Molecular and immunological characteristics of postoperative relapse in lymph node-positive esophageal squamous cell cancer.

BACKGROUND: The molecular and immunological characteristics of primary tumors and positive lymph nodes in esophageal squamous cell carcinoma (ESCC) are unknown and the relationship with recurrence is unclear, which this study attempted to explore. METHODS: A total of 30 ESCC patients with lymph node positive (IIB-IVA) were enrolled. Among them, primary tumor and lymph node specimens were collected from each patient, and subjected to 551-tumor-targeted DNA sequencing and 289-immuno-oncology RNA panel sequencing to identify the different molecular basis and immunological features, respectively. RESULTS: The primary tumors exhibited a higher mutation burden than lymph nodes (p < 0.001). One-year recurrent ESCC exhibited a higher Mucin16 (MUC16) mutation rate (p = 0.038), as well as univariate and multivariate analysis revealed that MUC16 mutation is independent genetic factor associated with reduced relapse-free survival (univariate, HR: 5.39, 95% CI: 1.67-17.4, p = 0.005; multivariate, HR: 7.36, 95% CI: 1.79-30.23, p = 0.006). Transcriptomic results showed non-relapse group had higher cytolytic activity (CYT) score (p = 0.025), and was enriched in the IFN-α pathway (p = 0.036), while those in the relapsed group were enriched in the TNF-α/NF-κB (p = 0.001) and PI3K/Akt pathway (p = 0.014). CONCLUSION: The difference in molecular characteristics between primary lesions and lymph nodes may be the cause of the inconsistent clinical outcomes. Mutations of MUC16 and poor immune infiltration are associated with rapid relapse of nodes-positive ESCC.

Liver X Receptor LXRα Promotes Grass Carp Reovirus Infection by Attenuating IRF3-CBP Interaction and Inhibiting RLR Antiviral Signaling.

Liver X receptors (LXRs) are nuclear receptors involved in metabolism and the immune response. Different from mammalian LXRs, which include two isoforms, LXRα and LXRß, only a single LXRα gene exists in the piscine genomes. Although a study has suggested that piscine LXR inhibits intracellular bacterial survival, the functions of piscine LXRα in viral infection are unknown. In this study, we show that overexpression of LXRα from grass carp (Ctenopharyngodon idellus), which is named as gcLXRα, increases host susceptibility to grass carp reovirus (GCRV) infection, whereas gcLXRα knockdown in CIK (C. idellus kidney) cells inhibits GCRV infection. Consistent with these functional studies, gcLXRα knockdown promotes the transcription of antiviral genes involved in the RIG-I-like receptor (RLR) antiviral signaling pathway, including IFN regulatory factor (IRF3) and the type I IFN IFN1. Further results show that gcLXRα knockdown induces the expression of CREB-binding protein (CBP), a transcriptional coactivator. In the knockdown of CBP, the inhibitory effect of gcLXRα knockdown in limiting GCRV infection is completely abolished. gcLXRα also interacts with IRF3 and CBP, which impairs the formation of the IRF3/CBP transcription complex. Moreover, gcLXRα heterodimerizes with RXRg, which cooperatively impair the transcription of the RLR antiviral signaling pathway and promote GCRV infection. Taken together, to our knowledge, our findings provide new insight into the functional correlation between nuclear receptor LXRα and the RLR antiviral signaling pathway, and they demonstrate that gcLXRα can impair the RLR antiviral signaling pathway and the production of type I IFN via forming gcLXRα/RXRg complexes and attenuating IRF3/CBP complexes.

Piscine Vitamin D Receptors Vdra/Vdrb in the Absence of Vitamin D Are Utilized by Grass Carp Reovirus for Promoting Viral Replication.

