast, and hepatocyte genes showed significantly higher expression in the 3D culture group. Three-dimensional-cultured HLCs also had higher albumin expression, CYP3A4 activity, urea synthesis, and ammonium metabolism, and much higher expression of ion transporter, blood coagulation, and cell communication genes. HLC transplantation improved serum liver function, especially in T-Bil levels, and engrafted into immunodeficient mice with HLA class I positive staining. Conclusion Our new 3D culture protocol is effective to improve hepatocyte functions. Our HLCs might be promising for clinical cell transplantation to treat metabolic disease.

To clarify whether the preoperative lymphocyte/C-reactive protein (CRP) ratio (LCR) is a prognostic factor for patients with intrahepatic cholangiocarcinoma (IHCC), and investigate its mechanism via tumor-infiltrating lymphocytes. The subjects of this retrospective study were 42 patients who had undergone hepatectomy for IHCC. We divided the patients into low LCR and high LCR groups (cutoff value: 8780) and analyzed their overall survival (OS) and disease-free survival (DFS) with respect to LCR and other clinicopathological factors. We also investigated the levels of stromal tumor-infiltrating lymphocytes (TILs) and CD8+ TILs in surgical specimens, and the relationship between LCR and TILs. A low LCR was identified in 21 patients and was significantly correlated with older age, a high CRP-albumin ratio, and advanced disease stage, and was a prognostic factor for OS and DFS. Multivariate analysis revealed that a low LCR was an independent prognostic factor for worse OS (HR 10.40, P = 0.0077). Although the LCR and levels of stromal TILs were not significantly related, LCR and levels of CD8+ TILs were significantly related (P = 0.0297). The preoperative LCR may predict the postsurgical prognosis of patients with IHCC and reflect the CD8+ TILs.

Background No universal classification method for intrahepatic cholangiocarcinoma (IHCC) has been reported based on the embryological origin of biliary epithelial cells. The aim of this study was to classify IHCC according to protein expression levels of somatostatin receptor 2 (SSTR2) and b-cell leukemia/lymphoma 2 (Bcl2) and to elucidate the clinicopathological features of each group. Methods Fifty-two IHCC patients who underwent hepatic resection were enrolled in this study. Protein expression levels of SSTR2 and Bcl2 were examined using immunohistochemistry. Clinicopathological factors were compared between the three groups and prognostic factors were investigated. Results The patients were divided into three groups: SSTR2 positive and Bcl2 negative (p-Group H, n = 21), SSTR2 negative and Bcl2 positive (p-Group P, n = 14), and the indeterminate group (p-Group U, n = 17) for cases where SSTR2 and Bcl2 were both positive or both negative. All p-Group P cases displayed curability A or B. The 5-year survival rates of p-Group H and U patients were worse than those in p-Group P. p-Group H had higher T-factor, clinical stage, and incidence of periductal infiltration than p-Group P. Conclusions This method could be used to classify IHCC into peripheral and perihilar type by embryological expression patterns of SSTR2 and Bcl2.

Background Daikenchuto (TU‐100), a Japanese herbal medicine, is widely used for various gastrointestinal diseases. We have previously reported that TU‐100 suppresses CPT‐11‐induced bacterial translocation (BT) by maintaining the diversity of the microbiome. In this study we show that TU‐100 modulates the immune response during BT by inducing PD‐1 expression in Peyer's patches. Methods Eighteen male Wistar rats were divided into four groups: a control group; a control + TU‐100 group, given TU‐100 1000 mg/kg orally for 5 d; a BT group, given CPT‐11 250 mg/kg intra‐peritoneal for 2 d; and a TU‐100 group, given TU‐100 1000 mg/kg orally for 5 d with CPT‐11 250 mg/kg intra‐peritoneal on days 4 and 5. Results The size of Peyer's patch was significantly bigger in the BT group compared to the control group (9.0 × 104 µm2 vs 29.4 × 104 µm2, P, Daikenchuto (TU‐100), a Japanese herbal medicine, is widely used for various gastrointestinal diseases. We have previously reported that TU‐100 suppresses CPT‐11‐induced bacterial translocation (BT) by maintaining the diversity of the microbiome. In this study, we show that TU‐100 modulates the immune response during BT by inducing PD‐1 expression in Peyer's patches.

