Neural-net-based cell deconvolution from DNA methylation reveals tumor microenvironment associated with cancer prognosis.

DNA methylation is a pivotal epigenetic modification that defines cellular identity. While cell deconvolution utilizing this information is considered useful for clinical practice, current methods for deconvolution are limited in their accuracy and resolution. In this study, we collected DNA methylation data from 945 human samples derived from various tissues and tumor-infiltrating immune cells and trained a neural network model with them. The model, termed MEnet, predicted abundance of cell population together with the detailed immune cell status from bulk DNA methylation data, and showed consistency to those of flow cytometry and histochemistry. MEnet was superior to the existing methods in the accuracy, speed, and detectable cell diversity, and could be applicable for peripheral blood, tumors, cell-free DNA, and formalin-fixed paraffin-embedded sections. Furthermore, by applying MEnet to 72 intrahepatic cholangiocarcinoma samples, we identified immune cell profiles associated with cancer prognosis. We believe that cell deconvolution by MEnet has the potential for use in clinical settings.

High expression of autotaxin is associated with poor recurrence-free survival in cholangiocarcinoma.

BACKGROUND AND AIM: Autotaxin (ATX) is an extracellular lysophospholipase D that catalyzes the hydrolysis of lysophosphatidylcholine into lysophosphatidic acid (LPA). Recent accumulating evidence indicates the biological roles of ATX in malignant tumors. However, the expression and clinical implications of ATX in human cholangiocarcinoma (CCA) remain elusive. METHODS: In this study, the expression of ATX in 97 human CCA tissues was evaluated by immunohistochemistry. Serum ATX levels were determined in CCA patients (n = 26) and healthy subjects (n = 8). Autotaxin expression in cell types within the tumor microenvironment was characterized by immunofluorescence staining. RESULTS: High ATX expression in CCA tissue was significantly associated with a higher frequency of lymph node metastasis (p = 0.050). High ATX expression was correlated with shorter overall survival (p = 0.032) and recurrence-free survival (RFS) (p = 0.001) than low ATX expression. In multivariate Cox analysis, high ATX expression (p = 0.019) was an independent factor for shorter RFS. Compared with low ATX expression, high ATX expression was significantly associated with higher Ki-67-positive cell counts (p < 0.001). Serum ATX levels were significantly higher in male CCA patients than in healthy male subjects (p = 0.030). In the tumor microenvironment of CCA, ATX protein was predominantly expressed in tumor cells, cancer-associated fibroblasts, plasma cells, and biliary epithelial cells. CONCLUSIONS: Our study highlights the clinical evidence and independent prognostic value of ATX in human CCA.

Ferroptosis-related gene glutathione peroxidase 4 promotes reprogramming of glucose metabolism via Akt-mTOR axis in intrahepatic cholangiocarcinoma.

The role of the ferroptosis-related gene glutathione peroxidase 4 (GPX4) in oncology has been extensively investigated. However, the clinical implications of GPX4 in patients with intrahepatic cholangiocarcinoma (ICC) remain unknown. This study aimed to evaluate the prognostic impact of GPX4 and its underlying molecular mechanisms in patients with ICC. Fifty-seven patients who underwent surgical resection for ICC between 2010 and 2017 were retrospectively analyzed. Based on the immunohistochemistry, patients were divided into GPX4 high (n = 15) and low (n = 42) groups, and clinical outcomes were assessed. Furthermore, the roles of GPX4 in cell proliferation, migration and gene expression were analyzed in ICC cell lines in vitro and in vivo. The results from clinical study showed that GPX4 high group showed significant associations with high SUVmax on 18F-fluorodeoxyglucose-positron emission tomography (≥8.0, P = 0.017), multiple tumors (P = 0.004), and showed glucose transporter 1 (GLUT1) high expression with a trend toward significance (P = 0.053). Overall and recurrence-free survival in the GPX4 high expression group were significantly worse than those in the GPX4 low expression group (P = 0.038 and P < 0.001, respectively). In the experimental study, inhibition of GPX4 attenuated cell proliferation and migration in ICC cell lines. Inhibition of GPX4 also decreased the expression of glucose metabolism-related genes, such as GLUT1 or HIF1α. Mechanistically, these molecular changes are regulated in Akt-mechanistic targets of rapamycin axis. In conclusion, this study suggested the pivotal value of GPX4 serving as a prognostic marker for patients with ICC. Furthermore, GPX4 can mediate glucose metabolism of ICC.