The vitamin D receptor (VDR) plays a pivotal role in the biological actions of vitamin D (VitD). However, little is known about the functions of VDR in the production of viral inclusion bodies (VIBs). Using a representative strain of grass carp reovirus (GCRV) genotype I, GCRV-873, we show that GCRV-873 recruits grass carp Vdrs for promoting the production of VIBs in the absence of VitD. Inhibition of cholesterol synthesis by lovastatin impairs the production of VIBs and blocks the effects of grass carp Vdrs in promoting the production of VIBs in the absence of VitD. Furthermore, grass carp Vdrs are found to form the Vdra-Vdrb heterodimer, which is vital for 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hmgcr)-dependent cholesterol synthesis and GCRV replication. Intriguingly in the presence of VitD, grass carp Vdra but not Vdrb forms the heterodimer with the retinoid X receptor beta b (Rxrbb), which induces the transcription of those genes involved in the RIG-I-like receptor (RLR) antiviral signaling pathway for inhibiting GCRV infection. Furthermore, the VitD-activated Vdra-Vdrb heterodimer attenuates the transcription of the RLR antiviral signaling pathway induced by VitD. In the presence of VitD, a balance between the Vdra-Rxrbb heterodimers as coactivators and Vdra-Vdrb heterodimers as corepressors in affecting the transcriptional regulation of the RLR antiviral signaling pathway may eventually determine the outcome of GCRV infection. Transfection with VitD can abolish the effect of grass carp Vdrs in promoting GCRV replication in a dose-dependent manner. Taken together, these findings demonstrate that GCRV utilizes host Vdrs to increase hmgcr-dependent cholesterol synthesis for promoting its replication, which can be prevented by VitD treatment. IMPORTANCE Grass carp reovirus (GCRV) is the causative agent of grass carp hemorrhagic disease, which seriously harms freshwater fish. Although many positive or negative regulators of GCRV infection have been identified in teleosts, little is known about the molecular mechanisms by which GCRV utilizes host factors to generate its infectious compartments beneficial for viral replication and infection. Here, we show that in the absence of VitD, the GCRV-873 strain utilizes host vitamin D receptors Vdra/Vdrb to increase hmgcr-dependent cholesterol synthesis for promoting the production of VIBs, which are important functional sites for aquareovirus replication and assembly. The negative regulation of Vdrs during viral infection can be prevented by VitD treatment. Thus, this present work broadens understanding of the pivotal roles of Vdrs in the interaction between the host and GCRV in the absence or presence of VitD, which might provide a rational basis for developing novel anti-GCRV strategies.

Clinical and biomarker analyses of sintilimab plus gemcitabine and cisplatin as first-line treatment for patients with advanced biliary tract cancer.

The prognosis of biliary tract cancer (BTC) remains unsatisfactory. This single-arm, phase II clinical trial (ChiCTR2000036652) investigated the efficacy, safety, and predictive biomarkers of sintilimab plus gemcitabine and cisplatin as the first-line treatment for patients with advanced BTCs. The primary endpoint was overall survival (OS). Secondary endpoints included toxicities, progression-free survival (PFS), and objective response rate (ORR); multi-omics biomarkers were assessed as exploratory objective. Thirty patients were enrolled and received treatment, the median OS and PFS were 15.9 months and 5.1 months, the ORR was 36.7%. The most common grade 3 or 4 treatment-related adverse events were thrombocytopenia (33.3%), with no reported deaths nor unexpected safety events. Predefined biomarker analysis indicated that patients with homologous recombination repair pathway gene alterations or loss-of-function mutations in chromatin remodeling genes presented better tumor response and survival outcomes. Furthermore, transcriptome analysis revealed a markedly longer PFS and tumor response were associated with higher expression of a 3-gene effector T cell signature or an 18-gene inflamed T cell signature. Sintilimab plus gemcitabine and cisplatin meets pre-specified endpoints and displays acceptable safety profile, multiomics potential predictive biomarkers are identified and warrant further verification.

Immune-related RNA signature predicts outcome of PD-1 inhibitor-combined GEMCIS therapy in advanced intrahepatic cholangiocarcinoma.

Background: Immune checkpoint inhibitor (ICI)-combined chemotherapy in advanced intrahepatic cholangiocarcinoma has been proved to have more efficacy in a series of clinical trials. However, whether the tumor microenvironment (TME) plays a vital role in immune-combined therapy has not been rigorously evaluated. Methods: Firstly, we assayed the immunogenic properties of GEM-based chemotherapy. Then, 12 ICC patients treated with PD-1 inhibitor (sintilimab) combined with gemcitabine and cisplatin (GemCis) from a phase 2 clinical trial (ChiCTR2000036652) were included and their immune-related gene expression profiles were analyzed using RNA from baseline tumor samples. Immune-related signature correlating with clinical outcome was identified according to the 12 ICC patients, and its predictive value was validated in an ICC cohort with 26 patients. Multiplexed immunofluorescence (mIF) and flow cytometry (FCM) analysis were performed to evaluate the immune-related molecules with therapeutic outcomes. Results: GEM-based chemotherapy induced immunogenic cell death of cholangiocarcinoma cells, together with increased CD274 expression. In an ICC cohort, we found that upregulation of immune-checkpoint molecules and immune response-related pathways were significantly related to better clinical outcome. On the contrary, baseline immune-cell proportions in tumor tissues did not show any correlation with clinical benefit between responders and non-responders. Immune-related signature (including six genes) correlating with clinical outcome was identified according to the 12 ICC patients, and its predictive value was validated in a small ICC cohort with 26 patients. Conclusion: Immune-related RNA signature predicts the outcome of PD-1 inhibitor-combined GEMCIS therapy in advanced intrahepatic cholangiocarcinoma, which could be tested as a biomarker for immune-chemotherapy in the future.

