C-reactive Protein-to-Albumin Ratio: A Useful Predictor for Biliary Fistula After Hepatectomy.

Introduction Postoperative bile leakage (POBL) has emerged as a complication following hepatectomy. POBL is associated with an elevated risk of liver failure and surgical death. This study aimed to examine risk factors for POBL in primary hepatocellular carcinoma (HCC) patients. Methods A total of 296 patients who had surgical resection for a preoperative diagnosis of primary HCC from January 2013 to December 2022 at Ehime Prefectural Central Hospital were included in this study. The patients were categorized into two groups based on the presence of POBL. The preoperative, operative, and histopathological findings were analyzed between the two groups. Risk factors were determined using multivariable analysis. Results Regarding preoperative findings, statistically significant differences were observed in white blood cell count, platelet count, C-reactive protein (CRP) level, and CRP-to-Albumin ratio (CAR) between the two groups (p = 0.023, p = 0.025, p = 0.011, and p = 0.012, respectively). As for intraoperative variables, only operation time (p = 0.017) was statistically correlated with the risk of POBL. Regarding pathological variables, there were no statistically significant differences between the two groups. The optimal cut-off value of CAR, as determined by ROC curve analysis, was 0.053. This value had a sensitivity of 80.0% and a specificity of 72.8%. Multivariate logistic regression analysis indicated that CAR ≥ 0.053 (p = 0.030) and operation time ≥ 308 min (p = 0.023) were independent potential markers for POBL after hepatectomy. Conclusion A high CAR level can be an effective predictor for POBL following hepatectomy.

Propensity score-matched analysis of the short-term outcomes of robotic versus laparoscopic surgery for rectal cancer.

PURPOSE: The advantages of robot-assisted rectal surgery (RARS) over conventional laparoscope-assisted rectal surgery (LARS) remain controversial. This study was performed to compare the short-term outcomes of RARS and LARS. METHODS: We retrospectively analyzed data of 207 patients who had undergone either RARS (n = 97) or LARS (n = 110) for rectal cancer (RC) from 2018 to 2020. A 1:1 matched propensity score-matched analysis was performed and the surgical outcomes of the two groups compared. RESULTS: After matching, a well-balanced cohort of 136 patients was analyzed (n = 68 in each group), and there was no significant difference in the median operative time. The RARS group had less intraoperative blood loss than the LARS group. There were no significant differences in length of postoperative hospital stay or complication rate between the two groups. In the subgroup of lower RC, defined as the inferior edge of the tumor being within the rectum distal to the peritoneal reflection, the rate of sphincter preservation was higher in the RARS group (81.8% vs. 44.4%, p = 0.021). CONCLUSION: This study shows that RARS is a safe and feasible approach for RC compared with LARS, RARS having the advantage of more often preserving the sphincter.

Syngeneically transplanted insulin producing cells differentiated from adipose derived stem cells undergo delayed damage by autoimmune responses in NOD mice.

Insulin-producing cells (IPCs) generated by our established protocol have reached the non-clinical 'proof of concept' stage. Our strategy for their clinical application is the autotransplantation of IPCs into patients with type 1 diabetes mellitus (T1DM). In this context, the autoimmunity that characterized T1DM is important, rather than allorejection. We aimed to determine how these IPCs respond to T1DM autoimmunity. IPCs were generated from the subcutaneous fat tissue of non-obese diabetic (NOD) mice using our protocol. IPCs derived from NOD mice were transplanted under the kidney capsules of NOD mice at the onset of diabetes and the subsequent changes in blood glucose concentration were characterized. Blood glucose decreased within 30 days of transplantation, but increased again after 40-60 days in three of four recipient NOD mice. In tissue samples, the numbers of CD4+ and CD8+ T cells were significantly higher 60 days after transplantation than 30 days after transplantation. In conclusion, IPCs significantly ameliorate the diabetes of mice in the short term, but are damaged by autoimmunity in the longer term, as evidenced by local T cells accumulation. This study provides new insights into potential stem cell therapies for T1DM.

Defective endoplasmic reticulum stress response via X box-binding protein 1 is a major cause of poor liver regeneration after partial hepatectomy in mice with non-alcoholic steatohepatitis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Dysfunction of the ER stress response might be a cause of poor liver regeneration in NASH.