Background: Patients with unresectable advanced hepatocellular carcinoma (HCC) are often treated with systemic therapies, and lenvatinib is currently used as one of the first-line therapies. However, the overall response rate from lenvatinib treatment is only 24.1%, and even the patients who initially respond to this treatment often subsequently acquire therapeutic resistance to this drug. Although the mechanism of lenvatinib resistance remains unclear, recent studies have suggested the involvement of epidermal growth factor receptor (EGFR) activation, indicating that a combination of lenvatinib and EGFR inhibitors might have potential clinical applications in HCC. Curcumin is one of the most widely studied natural phytochemicals as an anti-cancer compound. Many previous studies have revealed that it also functions as a potent tyrosine kinase inhibitor that targets EGFR. Accordingly, herein we hypothesized that the anti-EFGR potential of curcumin might help overcome lenvatinib resistance in HCC. Methods: We first developed two lenvatinib-resistant cell lines and performed a series of cell line experiments, including cell proliferation, invasion, sphere formation, and apoptosis assays, to determine the impact of curcumin in overcoming this resistance. We evaluated EGFR and its downstream signaling pathway to clarify its mechanistic role in lenvatinib resistance and its subsequent reversal mediated by curcumin. Results: Among five HCC cell lines, the most sensitive and relatively insensitive cells were Huh-7 (IC50: 2.0 µM) and PLC-PRF-5 (IC50: 50.0 µM), respectively. Accordingly, we successfully established lenvatinib-resistant cells, which had significantly higher IC50 concentrations; resistant Huh-7 (IC50: 50.0 µM) and resistant PLC-PRF-5 (IC50: >100.0 µM). We noted that co-administration of curcumin with lenvatinib inhibited cell proliferation and invasion compared to lenvatinib or curcumin monotherapy. Combination therapy also promoted apoptosis in resistant Huh-7 (lenvatinib, curcumin vs. combination (9.7%, 21.8%, vs. 30.2%) and resistant PLC-PRF-5 (12.2%, 23.1%, vs. 38.2%). This combination treatment also significantly diminished the spheroid-forming ability of cancer cells via suppression of cancer stemness markers such as CD44 (p Conclusions: We provide the first evidence that curcumin could overcome lenvatinib resistance in HCC cells via suppression of EGFR and its downstream targets, which can offer a novel therapeutic strategy in HCC. Citation Format: Katsuki Miyazaki, Yuji Morine, Caiming Xu, Mitsuo Shimada, Ajay Goel. Reversal of lenvatinib resistance by curcumin via EGFR signaling pathway in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1739.

Background: Cancer-tumor associated macrophage (TAM)-cancer associated fibroblast (CAF) interactions are an important factor in the tumor microenvironment of hepatocellular carcinoma. Materials and Methods: Hepatic stellate cells (HSCs) were cultured with cancer cell-conditioned medium (Ca.-CM), TAM-CM and CAF-CM, and the expression of CAF markers were evaluated by RT-PCR. Whether HSCs cultured with Ca.-CM, TAM-CM and CAF-CM contributed to the enhanced malignancy of cancer cells was examined using proliferation, invasion and migration assays. Furthermore, the differences between these three types of CM were evaluated using cytokine arrays. Results: HSCs cultured with Ca.-CM, TAM-CM and CAF-CM showed significantly increased mRNA expression of αSMA, FAP and IL-6. All HSCs cultured with each CM exhibited significantly increased proliferation, invasion and migration of cancer cells. The osteopontin concentration was significantly higher in HSCs cultured with TAM-CM than the other CAF-CMs. Osteopontin inhibition significantly reduced osteopontin secretion from HSCs cultured with TAM-CM and suppressed the proliferation and invasion of cancer cells enhanced by HSCs cultured with TAM-CM. Conclusions: We observed enhanced osteopontin secretion from TAMs, and this increased osteopontin further promoted osteopontin secretion from HSCs cultured with TAM-CM, leading to increased malignancy. For the first time, we demonstrated the importance of cancer-TAM-CAF interactions via osteopontin in hepatocellular carcinoma.

Background : The C-reactive protein (CRP)-albumin ratio (CAR) was reported as a prognostic factor of resectable hepatocellular carcinoma. The aim of this study was to analyse the significance of CAR in resectable pancreatic cancer. Patients and Methods : 163 patients with curative resection for pancreatic cancer were enrolled in this retrospective study. Cases of non-curative resection were excluded. The CAR was calculated with the preoperative plasma CRP and albumin values, with a cut-off value of 0.06, as calculated in a previous report. Results : Patients in the low CAR group had significantly better overall survival (OS) and disease-free survival (DFS) compared with the high CAR group (P 0.05). On multivariate analysis, for high CAR, CA19-9 300 U / ml and receipt of adjuvant chemotherapy were independent risk factors for OS and DFS. High CAR was significantly associated with advanced T stage. Conclusion : The CAR might be a prognostic factor for patients with resectable pancreatic cancer. J. Med. Invest. 68 : 244-248, August, 2021.