NOTCH1 mutation associates with impaired immune response and decreased relapse-free survival in patients with resected T1-2N0 laryngeal cancer.

Background: Patients with early-stage laryngeal cancer, even stage T1-2N0, are at considerable risk of recurrence and death. The genetic and immunologic characteristics of recurrent laryngeal cancer remain unclear. Methods: A total of 52 T1-2N0 laryngeal cancer patients were enrolled. Of these, 42 tissue samples were performed by targeted DNA sequencing, and 21 cases were performed by NanoString immuno-oncology targeted RNA sequencing to identify the distinct molecular bases and immunologic features associated with relapse in patients with early laryngeal cancer, respectively. Results: To the best to our knowledge, we present for the first time an overview of the genomic mutation spectrum of early-stage laryngeal cancers. A total of 469 genomic alterations were detected in 211 distinct cancer-relevant genes, and the genes found to be mutated in more than five patients (>10%) included tumor protein p53 (TP53, 78.5%), FAT atypical cadherin 1 (FAT1, 26%), LDL receptor related protein 1B (LRP1B, 19%), cyclin dependent kinase inhibitor 2A (CDKN2A, 17%), tet methylcytosine dioxygenase 2 (TET2, 17%), notch receptor 1 (NOTCH1, 12%) and neuregulin 1 (NRG1, 12%). Recurrent laryngeal cancer demonstrated a higher tumor mutation burden (TMB), as well as higher LRP1B mutation and NOTCH1 mutation rates. Univariate and multivariate analyses revealed that high TMB (TMB-H) and NOTCH1 mutation are independent genetic factors that are significantly associated with shorter relapse-free survival (RFS). Simultaneously, the results of the transcriptome analysis presented recurrent tumors with NOTCH1 mutation displayed upregulation of the cell cycle pathway, along with decreased B cells score, T cells score, immune signature score and tumor-infiltrating lymphocytes (TILs) score. The Cancer Genome Atlas (TCGA)-laryngeal cancer dataset also revealed weakened immune response and impaired adhesion functions in NOTCH1-mutant patients. Conclusions: Genomic instability and impaired immune response are key features of the immunosurveillance escape and recurrence of early laryngeal cancer after surgery. These findings revealed immunophenotypic attenuation in recurrent tumors and provided valuable information for improving the management of these high-risk patients. Due to the small number of patients in this study, these differences need to be further validated in a larger cohort.

Effects and Molecular Regulation Mechanisms of Salinity Stress on the Health and Disease Resistance of Grass Carp.

Though some freshwater fish have been successfully cultivated in saline-alkali water, the survival rates of freshwater fish are greatly affected by different saline-alkali conditions. The mechanisms of immune adaptation or immunosuppression of freshwater fish under different saline-alkali stress remain unclear. Here, grass carp were exposed to 3‰ and 6‰ salinity for 30 days. It was observed that salinity treatments had no obvious effects on survival rates, but significantly increased the percent of unhealthy fish. Salinity treatments also increased the susceptibility of grass carp against Flavobacterium columnare infection. The fatality rate (16.67%) of grass carp treated with 6‰ salinity was much lower than that treated with 3‰ salinity (40%). In the absence of infection, higher numbers of immune-related DEGs and signaling pathways were enriched in 6‰ salinity-treated asymptomatic fish than in 3‰ salinity-treated asymptomatic fish. Furthermore different from salinity-treated symptomatic fish, more DEGs involved in the upstream sensors of NOD-like receptor signaling pathway, such as NLRs, were induced in the gills of 6‰ salinity-treated asymptomatic fish. However in the case of F. columnare infection, more immune-related signaling pathways were impaired by salinity treatments. Among them, only NOD-like receptor signaling pathway was significantly enriched at early (1 and/or 2 dpi) and late (7 dpi) time points of infection both for 3‰ salinity-treated and 6‰ salinity-treated fish. Besides the innate immune responses, the adaptive immune responses such as the production of Ig levels were impaired by salinity treatments in the grass carp infected with F. columnare. The present study also characterized two novel NLRs regulated by salinity stress could inhibit bacterial proliferation and improve the survival rate of infected cells. Collectively, the present study provides the insights into the possible mechanisms why the percent of unhealthy fish in the absence of infection and mortality of grass carp in the case of F. columnare infection were much lower in the 6‰ salinity-treated grass carp than in 3‰ salinity-treated grass carp, and also offers a number of potential markers for sensing both environmental salinity stress and pathogen.