Periportal hepatocyte proliferation at midgestation governs maternal glucose homeostasis in mice.

The maternal liver is challenged by metabolic demands throughout pregnancy. However, hepatocyte dynamics and their physiological significance in pregnancy remain unclear. Here, we show in mice that hepatocyte proliferation is spatiotemporally regulated in each liver lobular zone during pregnancy, with transient proliferation of periportal and pericentral hepatocytes during mid and late gestation, respectively. Using adeno-associated virus (AAV)-8-mediated expression of the cell cycle inhibitor p21 in hepatocytes, we show that inhibition of hepatocyte proliferation during mid, but not late, gestation impairs liver growth. Transcriptionally, genes involved in glucose/glycogen metabolism are downregulated in late pregnancy when midgestational hepatocyte proliferation is attenuated. In addition, hepatic glycogen storage is abolished, with concomitant elevated blood glucose concentrations, glucose intolerance, placental glycogen deposition, and fetal overgrowth. Laser capture microdissection and RNA-seq analysis of each liver lobular zone show zone-specific changes in the transcriptome during pregnancy and identify genes that are periportally expressed at midgestation, including the hyaluronan-mediated motility receptor (Hmmr). Knockdown of Hmmr in hepatocytes by AAV8-shHmmr suppresses periportal hepatocyte proliferation at midgestation and induces impaired hepatic glycogen storage, glucose intolerance, placental glycogen deposition and fetal overgrowth. Our results suggest that periportal hepatocyte proliferation during midgestation is critical for maternal glycogen metabolism and fetal size.

Clinically relevant model of oxaliplatin-induced sinusoidal obstruction syndrome.

AIM: Sinusoidal obstruction syndrome (SOS) induced by oxaliplatin-including chemotherapies (OXCx) is associated with impaired hepatic reserve and higher morbidity after hepatic resection. However, in the absence of an appropriate animal experimental model, little is known about its pathophysiology. This study aimed to establish a clinically relevant reproducible model of FOLFOX-induced SOS and to compare the clinical/histopathological features between the clinical and animal SOS settings. METHODS: We performed clinical/pathological analyses of colorectal liver metastasis (CRLM) patients who underwent hepatectomy with/without preoperative treatment of FOLFOX (n = 22/18). Male micro-minipigs were treated with 50% of the standard human dosage of the FOLFOX regimen. RESULTS: In contrast to the monocrotaline-induced SOS model in rats, hepatomegaly, ascites, congestion, and coagulative necrosis of hepatocytes were absent in patients with CRLM with OXCx pretreatment and OXCx-treated micro-minipigs. In parallel to CRLM cases with OXCx pretreatment, OXCx-challenged micro-minipigs exhibited deteriorated indocyanine green clearance, morphological alteration of liver sinusoidal endothelial cells, and upregulated matrix metalloproteinase-9. Using our novel porcine SOS model, we identified the hepatoprotective influence of recombinant human soluble thrombomodulin in OXCx-SOS. CONCLUSIONS: With distinct differences between monocrotaline-induced rat SOS and human/pig OXCx-SOS, our pig OXCx-SOS model serves as a preclinical platform for future investigations to dissect the pathophysiology of OXCx-SOS and seek preventive strategies.

Suppression of intrahepatic cholangiocarcinoma cell growth by SKI via upregulation of the CDK inhibitor p21.

