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Intrahepatic cholangiocarcinoma (iCCA) exhibits extensive intratumoral heterogeneity and an extremely high mortality rate. Here, we performed whole-exome sequencing, RNA sequencing, T-cell receptor (TCR) sequencing, and multiplexed immunofluorescence on 207 tumor regions from 45 patients with iCCA. Over half of iCCA displayed intratumoral heterogeneity of immune infiltration, and iCCA were classified into sparsely, heterogeneously, and highly infiltrated subgroups with distinct immunogenomic characteristics. Sparsely infiltrated tumors displayed active copy-number loss of clonal neoantigens, and heterogeneous immune infiltration played an important role in the subclonal evolution across tumor subregions. Highly infiltrated tumors were characterized by extensive immune activation and a similar TCR repertoire across tumor subregions, but counteracted with T-cell exhaustion and pervasive antigen presentation defects. Notably, FGFR2 mutations and fusions correlated with low mutation burden and reduced immune infiltration. Our work delineated the dynamic tumor–immune interactions and developed a robust classification system to divide patients with iCCA into high and low immune evasion groups with different prognoses. Significance: This study elucidates the impact of spatial immune heterogeneity upon tumor evolution of iCCA and reveals distinct immune evasion mechanisms developed in different immune microenvironments, which can be exploited for the development of personalized immunotherapy strategies. This article is highlighted in the In This Issue feature, p. 2221

Conversion surgery is a treatment that aims for R0 resection of primary advanced gastric cancers (GCs) that have responded well to systemic chemotherapy. We investigated the role of conversion therapy in initially unresectable metastatic cancer with positive HER2 status that responded to chemotherapy plus trastuzumab.A total of 32 metastatic GC patients who underwent systemic chemotherapy plus trastuzumab sequenced by conversion surgery at Zhejiang Cancer Hospital between 2015 and 2020 were retrospectively reviewed.The observed overall survival (OS) and progression-free survival (PFS) for all the patients were 30.2 and 25.1 months, respectively. The 1-year survival rate was 81.25%, and the 1-year PFS rate was 78.13%. Univariate and multivariate analyses demonstrated that liver metastasis (P=0.021), peritoneal metastasis (P=0.047), para-aortic lymph node metastasis (16a1/b2) (P=0.048), macroscopic type 4 (P=0.027), number of noncurative factors (P=0.011), YoshidaHER2-positive patients with potentially resectable disease had a remarkably good prognosis after conversion gastrectomy following trastuzumab treatment. Adequate selection of metastatic GC patients for conversion surgery is recommended.

Intrahepatic cholangiocarcinoma (iCCA) exhibits extensive intratumoral heterogeneity and an extremely high mortality rate. Here, we performed whole-exome sequencing, RNA sequencing, T-cell receptor (TCR) sequencing, and multiplexed immunofluorescence on 207 tumor regions from 45 patients with iCCA. Over half of iCCA displayed intratumoral heterogeneity of immune infiltration, and iCCA were classified into sparsely, heterogeneously, and highly infiltrated subgroups with distinct immunogenomic characteristics. Sparsely infiltrated tumors displayed active copy-number loss of clonal neoantigens, and heterogeneous immune infiltration played an important role in the subclonal evolution across tumor subregions. Highly infiltrated tumors were characterized by extensive immune activation and a similar TCR repertoire across tumor subregions, but counteracted with T-cell exhaustion and pervasive antigen presentation defects. Notably, FGFR2 mutations and fusions correlated with low mutation burden and reduced immune infiltration. Our work delineated the dynamic tumor–immune interactions and developed a robust classification system to divide patients with iCCA into high and low immune evasion groups with different prognoses.Significance:This study elucidates the impact of spatial immune heterogeneity upon tumor evolution of iCCA and reveals distinct immune evasion mechanisms developed in different immune microenvironments, which can be exploited for the development of personalized immunotherapy strategies.This article is highlighted in the In This Issue feature, p. 2221