Peripheral nerve injury (PNI) after pelvic surgery is a common issue with a significant impact on patients. Autologous nerve grafting is the gold standard treatment for PNI, but this technique cannot be applied to fine nerve fibers in the pelvis. Schwann-like cell (SLC) differentiation is a novel therapeutic strategy for this clinical condition. However, the efficiency of SLC differentiation remains unsatisfactory. We modified an SLC differentiation protocol using adipose-derived stem cells (ADSCs) and folic acid. Morphology, gene expression and secretion of neurotrophic factors were examined to assess the differentiation quality and phenotypic characteristics. Our new modified protocol effectively induced a Schwann cell (SC) phenotype in ADSCs as assessed by morphology and expression of SC markers [S100 calcium-binding protein B (S100B), P < 0.01 ; p75 neurotrophic receptor (p75NTR), P < 0.05]. SLCs produced by the new protocol displayed a repair phenotype with decreased expression of ERBB2 and early growth response protein 2 (EGR2) / KROX20 (P < 0.01). Furthermore, our new protocol enhanced both mRNA expression and secretion of nerve growth factors by SLCs (P < 0.01). This protocol enhanced the SC characteristics and functions of ADSC-derived SLCs. This promising protocol requires further research and may contribute to SC-based nerve regeneration. J. Med. Invest. 68 : 347-353, August, 2021.

Aim Diffusion‐weighted magnetic resonance imaging (DWI‐MRI) is used to predict tumor malignancy. Here we explored the role of apparent diffusion coefficient (ADC) values in the treatment of patients with resectable colorectal liver metastasis (CRLM). Methods Magnetic resonance imaging (MRI) scans were conducted using a Signa HDe or Signa Explorer 1.5‐T scanner (GE Healthcare). ADC maps were calculated using DWI with b values of 0, 20, and 800 s/mm2. We enrolled 60 patients who underwent upfront hepatic resection for CRLM and divided them into ADC‐high (n = 30) and ADC‐low (n = 30) groups. Clinicopathological variables of the groups were compared. Immunohistochemical analysis of HIF‐1α expression in tumor tissues was performed, and the relationship between the ADC value and HIF‐1α expression was evaluated. Results The disease‐free survival rate of the ADC‐low group was significantly lower than that of the ADC‐high group (P, Low ADC values serve as an independent prognostic factor and correlate with HIF‐1α expression in CRLM.

Aim Pancreatectomy causes both hyperglycemia, secondary to surgical stress, and pancreatic diabetes, which leads to difficult‐to‐control postoperative blood glucose levels. We investigated whether using an artificial pancreas perioperatively to provide appropriate blood glucose control could reduce postoperative complications following pancreatectomy. Methods We retrospectively enrolled 52 patients who underwent pancreatectomy at Tokushima University Hospital from 2015 to 2019. The most recent 26/52 patients received perioperative blood glucose control using an artificial pancreas. Postoperative blood glucose control with manual insulin injections based on a sliding scale was performed in the earlier 26 patients (controls). We compared surgical outcomes between the artificial pancreas group and the control group. Results There was no significant difference in patients' white blood cell or neutrophil counts, prognostic nutritional index, neutrophil‐lymphocyte ratio, and C‐reactive protein‐to‐albumin ratio on postoperative day 1; however, lymphocyte counts were higher in the artificial pancreas group. The number of serious complications of Clavien‐Dindo grade >IIIa was significantly lower in the artificial pancreas group (P, Using an artificial pancreas for perioperative blood glucose control in patients undergoing pancreatectomy decreased the number of serious complications through proper management of blood glucose levels without hypoglycemia, and may influence peripheral lymphocytes.

BackgroundCeliac axis compression syndrome (CACS) is a relatively rare disease. Because of the nature of the blood flow in the celiac region when a pancreatoduodenectomy (PD) is performed for CACS, the celiac region can become ischemic. The aim of this study is to report on the importance of pre-operative diagnosis of CACS in terms of the outcomes for patients post-operatively. In this study, three 3 cases of PD co-morbid with CACS are reported: one intra-operative diagnosis case and two pre-operative diagnosis cases.Case presentationThe one case, not diagnosed with CACS prior to the operation, had a hard post-operative course because of complication caused by ischemia of the celiac region compared with the two cases diagnosed prior to the operation, who had a good post-operative course because of pre-operative or intra-operative intervention.ConclusionsPost-operative complications due to CACS are preventable by pre-operative diagnosis and appropriate interventions.