NLRC3-like 1 inhibits NOD1-RIPK2 pathway via targeting RIPK2.

Both NLRC3 and NOD1 belong to regulatory NLR subfamily based on their best-characterized function. In mammals, NLRC3 was reported to function by attenuating signaling cascades initiated by other families of PRRs. In teleosts, multiple NLRC3-like genes were identified through transcriptome sequencing. However, the functions of many NLRC3-like genes, especially the fish-specific NLRC3-like genes, remain unclear. In the present study, we report the functional characterization of a novel category of NLRC3-like proteins (named as NLRC3-like 1) from the zebrafish, which consists of a fish-specific FISNA, a conserved NACHT and five C-terminal LRRs domains. The expression of zebrafish NLRC3-like 1 was inducible in response to Edwardsiella piscicida infection. During bacterial infection, the in vitro and in vivo studies revealed that zebrafish NLRC3-like 1 overexpression facilitated bacterial growth and dissemination, together with the decreased survival rate of zebrafish larvae infected with E. piscicida. The attenuated response by zebrafish NLRC3-like 1 in response to bacterial infection were characterized by the impaired expression of antibacterial genes, proinflammatory cytokines and Nox genes. Furthermore, zebrafish NLRC3-like 1 interacted with the adaptor protein RIPK2 of NODs signaling via the FISNA (Fish-specific NACHT associated domain) and NACHT domains. However, the interaction between zebrafish NLRC3-like 1 and RIPK2 inhibited the assembly of the NOD1-RIPK2 complex. Importantly, zebrafish NLRC3-like 1 inhibited NOD1-mediated antibacterial activity, NF-κB and MAPK pathways and proinflammatory cytokine production. All together, these results firstly demonstrate that zebrafish NLRC3-like 1 inhibits NOD1-RIPK2 antibacterial pathway via targeting the adaptor protein RIPK2.

Effects of amlodipine on TGF-ß-induced Smad2, 4 expressions in adriamycin toxicity of rat mesangial cells.

Transforming growth factor-ß (TGF-ß) is closely associated with progressive renal fibrosis. A central component of TGF-ß-stimulated mesangial cell fibrogenesis is the TGF-ß family-specific Smad signal transduction pathway. This study investigated the expression of TGF-ß-receptor--activated Smad2, its common partner Smad4, and the phosphorylated Smad2 (p-Smad2) in adriamycin-induced toxicity of cultured rat mesangial cells. This in vitro study showed that amlodipine (10(-9) to 10(-5) mol/l) had no effect on the toxicity of rat mesangial cells induced by adriamycin in the absence of TGF-ß1. However, amlodipine (10(-7) to 10(-5) mol/l) reduced the toxicity of rat mesangial cells induced by TGF-ß1 in the absence of adriamycin; moreover, amlodipine (10(-8) to 10(-5) mol/l) significantly reduced adriamycin-induced cytotoxicity when it was given in combination with TGF-ß1; amlodipine (10(-6), 10(-5) mol/l) had no effect on Smad2 mRNA and protein expression induced by adriamycin + TGF-ß1, but it (10(-6), 10(-5) mol/l) dramatically inhibited the down-regulation of p-Smad2 protein expression as well as Smad4 mRNA and protein expression induced by adriamycin + TGF-ß1 in rat mesangial cells. Present study shows that amlodipine exerts a significant inhibition on adriamycin-induced toxicity in rat mesangial cells by affecting the expression of TGF-ß/Smad signaling intermediates p-Smad2 and Smad4.