Cholangiocarcinoma (CC) has a poor prognosis and different driver genes depending on the site of onset. Intrahepatic CC is the second-most common liver cancer after hepatocellular carcinoma, and novel therapeutic targets are urgently needed. The present study was conducted to identify novel therapeutic targets by exploring differentially regulated genes in human CC. MicroRNA (miRNA) and mRNA microarrays were performed using tissue and serum samples obtained from 24 surgically resected hepatobiliary tumor cases, including 10 CC cases. We conducted principal component analysis to identify differentially expressed miRNA, leading to the identification of miRNA-3648 as a differentially expressed miRNA. We used an in silico screening approach to identify its target mRNA, the tumor suppressor Sloan Kettering Institute (SKI). SKI protein expression was decreased in human CC cells overexpressing miRNA-3648, endogenous SKI protein expression was decreased in human CC tumor tissues, and endogenous SKI mRNA expression was suppressed in human CC cells characterized by rapid growth. SKI-overexpressing OZ cells (human intrahepatic CC cells) showed upregulation of cyclin-dependent kinase inhibitor p21 mRNA and protein expression and suppressed cell proliferation. Nuclear expression of CDT1 (chromatin licensing and DNA replication factor 1), which is required for the G1/S transition, was suppressed in SKI-overexpressing OZ cells. SKI knockdown resulted in the opposite effects. Transgenic p21-luciferase was activated in SKI-overexpressing OZ cells. These data indicate SKI involvement in p21 transcription and that SKI-p21 signaling causes cell cycle arrest in G1, suppressing intrahepatic CC cell growth. Therefore, SKI may be a potential therapeutic target for intrahepatic CC.

In situ vaccination using unique TLR9 ligand K3-SPG induces long-lasting systemic immune response and synergizes with systemic and local immunotherapy.

Although checkpoint inhibitors (CPIs) have changed the paradigm of cancer therapy, low response rates and serious systemic adverse events remain challenging. In situ vaccine (ISV), intratumoral injection of immunomodulators that stimulate innate immunity at the tumor site, allows for the development of vaccines in patients themselves. K3-SPG, a second-generation nanoparticulate Toll-like receptor 9 (TLR9) ligand consisting of K-type CpG oligodeoxynucleotide (ODN) wrapped with SPG (schizophyllan), integrates the best of conventional CpG ODNs, making it an ideal cancer immunotherapy adjuvant. Focusing on clinical feasibility for pancreaticobiliary and gastrointestinal cancers, we investigated the antitumor activity of K3-SPG-ISV in preclinical models of pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC). K3-SPG-ISV suppressed tumor growth more potently than K3-ISV or K3-SPG intravenous injections, prolonged survival, and enhanced the antitumor effect of CPIs. Notably, in PDAC model, K3-SPG-ISV alone induced systemic antitumor effect and immunological memory. ISV combination of K3-SPG and agonistic CD40 antibody further enhanced the antitumor effect. Our results imply that K3-SPG-based ISV can be applied as monotherapy or combined with CPIs to improve their response rate or, conversely, with CPI-free local immunotherapy to avoid CPI-related adverse events. In either strategy, the potency of K3-SPG-based ISV would provide the rationale for its clinical application to puncturable pancreaticobiliary and gastrointestinal malignancies.

Alteration of the immune environment in bone marrow from children with recurrent B cell precursor acute lymphoblastic leukemia.

Due to the considerable success of cancer immunotherapy for leukemia, the tumor immune environment has become a focus of intense research; however, there are few reports on the dynamics of the tumor immune environment in leukemia. Here, we analyzed the tumor immune environment in pediatric B cell precursor acute lymphoblastic leukemia by analyzing serial bone marrow samples from nine patients with primary and recurrent disease by mass cytometry using 39 immunophenotype markers, and transcriptome analysis. High-dimensional single-cell mass cytometry analysis elucidated a dynamic shift of T cells from naïve to effector subsets, and clarified that, during relapse, the tumor immune environment comprised a T helper 1-polarized immune profile, together with an increased number of effector regulatory T cells. These results were confirmed in a validation cohort using conventional flow cytometry. Furthermore, RNA transcriptome analysis identified the upregulation of immune-related pathways in B cell precursor acute lymphoblastic leukemia cells during relapse, suggesting interaction with the surrounding environment. In conclusion, a tumor immune environment characterized by a T helper 1-polarized immune profile, with an increased number of effector regulatory T cells, could contribute to the pathophysiology of recurrent B cell precursor acute lymphoblastic leukemia. This information could contribute to the development of effective immunotherapeutic approaches against B cell precursor acute lymphoblastic leukemia relapse.

Sinusoidal Obstruction Syndrome Promotes Liver Metastatic Seeding of Colorectal Cancer Cells in a Rat Model.