BACKGROUND We evaluated apparent diffusion coefficient (ADC) of diffusion-weighted image MRI as a prognostic factor for mass-forming intrahepatic cholangiocarcinoma (IHCC). METHODS We enrolled 26 patients who had undergone hepatic resections for mass-forming-type IHCC in this study, and calculated their mean ADC, using diffusion-weighted image MRI (b: 0, 20, 800 seconds/mm2 ; 1.5 T MRI). Patients were divided into the ADCHigh and the ADCLow groups at the median ADC value (n = 13 for both). We also immunohistochemically evaluated hypoxia-inducible factor (HIF)-1α in tumor tissue. RESULTS Median age in the ADCLow was older (P = .03), and showed significant higher rate of scirrhous tumor (P = .02). The 5-year overall survival rate in the ADCLow group was significantly worse than in the ADCHigh group (P = .04). In multivariate analysis, hilar tumor, portal vein invasion and low ADC were independent prognostic factors (P

Peripheral nerve injury (PNI) after pelvic surgery is a common issue with a significant impact on patients. Autologous nerve grafting is the gold standard treatment for PNI, but this technique cannot be applied to fine nerve fibers in the pelvis. Schwann-like cell (SLC) differentiation is a novel therapeutic strategy for this clinical condition. However, the efficiency of SLC differentiation remains unsatisfactory. We modified an SLC differentiation protocol using adipose-derived stem cells (ADSCs) and folic acid. Morphology, gene expression and secretion of neurotrophic factors were examined to assess the differentiation quality and phenotypic characteristics. Our new modified protocol effectively induced a Schwann cell (SC) phenotype in ADSCs as assessed by morphology and expression of SC markers [S100 calcium-binding protein B (S100B), P 0.01 ; p75 neurotrophic receptor (p75NTR), P 0.05]. SLCs produced by the new protocol displayed a repair phenotype with decreased expression of ERBB2 and early growth response protein 2 (EGR2) / KROX20 (P 0.01). Furthermore, our new protocol enhanced both mRNA expression and secretion of nerve growth factors by SLCs (P 0.01). This protocol enhanced the SC characteristics and functions of ADSC-derived SLCs. This promising protocol requires further research and may contribute to SC-based nerve regeneration. J. Med. Invest. 68 : 347-353, August, 2021.

Generation of hepatocytes from human adipose-derived mesenchymal stem cells (hADSCs) could be a promising alternative source of human hepatocytes. However, mechanisms to differentiate hepatocytes from hADSCs are not fully elucidated. In this study, we investigated the role of nuclear factor erythroid-2 related factor 2 (Nrf2) in differentiation of hepatocyte-like cells (HLCs). We used our established three-step differentiation protocol to develop HLCs from hADSCs. Significant nuclear translocation of Nrf2 occurred from day 11 (Step 2) until the end of HLC differentiation. There were no significant differences in Nrf2 translocation rates among the four experimental groups (activin-A, GSK3 inhibitor, Nrf2 siRNA, and control) at day 6 (end of Step 1). Nuclear translocation of Nrf2 in the GSK3 inhibitor-treated group was obviously higher than the other groups at day 11 (Step 2). Moreover, nuclear translocation of Nrf2 in the GSK3 inhibitor-treated group was notably higher than the other groups during Step 3. CYP3A4 activity (Luciferin-IPA assay) of the GSK3 inhibitor-treated group was significantly higher than the other three groups. Nrf2 was activated during differentiation of HLCs, and inhibition of Nrf2 delayed maturation and impaired the function of HLCs. Thus, Nrf2 might be a notable target for developing highly functional human HLCs.