Inhibition of the VEGF signaling pathway attenuates tumor­associated macrophage activity in liver cancer.

Tumor­associated macrophage (TAMs) are paramount for tumor progression and immune tolerance in the tumor microenvironment of various types of cancer, including liver cancer. The aim of the present study was to investigate the effect of vascular endothelial growth factor (VEGF) inhibition on TAM polarization and function during their interactions with macrophages and liver cancer cells. TAMs were induced by culturing M0 macrophages with cancer cell­conditioned medium. TAMs cultured with cancer cell­conditioned medium and vascular endothelial growth factor (VEGF) inhibitor were defined as modified TAMs, and the expression levels of TAM­associated markers and VEGF receptor 2 were evaluated using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). The effects of TAMs and modified TAMs on cancer cell proliferation and migration were investigated using conditioned medium. Programmed death­ligand 1 (PD­L1) mRNA expression in modified TAMs and cancer cells cultured in modified TAM­conditioned medium (TAM­CM) for 48 h was examined using RT­qPCR. In order to investigate signaling pathways in macrophages, western blot analysis was performed. CD163 and CD206 and M2 macrophage marker expression was upregulated in TAMs and modified TAMs. Modified TAM­CM exhibited a decreased ability to promote cancer cell proliferation and migration in comparison with the use of TAM­CM. The VEGF concentration was significantly higher in the TAMs than in M0 macrophages; however, the modified TAMs displayed a significantly lower VEGF secretion than TAMs. PD­L1 expression was decreased in modified TAMs as compared with TAMs. Western blot analysis revealed that the Akt/mTOR signaling pathway was significantly suppressed in the modified TAMs compared with TAMs. It was observed that TAMs cultured in a VEGF­depleted environment displayed lower secretion levels of cytokines involved in tumor progression and a decreased immune tolerance­inducing ability. On the whole, the results of the present study suggested that VEGF inhibition in TAMs may be a potential therapeutic target for liver cancer.

Effective in vitro differentiation of adipose-derived stem cells into Schwann-like cells with folic acid supplementation.

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.

Stromal tumor-infiltrating lymphocytes level as a prognostic factor for resected intrahepatic cholangiocarcinoma and its prediction by apparent diffusion coefficient.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 < 0.05). In the multivariate analysis, marginal low TILs were an independent prognostic factor for both overall- and disease-free survival (P < 0.05), and marginal low TILs were significantly associated with lower minimum ADC values (P < 0.02). CONCLUSIONS: Stromal TILs, especially in the marginal area, might demonstrate prognostic impact in patients with IHCC. Moreover, the ADC values from MRI may predict TILs in IHCC tumor tissue.

Frailty Can Predict Prognosis After Hepatectomy in Patients With Colorectal Liver Metastasis.

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.

Nrf2 signaling promotes cancer stemness, migration, and expression of ABC transporter genes in sorafenib-resistant hepatocellular carcinoma cells.

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.

Effective three-dimensional culture of hepatocyte-like cells generated from human adipose-derived mesenchymal stem cells.

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.

Cancer­associated fibroblast­induced M2­polarized macrophages promote hepatocellular carcinoma progression via the plasminogen activator inhibitor­1 pathway.

Targeting the tumor stroma is an important strategy in cancer treatment. Cancer­associated fibroblasts (CAFs) and tumor­associated macrophages (TAMs) are two main components in the tumor microenvironment (TME) in hepatocellular carcinoma (HCC), which can promote tumor progression. Plasminogen activator inhibitor­1 (PAI­1) upregulation in HCC is predictive of unfavorable tumor behavior and prognosis. However, the crosstalk between cancer cells, TAMs and CAFs, and the functions of PAI­1 in HCC remain to be fully investigated. In the present study, macrophage polarization and key paracrine factors were assessed during their interactions with CAFs and cancer cells. Cell proliferation, wound healing and Transwell and Matrigel assays were used to investigate the malignant behavior of HCC cells in vitro. It was found that cancer cells and CAFs induced the M2 polarization of TAMs by upregulating the mRNA expression levels of CD163 and CD206, and downregulating IL­6 mRNA expression and secretion in the macrophages. Both TAMs derived from cancer cells and CAFs promoted HCC cell proliferation and invasion. Furthermore, PAI­1 expression was upregulated in TAMs after being stimulated with CAF­conditioned medium and promoted the malignant behavior of the HCC cells by mediating epithelial­mesenchymal transition. CAFs were the main producer of C­X­C motif chemokine ligand 12 (CXCL12) in the TME and CXCL12 contributed to the induction of PAI­1 secretion in TAMs. In conclusion, the results of the present study suggested that CAFs promoted the M2 polarization of macrophages and induced PAI­1 secretion via CXCL12. Furthermore, it was found that PAI­1 produced by the TAMs enhanced the malignant behavior of the HCC cells. Therefore, these factors may be targets for inhibiting the crosstalk between tumor cells, CAFs and TAMs.