BACKGROUND/AIM: Sinusoidal obstruction syndrome (SOS) after neoadjuvant chemotherapy with oxaliplatin for colorectal liver metastases (CRLM) has been reported to lead to early recurrence. This study investigated the effects of SOS on the development of CRLM in a rat model. MATERIALS AND METHODS: RCN-H4 cells were injected into the spleen or liver of ten monocrotaline-treated (SOS group) and ten untreated (control group) rats. The number and size of liver tumors were compared between the groups. RESULTS: The number of liver tumors in the splenic RCN-H4 injection model was significantly higher in the SOS group than in the control group (332±213 vs. 16±5, p=0.029); however, the largest tumor diameter in the hepatic model was similar between groups (6.2±1.8 vs. 6.4±2.4 mm, p=0.87). CONCLUSION: SOS promotes CRLM development by splenic RCN-H4 cell injection. This might be due to the higher incidence of cancer cell implantation into the liver.

On-tissue polysulfide visualization by surface-enhanced Raman spectroscopy benefits patients with ovarian cancer to predict post-operative chemosensitivity.

Chemosensitivity to cisplatin derivatives varies among individual patients with intractable malignancies including ovarian cancer, while how to unlock the resistance remain unknown. Ovarian cancer tissues were collected the debulking surgery in discovery- (n = 135) and validation- (n = 47) cohorts, to be analyzed with high-throughput automated immunohistochemistry which identified cystathionine γ-lyase (CSE) as an independent marker distinguishing non-responders from responders to post-operative platinum-based chemotherapy. We aimed to identify CSE-derived metabolites responsible for chemoresistant mechanisms: gold-nanoparticle (AuN)-based surface-enhanced Raman spectroscopy (SERS) was used to enhance electromagnetic fields which enabled to visualize multiple sulfur-containing metabolites through detecting scattering light from Au-S vibration two-dimensionally. Clear cell carcinoma (CCC) who turned out less sensitive to cisplatin than serous adenocarcinoma was classified into two groups by the intensities of SERS intensities at 480 cm-1; patients with greater intensities displayed the shorter overall survival after the debulking surgery. The SERS signals were eliminated by topically applied monobromobimane that breaks sulfane-sulfur bonds of polysulfides to result in formation of sulfodibimane which was detected at 580 cm-1, manifesting the presence of polysulfides in cancer tissues. CCC-derived cancer cell lines in culture were resistant against cisplatin, but treatment with ambroxol, an expectorant degrading polysulfides, renders the cells CDDP-susceptible. Co-administration of ambroxol with cisplatin significantly suppressed growth of cancer xenografts in nude mice. Furthermore, polysulfides, but neither glutathione nor hypotaurine, attenuated cisplatin-induced disturbance of DNA supercoiling. Polysulfide detection by on-tissue SERS thus enables to predict prognosis of cisplatin-based chemotherapy. The current findings suggest polysulfide degradation as a stratagem unlocking cisplatin chemoresistance.

Gene expression profiles of liver cancer cell lines reveal two hepatocyte-like and fibroblast-like clusters.

Cancer cell lines are widely used in basic research to study cancer development, growth, invasion, or metastasis. They are also used for the development and screening of anticancer drugs. However, there are no clear criteria for choosing the most suitable cell lines among the wide variety of cancer cell lines commercially available for research, and the choice is often based on previously published reports. Here, we investigated the characteristics of liver cancer cell lines by analyzing the gene expression data available in the Cancer Cell Line Encyclopedia. Unsupervised clustering analysis of 28 liver cancer cell lines yielded two main clusters. One cluster showed a gene expression pattern similar to that of hepatocytes, and the other showed a pattern similar to that of fibroblasts. Analysis of hepatocellular carcinoma gene expression profiles available in The Cancer Genome Atlas showed that the gene expression patterns in most hepatoma tissues were similar to those in the hepatocyte-like cluster. With respect to liver cancer research, our findings may be useful for selecting an appropriate cell line for a specific study objective. Furthermore, our approach of utilizing a public database for comparing the properties of cell lines could be an attractive cell line selection strategy that can be applied to other fields of research.

Utility of Mac-2 Binding Protein Glycosylation Isomer to Evaluate Graft Status After Liver Transplantation.