Supplementary Table from Geospatial Immune Heterogeneity Reflects the Diverse Tumor–Immune Interactions in Intrahepatic Cholangiocarcinoma

Supplementary Figure from Geospatial Immune Heterogeneity Reflects the Diverse Tumor–Immune Interactions in Intrahepatic Cholangiocarcinoma

PDF file - 130K, Univariate analyses of factors associated with survival and recurrence in cohort 2

PDF file - 153K, Clinical characteristics of the 323 HCC Patients used in cohort 2

PDF file - 73K, Supplemental Figure S1: The relationship of SBP1 Expression level and Prognosis. The expression levels of SBP1 in 323 HCC tissues were divided by a 4-point scale (0 - +++) system based on the number of positive cells and the intensity of staining. Kapl

PDF file - 129K, Multivariate analyses of factors associated with survival and recurrence in cohort 2

PDF file - 162K

Purpose: We aimed to characterize the role of selenium-binding protein 1 (SBP1) in hepatocellular carcinoma (HCC) invasiveness and underlying clinical significance.Experimental Design: SBP1 expression was measured in stepwise metastatic HCC cell lines by Western blotting. The role of SBP1 in HCC was investigated using siRNA. Immunofluorescence analyses were used to detect the interaction between SBP1 and glutathione peroxidase 1 (GPX1). Nineteen fresh tumor tissues and 323 paraffin-embedded samples were used to validate in vitro findings and to detect the prognostic significance of SBP1, respectively.Results: Inhibition of SBP1 effectively increased cell motility, promoted cell proliferation, and inhibited apoptosis only under oxidative stress; it also greatly enhanced GPX1 activity without altering GPX1 expression and downregulated hypoxia-inducible factor-1α (HIF-1α) expression. SBP1 and GPX1 formed nuclear bodies and colocalized under oxidative stress. In freshly isolated clinical HCC tissues, decreased SBP1 was linked with increased GPX1 activity and correlated with vascular invasion. Tumor tissue microarrays indicated that SBP1 was an independent risk factor for overall survival and disease recurrence; patients with lower SBP1 expression experienced shorter overall survival periods and higher rates of disease recurrence (P < 0.001). Further analyses indicated that the predictive power of SBP1 was more significant for patients beyond the Milan criteria than patients within the Milan criteria.Conclusions: Decreased expression of SBP1 could promote tumor invasiveness by increasing GPX1 activity and diminishing HIF-1α expression in HCC; SBP1 could be a novel biomarker for predicting prognosis and guiding personalized therapeutic strategies, especially in patients with advanced HCC. Clin Cancer Res; 18(11); 3042–53. ©2012 AACR.

PDF file - 66K, Clinical characteristics of the 19 HCC patients used in cohort 1

Remote sensing data generated by satellites have important research and application values. However, traditional detection models have poor adaptability and generalization ability. In order to solv...

Background: SQSTM1/p62, as a selective autophagy receptor, regulates multiple signaling pathways participating in the initiation and progression of tumors. Since metastasis is still a main cause for intrahepatic cholangiocarcinoma (ICC)-associated mortality, this study aimed to explore the mechanism of p62 promoting progression of ICC.Methods: Western blotting and immunohistochemical analysis were conducted to detect the expression level of protein p62 in ICC tissues. Subsequently, loss of function experiments was applied to define the role of p62 in the progression of ICC in vitro and in vivo. Mitochondrial function and mitophagy was evaluated by measuring oxygen consumption rates (OCR) and immunofluorescence detection respectively.Results: Here we identified expression of p62 was significantly upregulated in ICC specimens compared to normal tissue. And we further illustrated that p62 expression was positively correlated with lymph-node metastasis and poor prognosis. Loss of function assays revealed that p62 not only promoted ICC cells proliferation, migration and invasive capacity in vitro, but also induced lung metastasis in xenograft mouse model. Mechanistically, high expression of p62 induced epithelial-mesenchymal transition (EMT) with upregulation of Snail1, Vimentin and down-regulation of E-Cadherin. Moreover, OCR assays and immunofluorescence cell staining demonstrated that the autophagy-dependent function of p62 may play a vital role in maintaining mitochondrial function of ICC by mitophagy.Conclusions: These data provide new evidence and feasible mechanism that abundant p62 expression promote ICC progression, suggesting a promising therapeutic target for anti-metastatic strategies in ICC patients.