The tumor microenvironment affects malignancy in hepatocellular carcinoma (HCC) cells, and cancer‐associated fibroblasts (CAFs) play an important role in the microenvironment. As recent studies indicated a difference between CAFs isolated from chemoresistant and non‐resistant cancer tissues, therefore we investigated the intracellular mechanism in resistant HCC co‐cultured CAFs and interactions between these CAFs with cancer cells. We established a sorafenib‐resistant (SR) Huh7 (human HCC) cell line, and characterized it with cytokine assays, then developed CAFs by co‐culturing human hepatic stellate cells with resistant or parental Huh7 cells. The 2 types of CAFs were co‐cultured with parental Huh7 cells, thereafter the cell viability of these Huh7 cells was checked under sorafenib treatment. The SR Huh7 (Huh7SR) cells expressed increased B‐cell activating factor (BAFF), which promoted high expression of CAF‐specific markers in Huh7SR‐co‐cultured CAFs, showed activated BAFF, BAFF‐R, and downstream of the NFκB‐Nrf2 pathway, and aggravated invasion, migration, and drug resistance in co‐cultured Huh7 cells. When we knocked down BAFF expression in Huh7SR cells, the previously increased malignancy and BAFF/NFκB axis in Huh7SR‐co‐cultured CAFs reversed, and enhanced chemoresistance in co‐cultured Huh7 cells returned as well. In conclusion, the BAFF/NFκB pathway was activated in CAFs co‐cultured with cell‐culture medium from resistant Huh7, which promoted chemoresistance, and increased the malignancy in co‐cultured non‐resistant Huh7 cells. This suggests that the BAFF/NFκB axis in CAFs might be a potential therapeutic target in chemoresistance of HCC., In this study, we established a sorafenib‐resistant (SR) Huh7 (human hepatocellular carcinoma [HCC]) cell line, and developed cancer‐associated fibroblasts (CAFs) induced from resistant or parental Huh7 cells. We found that the B‐cell activating factor (BAFF)‐NFκB pathway was activated in sorafenib‐resistant Huh7‐co‐cultured CAFs, which promoted chemoresistance and increased malignancy in co‐cultured non‐resistant Huh7 cells. This suggests that the BAFF‐NFκB axis plays a crucial role between the interaction of SR HCC and CAFs, and it might be a potential therapeutic target in CAFs that caused chemoresistance of HCC.

Background Frailty is an important consideration for older patients undergoing surgery. We aimed to investigate whether frailty could be a prognostic factor in patients with pancreatic ductal adenocarcinoma who underwent pancreatic resection. Methods One hundred and twenty patients who underwent pancreatic resection for pancreatic ductal adenocarcinoma were enrolled. Frailty was defined as a clinical frailty scale score ≥4. Patients were divided into frailty (n = 29) and non-frailty (n=91) groups, and clinicopathological factors were compared between the two groups. Results The frailty group showed an older age, lower serum albumin concentration, lower prognostic nutritional index, larger tumor diameter, and higher rate of lymph node metastasis than the non-frailty group (p < 0.05). Neutrophil–lymphocyte ratio and modified Glasgow prognostic score tended to be higher in the frailty group. Cancer-specific and disease-free survival rates were significantly poor in the frailty group (p < 0.05). With a multivariate analysis, frailty was an independent prognostic factor of cancer-specific survival. Conclusions Frailty can predict the prognosis of patients with pancreatic ductal adenocarcinoma who undergo pancreatic resection.

To clarify whether the preoperative lymphocyte/C-reactive protein (CRP) ratio (LCR) is a prognostic factor for patients with intrahepatic cholangiocarcinoma (IHCC), and investigate its mechanism via tumor-infiltrating lymphocytes.The subjects of this retrospective study were 42 patients who had undergone hepatectomy for IHCC. We divided the patients into low LCR and high LCR groups (cutoff value: 8780) and analyzed their overall survival (OS) and disease-free survival (DFS) with respect to LCR and other clinicopathological factors. We also investigated the levels of stromal tumor-infiltrating lymphocytes (TILs) and CD8A low LCR was identified in 21 patients and was significantly correlated with older age, a high CRP-albumin ratio, and advanced disease stage, and was a prognostic factor for OS and DFS. Multivariate analysis revealed that a low LCR was an independent prognostic factor for worse OS (HR 10.40, P = 0.0077). Although the LCR and levels of stromal TILs were not significantly related, LCR and levels of CD8The preoperative LCR may predict the postsurgical prognosis of patients with IHCC and reflect the CD8

Background Lymphocyte/C-reactive protein (CRP) ratio (LCR), is a promising prognostic factor that can reflect tumor inflammation and systemic patient condition. The aim of this study was to investigate whether preoperative LCR can be a prognostic factor for post-surgical outcomes among patients with intrahepatic cholangiocarcinoma (IHCC). We also investigated the relationship between LCR and tumor infiltrating lymphocytes (TILs) to clarify whether systemic host immune parameters reflect local tumor immunity.Methods We enrolled 45 patients who had undergone hepatectomy for IHCC. Patients were divided into low LCR and high LCR groups, according to reactive operating characteristic curve (Cut-off value: 8780). We analyzed their overall survival (OS) and disease-free survival (DFS) with respect to LCR and other clinicopathological factors. We also investigated stromal TILs and numbers of CD8+ TILs in surgical specimens, and the relationship between LCR and TILs.Results Twenty-one patients (46.7%) were associated with low LCR. Low LCR was significantly correlated with older age, high CRP and advanced disease stage, and was a prognostic factor for OS and DFS. And multivariate analysis revealed that low LCR was an independent prognostic factor for worse OS (HR: 2.81 P+ TILs were significantly related. Conclusions Preoperative LCR levels could predict the post-surgical prognosis of patients with IHCC, and reflected numbers of intra-tumoral CD8+ TILs.Trial registration This study was approved by Tokushima University Hospital ethics committee and with the approval of corresponding regulatory agencies, and all the experiments were carried out in accordance with the approved guidelines (Tokushima Clinical Trial Management System Number; 3215). All the patients involved in this study signed informed consent forms and agreed to participate.