Mac-2 binding protein glycosylation isomer (M2BPGi) is a novel liver fibrosis biomarker, but there are few studies on M2BPGi in liver transplantation (LT) recipients. This study aimed to evaluate the utility of M2BPGi measurement in LT recipients. We collected the clinicopathological data of 233 patients who underwent a liver biopsy at Kyoto University Hospital after LT between August 2015 and June 2019. The median values of M2BPGi in patients with METAVIR fibrosis stages F0, F1, F2, and ≥F3 were 0.61, 0.76, 1.16, and 1.47, respectively, whereas those in patients with METAVIR necroinflammatory indexes A0, A1, and ≥A2 were 0.53, 1.145, and 2.24, respectively. Spearman rank correlation test suggested that the necroinflammatory index had a stronger correlation to the M2BPGi value than the fibrosis stage. The area under the receiver operating characteristic curve of M2BPGi to predict ≥A1 was 0.75, which was significantly higher than that of any other liver fibrosis and inflammation marker. Patients with a rejection activity index (RAI) of ≥3 had a higher M2BPGi value than those with RAI ≤ 2 (P = 0.001). Patients with hepatitis C virus viremia had a higher M2BPGi value than sustained virological responders or those with other etiologies. In conclusion, the present study demonstrated that M2BPGi values are more strongly influenced by necroinflammatory activity and revealed M2BPGi, which has been thought to be a so-called fibrosis marker, as a disease activity marker in transplant recipients. M2BPGi measurement may be useful to detect early stage liver inflammation that cannot be detected by routine blood examination of LT recipients.

Extent of liver resection is associated with incomplete liver restoration and splenomegaly a long period after liver resection.

BACKGROUND: Little is known about the clinical significance and risk factors for incomplete liver restoration after partial hepatectomy, which is defined by a liver volume restoration of less than 100% of the original volume. METHODS: We retrospectively analyzed patients who underwent hepatic resection for liver tumors at the Kyoto University Hospital between January 2011 and October 2015 and survived without recurrence for more than 3 years. The preoperative and postoperative data, as well as liver and splenic volume after 3 postoperative years, were assessed. RESULTS: The percentage of resected liver was higher in the incomplete liver restoration group (n = 52, 41.6%) than in the complete liver restoration group (n = 73, 58.4%) (28 [3-78]% vs 14.5 [2-63]%, P = .0226). The percentage of resected liver was also higher in the splenomegaly group (defined by spleen volume increases of more than 35% of the original volume) than in the nonsplenomegaly group (40 [4-63]% vs 16.5 [2-78]%, P = .0002). Multivariate analysis demonstrated that the percentage of resected liver was a significant predictor of incomplete liver restoration (odds ratio = 9.75, P = .0043) and splenomegaly (odds ratio = 74.4, P = .0006). Incomplete liver restoration 3 years after hepatectomy was associated with lower serum albumin levels (4.0 [2.4-4.7] g/dL compared with 4.2 [2.6-4.8] g/dL in the complete liver restoration group, P = .0032). Splenomegaly was associated with a lower platelet count (109.9 ± 49.8 x103/µL vs 163 ± 58.1 × 103/µL,P = .0007) and lower serum albumin level (3.6 [2.6-4.4] g/dL vs 4.1 [2.4-4.8] g/dL, P = .0002). CONCLUSION: An extensive resection of the liver parenchyma results in an increased risk for incomplete liver restoration and splenomegaly long after hepatectomy, which is associated with the clinical consequences of hypoalbuminemia and thrombocytopenia.

Cytotoxic T Lymphocytes Regenerated from iPS Cells Have Therapeutic Efficacy in a Patient-Derived Xenograft Solid Tumor Model.

Current adoptive T cell therapies conducted in an autologous setting are costly, time consuming, and depend on the quality of the patient's T cells. To address these issues, we developed a strategy in which cytotoxic T lymphocytes (CTLs) are regenerated from iPSCs that were originally derived from T cells and succeeded in regenerating CTLs specific for the WT1 antigen, which exhibited therapeutic efficacy in a xenograft model of leukemia. In this study, we extended our strategy to solid tumors. The regenerated WT1-specific CTLs had a strong therapeutic effect in orthotopic xenograft model using a renal cell carcinoma (RCC) cell line. To make our method more generally applicable, we developed an allogeneic approach by transducing HLA-haplotype homozygous iPSCs with WT1-specific TCR α/ß genes that had been tested clinically. The regenerated CTLs antigen-specifically suppressed tumor growth in a patient-derived xenograft model of RCC, demonstrating the feasibility of our strategy against solid tumors.