Intrahepatic cholangiocarcinoma (ICC) is a highly metastatic cancer.Between August 2010 and August 2018, 660 consecutive ICC patients, without prior anti-tumor treatments nor other malignancies, were enrolled. The diagnostic performance of PET/CT on the N- and M-staging was compared with conventional imaging, and the preoperative staging accuracy and treatment re-allocation by PET/CT were retrospectively calculated. Survival difference was compared between patients receiving PET/CT or not after propensity score matching.Patients were divided into group A (n=291) and group B (n=369) according to whether PET/CT was performed. Among 291 patients with both PET/CT and conventional imaging for staging in group A, PET/CT showed significantly higher sensitivity (83.0%PET/CT was valuable for diagnosing regional lymph node metastasis and distant metastasis in ICC patients, and facilitated accurate tumor staging and optimal treatment allocation.

The acquisition of new metabolic activities is a major force driving evolution. We explored, from the perspectives of gene family expansion and the evolutionary adaptability of proteins, how new functions have arisen in which terpene synthases diverged. Monoterpenoids are diverse natural compounds that can be divided into cyclic and acyclic skeleton forms according to their chemical structure. We demonstrate, through phylogenetic reconstructions and genome synteny analyses, that the (E)-β-ocimene synthases, which are acyclic monoterpene synthases (mTPSs), appear to have arisen several times in independent lineages during plant evolution. Bioinformatics analyses and classical mutation experiments identified four sites (I388, F420, S446, and F485) playing important roles in the neofunctionalization of mTPSs. Incubation of neryl diphosphate with Salvia officinalis 1,8-cineole synthase (SCS) and mutated proteins show that these four sites obstruct the isomerization of geranyl diphosphate. Quantum mechanical/molecular mechanical molecular dynamics simulations of models of SCS, SCSY420F/I446S, and SCSN338I/Y420F/I446S/L485F with (3R)-linalyl diphosphate suggest that mutations changed the configuration of the intermediate to obtain new activities. These results provide new perspectives on the evolution of mTPSs, explain the convergent evolution of (E)-β-ocimene synthases at the molecular level, and identify key residues to control the specificity of engineered mTPSs.

Additional file 7:. Table S3. Performance of the 7-AAb panel and AFP in HBsAg+/HBsAg--HCC detection.

Additional file 2:. Fig. S2. One hundred proteins for HCC Focused Arrays. (A) A total of 100 proteins were selected to prepare HCC Focused Arrays using GST fusion proteins. Anti-GST antibody was employed for quality control of HCC Focused Array. (B) Two spots (spot 1 and spot 2) of each protein were printed onto array with correlation coefficient (R2) approximate of 0.99. (C) Function enrichment analysis identified biological processes, including focal adhesion, and negative regulation of cellular component organization and protein modification process.

Additional file 9:. Table S5. Performance of the 7-AAb panel and AFP to detect HCC with different Chinese HCC stages.

Additional file 8:. Table S4. Performance of the 7-AAb panel and AFP to detect HCC with different TNM stages.

Additional file 1:. Fig. S1. Boxplot of signals obtained from HuProtTM array. In discovery phase (I), boxplot of signal intensities (SNR = median foreground intensity/median background intensity) was shown after normalization. The red boxes represent 50 HCC samples and the other boxes represent 50 healthy controls. The antigens were recognized by human autoantibodies of the IgG (A) and IgM (B) isotypes, respectively.

Additional file 5:. Table S1. Summary of the study subjects.