Background: No universal classification method for intrahepatic cholangiocarcinoma (IHCC) has been reported based on the embryological origin of biliary epithelial cells. The aim of this study was to classify IHCC according to protein expression levels of somatostatin receptor 2 (SSTR2) and b-cell leukemia/lymphoma 2 (Bcl2) and to elucidate the clinicopathological features of each group.Methods: Fifty-two IHCC patients who underwent hepatic resection were enrolled in this study. Protein expression levels of SSTR2 and Bcl2 were examined using immunohistochemistry. Clinicopathological factors were compared between the three groups and prognostic factors were investigated. Results: The patients were divided into three groups: SSTR2 positive and Bcl2 negative (Group H, n=21), SSTR2 negative and Bcl2 positive (Group P, n=14), and the indeterminate group (Group U, n=17) for cases where SSTR2 and Bcl2 were both positive or both negative. All Group P cases displayed curability A or B. The 5-year survival rates of Group H and U patients were worse than those in Group P. Group H had higher T-factor, clinical stage, and incidence of periductal infiltration than Group P.Conclusions:This method could be used to classify IHCC into peripheral and perihilar type by embryological expression patterns of SSTR2 and Bcl2.

OBJECTIVES We aimed to determine whether responsive insulin-producing cells (IPCs) could be generated from adipose-derived stem cells (ADSCs) isolated from patients with type 1 diabetes mellitus (T1DM). METHODS We isolated ADSCs from adipose tissue of 4 patients (one patient with T1DM and 3 nondiabetic patients), who underwent surgery and differentiated them into IPCs with using a 2-step xeno-antigen free, 3-dimensional culture method. Characteristics of isolated ADSCs, in vitro cell quality, programmed cell death ligand-1 (PDL-1) expression, and transplantation into streptozotocin induced diabetic nude mice were investigated. RESULTS Adipose-derived stem cells from T1DM patients and commercially obtained ADSCs showed the same surface markers; CD31CD34CD45CD90CD105CD146. Moreover, the generated IPCs at day 21 demonstrated appropriate autonomous insulin secretion (stimulation index, 3.5; standard deviation, 0.8). Nonfasting blood glucose concentrations of IPC-transplanted mice were normal at 30 days. The normalized rate of IPC-transplanted mice was significantly higher than that of the sham-operated group (P < 0.05). Insulin-producing cells generated from T1DM adipose tissue expressed high levels of PDL-1. CONCLUSIONS Insulin-producing cells obtained from adipose tissue of T1DM patients are capable of secreting insulin long-term and achieve normoglycemia after transplantation. Expression of PDL-1 suggests the potential for immune circumvention.

The aim of our study is to determine whether insulin-producing cells (IPCs) differentiated from adipose-tissue-derived stem cells (ADSCs) can be cryopreserved. Human ADSCs were differentiated into IPCs using our two-step protocol encompassing a three-dimensional culture and xenoantigen-free method. Thereafter, IPCs were frozen using three different methods. First, IPCs were immediately frozen at −80°C (−80°C group). Second, IPCs were initially placed into a Bicell freezing container before freezing at −80°C (BICELL group). Third, a vitrification method for oocytes and embryos was used (CRYOTOP group). Cell counting kit-8 (CCK-8) assay showed that cell viability was decreased in all groups after cryopreservation ( P < 0.01). Corroboratively, the amount of adenosine triphosphate was markedly decreased after cryopreservation in all groups ( P < 0.01). Immunofluorescence staining showed a reduced positive staining area for insulin in all cryopreservation groups. Furthermore, 4′,6-diamidino-2-phenylindole and merged immunofluorescence images showed that cryopreserved cells appeared to be randomly reduced in the −80°C group and CRYOTOP group, while only the central region was visibly reduced in the BICELL group. Using immunohistochemical staining, IPCs after cryopreservation were shown to be positive for cleaved caspase-3 antibody in all groups. Finally, insulin secretion following glucose stimulation was significantly reduced in IPCs from all groups after cryopreservation ( P < 0.01). In conclusion, IPCs may be too fragile for cryopreservation with accomplished methods and further investigations for a suitable preservation method are required.