Tyrosine kinase inhibitor imatinib augments tumor immunity by depleting effector regulatory T cells.

This report addresses whether small molecules can deplete FoxP3-expressing regulatory T (T reg) cells, thereby augmenting antitumor immunity. Imatinib, a tyrosine kinase inhibitor of oncogenic BCR-ABL protein expressed by chronic myelogenous leukemia (CML) cells, possesses off-targets including LCK expressed in T cells. We showed that imatinib-treated CML patients in complete molecular remission (CMR) exhibited selective depletion of effector T reg (eT reg) cells and significant increase in effector/memory CD8+ T cells while non-CMR patients did not. Imatinib at CML-therapeutic concentrations indeed induced apoptosis specifically in eT reg cells and expanded tumor antigen-specific CD8+ T cells in vitro in healthy individuals and melanoma patients, and suppressed colon tumor growth in vivo in mice. Mechanistically, because of FoxP3-dependent much lower expression of LCK and ZAP-70 in T reg cells compared with other T cells, imatinib inhibition of LCK further reduced their TCR signal intensity, rendering them selectively susceptible to signal-deprived apoptotis. Taken together, eT reg cell depletion by imatinib is instrumental in evoking effective immune responses to various cancers.

High-resolution imaging mass spectrometry combined with transcriptomic analysis identified a link between fatty acid composition of phosphatidylinositols and the immune checkpoint pathway at the primary tumour site of breast cancer.

BACKGROUND: The fatty acid (FA) composition of phosphatidylinositols (PIs) is tightly regulated in mammalian tissue since its disruption impairs normal cellular functions. We previously found its significant alteration in breast cancer by using matrix-assisted laser desorption and ionisation imaging mass spectrometry (MALDI-IMS). METHODS: We visualised the histological distribution of PIs containing different FAs in 65 primary breast cancer tissues using MALDI-IMS and investigated its association with clinicopathological features and gene expression profiles. RESULTS: Normal ductal cells (n = 7) predominantly accumulated a PI containing polyunsaturated FA (PI-PUFA), PI(18:0/20:4). PI(18:0/20:4) was replaced by PIs containing monounsaturated FA (PIs-MUFA) in all non-invasive cancer cells (n = 12). While 54% of invasive cancer cells (n = 27) also accumulated PIs-MUFA, 46% of invasive cancer cells (n = 23) accumulated the PIs-PUFA, PI(18:0/20:3) and PI(18:0/20:4). The accumulation of PI(18:0/20:3) was associated with higher incidence of lymph node metastasis and activation of the PD-1-related immune checkpoint pathway. Fatty acid-binding protein 7 was identified as a putative molecule controlling PI composition. CONCLUSIONS: MALDI-IMS identified PI composition associated with invasion and nodal metastasis of breast cancer. The accumulation of PI(18:0/20:3) could affect the PD-1-related immune checkpoint pathway, although its precise mechanism should be further validated.

Serum Nardilysin, a Surrogate Marker for Epithelial-Mesenchymal Transition, Predicts Prognosis of Intrahepatic Cholangiocarcinoma after Surgical Resection.

PURPOSE: Few studies have investigated prognostic biomarkers in patients with intrahepatic cholangiocarcinoma (ICC). Nardilysin (NRDC), a metalloendopeptidase of the M16 family, has been suggested to play important roles in inflammation and several cancer types. We herein examined the clinical significance and biological function of NRDC in ICC.Experimental Design: We measured serum NRDC levels in 98 patients with ICC who underwent surgical resection in two independent cohorts to assess its prognostic impact. We also analyzed NRDC mRNA levels in cancerous tissue specimens from 43 patients with ICC. We investigated the roles of NRDC in cell proliferation, migration, gemcitabine sensitivity, and gene expression in ICC cell lines using gene silencing. RESULTS: High serum NRDC levels were associated with shorter overall survival and disease-free survival in the primary (n = 79) and validation (n = 19) cohorts. A correlation was observed between serum protein levels and cancerous tissue mRNA levels of NRDC (Spearman ρ = 0.413; P = 0.006). The gene knockdown of NRDC in ICC cell lines attenuated cell proliferation, migration, and tumor growth in xenografts, and increased sensitivity to gemcitabine. The gene knockdown of NRDC was also accompanied by significant changes in the expression of several epithelial-mesenchymal transition (EMT)-related genes. Strong correlations were observed between the mRNA levels of NRDC and EMT-inducing transcription factors, ZEB1 and SNAI1, in surgical specimens from patients with ICC. CONCLUSIONS: Serum NRDC, a possible surrogate marker reflecting the EMT state in primary tumors, predicts the outcome of ICC after surgical resection.