The BAFF/NFκB axis is crucial to interactions between sorafenib-resistant HCC cells and cancer-associated fibroblasts.

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.

Preoperative lymphocyte/C-reactive protein ratio and its correlation with CD8+ tumor-infiltrating lymphocytes as a predictor of prognosis after resection of intrahepatic cholangiocarcinoma.

PURPOSE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: The preoperative LCR may predict the postsurgical prognosis of patients with IHCC and reflect the CD8+ TILs.

A new pathological classification of intrahepatic cholangiocarcinoma according to protein expression of SSTR2 and Bcl2.

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.

Prognostic prediction of resectable colorectal liver metastasis using the apparent diffusion coefficient from diffusion-weighted magnetic resonance imaging.

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 < .05). Univariate analysis revealed that tumor number (more than five), synchronous metastasis, and low ADC were prognostic factors. Multivariate analysis identified low ADC as an independent prognostic factor. Furthermore, the ADC-low group more frequently expressed high levels of HIF-1α than the ADC-high group. CONCLUSION: Low ADC values were an independent prognostic factor of resectable CRLM and correlated with HIF-1α expression.

The interaction between cancer associated fibroblasts and tumor associated macrophages via the osteopontin pathway in the tumor microenvironment of hepatocellular carcinoma.

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.

The inhibitory effect of TU-100 on hepatic stellate cell activation in the tumor microenvironment.

INTRODUCTION: The tumor microenvironment is involved in acquiring tumor malignancies of colorectal liver metastasis (CRLM). We have reported that TU-100 (Daikenchuto) suppresses hepatic stellate cell (HSC) activation in obstructive jaundice. In this study, we report new findings as the direct and indirect inhibitory effects of TU-100 on cancer cell growth through the suppression of HSC activation. MATERIALS AND METHODS: The HSCs (LX2) were cultured in colon cancer cells (HCT116 and HT29)-conditioned medium (CM) with or without TU-100 treatment (90, 270, 900 µg/ml). Activated HSCs (aHSCs) were detected by α-SMA and IL-6 mRNA expressions and cytokine arrays of HSC's culture supernatants. Cancer cell growth was analyzed for proliferation and migration ability, compared with TU-100 treatment. To investigate the direct anti-tumor effect of TU-100, cancer cells were cultured in the presence of aHSC-CM and TU-100 (90, 270, 900) or aHSC-CM alone, and assessed autophagosomes, conversion to LC3-II protein, and Beclin-1 mRNA expression. RESULTS: Colon cancer-CM significantly increased α-SMA and IL-6 mRNA expressions of aHSC. α-SMA and IL-6 mRNA expressions of aHSC, and IL-6 secretions from aHSCs were significantly decreased with TU-100 (270, 900) treatment, compared to colon cancer-CM alone. Compared with normal culture medium, aHSC-CM led to a significantly increased cell number and modified HSC-CM (TU-100; 270, 900) significantly suppressed cancer cell growth and migration. TU-100 (900) treatment induced autophagy and significantly promoted the autophagic cell death. CONCLUSIONS: TU-100 inhibited colon cancer cell malignant potential by both suppressing HSC activation and inducing directly autophagy of cancer cells.

Adipose Tissue From Type 1 Diabetes Mellitus Patients Can Be Used to Generate Insulin-Producing Cells.

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.

Effectiveness of repeat surgery for recurrence after primary hepatectomy in patients with intrahepatic cholangiocarcinoma.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The Fragility of Cryopreserved Insulin-producing Cells Differentiated from Adipose-tissue-derived Stem Cells.

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.