Intrahepatic cholangiocarcinoma (IHCC) has a poor prognosis, and surgery remains the only effective treatment. However, tumor recurrence after primary hepatectomy is common. We examined the significance of repeat surgery for IHCC. We collected data for all patients with IHCC between 1992 and 2018 (n = 67) in our database. Fifty-three (79.1%) of all 67 patients experienced recurrence after primary hepatectomy and we analyzed data for those 53 recurrent patients. We divided recurrent patients into a repeat surgery group (n = 9), chemotherapy group (n = 19), and best supportive care group (n = 25). We analyzed differences in patients’ clinicopathological factors, including prognosis, between the three groups. The IHCC recurrence rate after hepatectomy in our institution was 79.1%. Of the 53 patients with recurrence, nine underwent repeat surgery (17.0%). The characteristics of the patients undergoing repeat surgery was lower stage at primary hepatectomy. Recurrence sites in the repeat surgery group were liver (n = 6), lung (n = 2), and adrenal gland (n = 1), as a single nodule. The period between primary hepatectomy and recurrence was comparatively longer in the repeat surgery group, at 1.8 years. The prognosis in patients undergoing repeat surgery was significantly better compared with the other groups. Not undergoing repeat surgery (hazard ratio: 5.506; p = 0.0077) and positive lymph node metastasis (hazard ratio: 2.207; p = 0.0242) were independent poor prognostic factors. Repeat surgery should be considered in patients with IHCC with a single recurrence site and negative lymph node metastasis at primary surgery and at least more than 6 months of disease-free period after primary surgery.

Background and aim As a multiple tyrosine kinase inhibitor, sorafenib is widely used to treat hepatocellular carcinoma (HCC), but patients frequently face resistance problems. Because the mechanism controlling sorafenib-resistance is not well understood, this study focused on the connection between tumor characteristics and the Nrf2 signaling pathway in a sorafenib-resistant HCC cell line. Methods A sorafenib-resistant HCC cell line (Huh7) was developed by increasing the dose of sorafenib in the culture medium until the target concentration was reached. Cell morphology, migration/invasion rates, and expression of stemness-related and ATP-binding cassette (ABC) transporter genes were compared between sorafenib-resistant Huh7 cells and parental Huh7 cells. Next, a small interfering RNA was used to knock down Nrf2 expression in sorafenib-resistant Huh7 cells, after which cell viability, stemness, migration, and ABC transporter gene expression were examined again. Results Proliferation, migration, and invasion rates of sorafenib-resistant Huh7 cells were significantly increased relative to the parental cells with or without sorafenib added to the medium. The expression levels of stemness markers and ABC transporter genes were up-regulated in sorafenib-resistant cells. After Nrf2 was knocked down in sorafenib-resistant cells, cell migration and invasion rates were reduced, and expression levels of stemness markers and ABC transporter genes were reduced. Conclusion Nrf2 signaling promotes cancer stemness, migration, and expression of ABC transporter genes in sorafenib-resistant HCC cells.

Background & Aim We have reported the effective 3D-culture system to differentiate insulin producing cell (IPC) from human adipose derived stem cell (hADSC) recombinant peptide micro-pieces (RCP μ-pieces, FUJIFILM, Tokyo, Japan) (Ikemoto T, et al. Pancreas. 2018, Ikemoto T, et al. Sci Rep 2019, Wada Y, et al. Sci Rep 2019). Although a successful short-term result was confirmed within 30 days with a single administration of IPCs, a long-term fate of IPCs remains unclear. Thus, the aim of this study is to clarify the long-term result of transplanted IPCs in mice. Methods, Results & Conclusion Methods IPCs were generated from hADSCs, which were isolated from patient's subcutaneous adipose, with our original 2-steop protocol which was a xeno-antien free, 3D-culture system as mentioned above (Pancreas. 2018, Sci Rep 2019). BALB/c nu-nu mice were induced to a diabetic state with 200mg/kg streptozotocin for the recipients. 2.0 × 106 IPCs were implanted to these mice under the kidney capsule (Tx group, n=4), same quantity normal saline were administered as control (Sham group, n=4). Non-fasting blood glucose and body weight were measured up to 180 days. Then transplanted mice were sacrificed for immunohistochemical evaluations. Results In Tx group, blood glucose levels in all recipients decreased below 200 mg/dl on day 10 and below 150 mg/dl around day 60 (100%), even though all mice in Sham group were dead in 40 days (100%). The normoglycemic state in Tx group continued until 180 days with IPCs single transplantation (Fig 1). No abnormal tumorous lesions or inflammations were observed at the transplanted sites. Transplanted IPCs were strongly stained by anti-insulin antibody, anti-C peptide antibody and anti-human leukocyte antigen class I antibody (Fig 2). Conclusions Our IPCs can be applied to clinical trial as a promising strategy for type 1 DM patients and totally pancreatectomized patients.