Necrostatin-7 suppresses RANK-NFATc1 signaling and attenuates macrophage to osteoclast differentiation.

Osteoclasts play a crucial role in osteolytic bone diseases, such as osteoporosis, rheumatoid arthritis, periodontitis, Paget's disease of bone and bone metastatic tumors. Therefore, controlling osteoclast differentiation and function has been considered a promising therapeutic strategy. Here, we show that necrostatin (Nec)-7, an inhibitor of programmed necrosis, strongly suppressed receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption, without compromising macrophage colony-stimulating factor (M-CSF)-supported survival and growth of osteoclast precursor cells. Accordingly, Nec-7 significantly decreased the levels of RANKL-induced osteoclastogenic marker genes, such as cathepsin K. Mechanistically, Nec-7 neither affected MAPK nor NF-κB activation; however, it strongly inhibited the RANKL receptor (RANK) to nuclear factor of activated T cells c1 (NFATc1) signaling. Lentiviral expression of RANK in bone marrow-derived macrophages significantly restored osteoclastogenesis and NFATc1 amplification in Nec-7-treated cells. In this study, we revealed that Nec-7-sensitive pathways are crucially involved in osteoclast formation and function. Investigation of the molecular mechanism(s) through which Nec-7 inhibits RANK-NFATc1 signaling axis may lead to the development of new therapeutic strategies for bone disease.

Preoperative metabolic tumor volume of intrahepatic cholangiocarcinoma measured by 18F-FDG-PET is associated with the KRAS mutation status and prognosis.

BACKGROUND: Surgical resection remains the mainstay of curative treatment for intrahepatic cholangiocarcinoma (ICC). Prognosis after surgery is unsatisfactory despite improvements in treatment and post-operative clinical management. Despite developments in the molecular profiling of ICC, the preoperative prediction of prognosis remains a challenge. This study aimed to identify clinical prognostic indicators by investigating the molecular profiles of ICC and evaluating the preoperative imaging data of 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET). METHODS: A retrospective analysis was performed on 50 consecutive patients with ICC who underwent curative hepatectomy after 18F-FDG-PET examination. To evaluate the molecular profiles of ICC, KRAS mutation status was assessed in resected specimens. For the assessment of glucose uptake, we observed the expression of glucose transporter-1 (GLUT-1) by immunohistochemistry. The data of 18F-FDG-PET were re-evaluated as follows: maximum standardized uptake value, metabolic tumor volume (MTV), and total lesion glycolysis (TLG). Cut-off values were determined using receiver operating characteristic (ROC) curve analysis. Cumulative overall survival (OS) was analyzed using the Kaplan-Meier analysis. RESULTS: Overall, 16 (32.0%) patients had mutations in KRAS. Patients with mutated KRAS exhibited shorter OS than those with wild-type KRAS (5-year OS, 0% vs. 35.1%, P < 0.001). GLUT-1 expression was significantly higher in tumors with mutated KRAS than in tumors with wild-type KRAS (median, 4.0 vs. 1.0, P < 0.001). Survival was significantly different when stratified by expression of GLUT-1 (5-year OS, 0% vs. 46.5%, P <0.001). Among the 18F-FDG-PET parameters, the MTV and TLG were significantly higher in the mutated KRAS group than in the wild-type KRAS group (P = 0.013 and P = 0.026, respectively). ROC curve analysis revealed a cut-off value of 38 for the MTV, with the highest accuracy (area under the curve = 0.789; 95% confidence interval, 0.581-0.902) for predicting KRAS mutation. This cut-off value permitted stratification of OS (high vs. low: 5-year OS, 13.1% vs. 36.7%, P = 0.008). CONCLUSIONS: High MTV is associated with KRAS mutation and poor postoperative outcomes in patients with ICC, suggesting that the MTV of ICC measured by 18F-FDG-PET may provide useful information for tumor molecular profiles and prognosis.