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BackgroundWe evaluated apparent diffusion coefficient (ADC) of diffusion‐weighted image MRI as a prognostic factor for mass‐forming intrahepatic cholangiocarcinoma (IHCC).MethodsWe 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.ResultsMedian 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 < .05). The ADCLow group also had a higher rate of high HIF‐1α expression than the ADCHigh group (P < .05). Representative case of ADCLow group showed rich stroma and high HIF‐1α expression.ConclusionsThe ADC values in MRIs can predict IHCC prognosis, and correlated with stromal density and HIF‐1α expression.

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 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.

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.

Tumor-associated 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 cell-conditioned medium. TAMs cultured with cancer cell-conditioned medium and vascular endothelial growth factor (VEGF) inhibitor were defined as modified TAMs, and the expression levels of TAM-associated markers and VEGF receptor 2 were evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The effects of TAMs and modified TAMs on cancer cell proliferation and migration were investigated using conditioned medium. Programmed death-ligand 1 (PD-L1) mRNA expression in modified TAMs and cancer cells cultured in modified TAM-conditioned medium (TAM-CM) for 48 h was examined using RT-qPCR. 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 TAM-CM exhibited a decreased ability to promote cancer cell proliferation and migration in comparison with the use of TAM-CM. 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. PD-L1 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 VEGF-depleted environment displayed lower secretion levels of cytokines involved in tumor progression and a decreased immune tolerance-inducing 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.

Background:The aim of this study was to clarify the effectiveness of a new threedimensional (3D) culture system for hepatocyte-like cells (HLCs) generated from human adipose-derived mesenchymal stem cells (ADSCs). Methods: Human ADSCs (2 × 10 4 ) 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 threestep 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 3Dcultured HLCs, and, DNA microarray analysis was also performed. Finally, HLCs were transplanted in to CCl 4 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.

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 nonfrailty 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.Survival in patients with colorectal cancer (CRC) has improved over the last decades, primarily due to efforts in screening and early detection, and the recent advances in systemic and local therapies. However, the main cause of mortality in CRC patients is metastasis. The liver is the most common anatomic site for CRC metastasis (1). Approximately 35%-55% of CRC patients have liver metastases as the disease progresses (2). Surgical resection is currently the treatment of choice for CRC liver metastases, and has been proven as the only curative treatment (3, 4).Due to aging, the number of elderly patients with colorectal liver metastasis (CRLM) will increase significantly in the near future. Age-related complications, such as cardiopulmonary complications, delirium, transfer to a rehabilitation facility, and dependency are major problems after hepatectomy in the elderly patients (5). Given the increasing number of elderly patients with comorbidities undergoing surgery, there is increased interest in preoperatively identifying patients who are at high risk of morbidity and mortality after hepatectomy (6). Frailty is a multidimensional and heterogeneous syndrome associated with instability that differs from disability or aging alone (7). Frailty is commonly measured using summative impairment lists and algorithms based on clinical assessment (8-10). Frailty is very important in predicting surgical outcomes in elderly patients (10). Recently, McIsaac et al. suggested that the clinical frailty scale (CFS) could be used to predict new disability or death after elective noncardiac surgery (11). The CFS is a nine-point global frailty scale based on a clinical evaluation in the domains of mobility, energy, physical activity, and function (12,13). The CFS is a highly acceptable, feasible, and convenient instrument for clinical perioperative use.There have been several reports on the relationship between hepatectomy and frailty (5,6,(14)(15)(16)(17)(18). Louwers et al. (14) reported th...