Background & Aim We have reported the possibility of non-clinical PoC for generating insulin-producing cells (IPCs) differentiated from adipose-derived stem cells (ADSCs). However, whether ADSC isolated from type 1 diabetes mellitus (T1DM) patients has the differentiation ability or not is still unclear. Thus, we aimed to determine whether appropriately responsive IPCs could be generated from ADSCs isolated from actual T1DM patients. Methods, Results & Conclusion Methods We isolated ADSCs from the discarded adipose tissue from four patients undergoing surgery at our institute and differentiated them into IPCs using our established two-step xeno-antigen free, three-dimensional culture method (UMIN000035546,IRB approved on Jan 10, 2018). Characters of isolated ADSCs, in vitro cell quality and cell function, programmed cell death ligand-1(PDL-1) expression and in vivo functional tests by transplantation to streptozotocin (STZ) induced diabetic nude mice were investigated. Results ADSCs from T1DM patients and commercially obtained ADSCs showed the same surface markers; CD31−CD34−CD45−CD90+CD105+CD146−. Moreover, the generated IPCs at day 21 demonstrated appropriate autonomous insulin secretion in vitro (stimulation index;3.5). The non-fasting blood glucose concentrations of STZ-induced diabetic nude mice were normal 30 days after IPC transplantation. The normalized rate of STZ-induced diabetic nude mice was significantly different to the sham-operated group (P Conclusions These findings suggest that IPCs obtained from the adipose tissue of T1DM patients are capable of secreting insulin long-term and achieving normoglycemia following a single auto-transplantation, and without the need for immunosuppression.

The concept of frailty becomes important for patients who undergo surgery in this recent aging society. The aim of this study is to investigate the frailty as a prognostic factor in elderly patients with hepatocellular carcinoma (HCC) who underwent hepatectomy. A total of 92 patients over 75 years old who underwent hepatectomy were enrolled in this study. Frailty was defined as clinical frailty scale (CFS) ≥ 4. Patients were divided into two groups, i.e., frailty group (n = 21) and no-frailty group (n = 71), and clinicopathological features were compared between them. The frailty group showed significant higher PIVKA-II level and larger tumor diameter (p

Background & Aim Background As a curative treatment for type 1 diabetes, we have established and reported a rapid and efficient protocol of insulin producing cell (IPC) differentiation from adipose derived stem cell (ADSC) which were isolated with a minimally invasive procurement (Wada Y, Ikemoto T, et al. Sci Rep. 2019). Experimental β cell transplantation is often performed under the kidney capsule in murine model, however this is not suitable for clinical application. Aim We examined the optimal transplantation site for clinical transplantation of IPCs. Methods, Results & Conclusion Methods ADSCs were differentiated into IPC using our developed xeno-antigen free, 3D cultured (scaffold; cellnest, provided by Fuji Film CO.,Ltd) and two-step protocol which we have reported (Ikemoto T, et al. Sci Rep. 2019). BALB/c nu-nu mice were induced to a diabetic state with 200mg/kg streptozotocin as the recipients. 2.0 × 106 generated IPCs were transplanted in the mesenteric space (n=4), under the kidney capsule (n=4), and subcutaneous space (n=2), respectively. Non-fasting blood glucose and body weight were measured and observed in every 3 days. Immunohistochemical evaluations were undergone after the transplanted mice were sacrificed sequentially. Results Under the kidney capsule group, blood glucose level decreased below 200mg/dl on day 10 and then gradually reached the normal range (4/4, 100%). In the mesenteric space group, blood glucose level approached the normal range more quickly and normalized below 150mg/dl around day 14 (4/4,100%). In both groups blood glucose level was normalized with single shot of IPC transplantation. As a preliminary data, the tendency of improving hyperglycemic state was observed in subcutaneous space group, however, that improvement did not reach the significance. Conclusion The optimal site for IPC transplantation was considered to be in the mesenteric space from our investigations. Simultaneously, we plan to research the usefulness of subcutaneous transplantation with administration of angiogenetic factor, for achieving less invasive transplantation of IPCs.