Additional file 4:. Fig. S4. The representative blots for both HCC and controls. (A) HCC Focused Arrays incubated with HCC, liver cirrhosis (Cirrhotic), and healthy control (Healthy), respectively. (B) Performance of CIAPIN1, EGFR, MAS1, SLC44A3, ASAH1, UBL7 and ZNF428 in the test phase (II).

Additional file 3: Fig. S3. Expression level for the 100 proteins. (A) Gene expression level of 100 proteins in the liver compared to other tissues using The Human Protein Atlas ( http://www.proteinatlas.org/ ). Based on this database, 7 proteins were absent in the liver, including MAS1, C3orf56, DCAF4L2, DEFB112, GPR78, PAGE1 and SCGB1C2. 16 genes showed increased mRNA expression level in the liver. Liver elevated proteins reported in the liver-specific proteome of The Human Protein Atlas were labeled with green. (B) Totally, 64 of these 100 proteins were found and further analyzed in paired HCC tumor and adjacent non-tumor liver tissues according to our previous proteomics (Ref. Cell. 179, 561-577 (2019)). (C) Among the 64 proteins, 10 were down-regulated and 8 were up-regulated significantly in HCC tumor, compared with adjacent liver tissues (adjusted p < 0.05 and∣log2 FC∣ > 0.5) according to our previous proteomics (Ref. Cell. 179, 561-577 (2019)). ( 4412 kb)

Biotic stresses induce the expression of mulberry cystatins. MaCPI-4 protein is stable in silkworm digestive fluid and accumulates in gut food debris and frass. Plant cystatins are considered to be involved in defense responses to insect herbivores though little is known about how cystatins from the natural host respond to a specialist herbivory and the following postingestive interaction is also poorly understood. Here, we studied the biotic stress-mediated inductions of cystatins from mulberry tree, and examined the stability of mulberry cystatin proteins in the gut of silkworm, Bombyx mori, a specialist insect feeding on mulberry leaf. First, we cloned and characterized six cystatin genes from a mulberry cultivar, Morus atropurpurea Roxb., named as MaCPI-1 to MaCPI-6. The recombinant MaCPI-1, MaCPI-3 and MaCPI-4 proteins, which showed inhibitory effects against papain in vitro, were produced. Silkworm herbivory as well as methyl jasmonate (MeJA) treatment induced the expression of five mulberry cystatin genes, and the highest inductions were observed from MaCPI-1 and MaCPI-6. Mechanical wounding led to the inductions of four cystatin genes. The differential induction occurred in MaCPI-2. The induced protein changes were detected from three mulberry cystatins comprising MaCPI-1, MaCPI-3 and MaCPI-4. In vivo and in vitro assays showed that MaCPI-1 and MaCPI-3 proteins were susceptible to silkworm digestive fluid and MaCPI-4 had an antidigestive stability, and was detected in silkworm gut and frass. Collectively, our data indicated that biotic stresses resulted in the transcriptional inductions and protein changes of mulberry cystatins (MaCPIs), and identified MaCPI-4 with stability in the gut of its specialist herbivore.

Although immune checkpoint blockade have demonstrated promising results, their effects on gastric cancer (GC) are under investigation. Understanding the clinical significance of PD1 and its ligands' expression, together with T cell infiltration might provide clues for biomarkers screening in GC immunotherapy. Immunohistochemistry were performed on a tissue microarray including 1,014 GC specimens using PD1, PDL1 and PDL2 antibodies. T cell markers CD3 and CD8 were also stained and quantified by automated image analysis. Correlation with clinical features and outcome were analyzed after controlling for potential confounders including EBV infection, HER2, C-met and PCNA expression. 37.8% of the cases showed membranous PD-L1 expression in tumor cells and 74.9% in infiltrating immune cells. PDL1 expression rate was rather higher in patients without metastasis, in EBV positive group and those with C-met and PCNA expression. GC patients with high level PDL1 expression exhibited better survival. GC Patients with higher T cell infiltration also showed elevated PDL1, PDL2 and PD1 expression and predict favorable outcome, indicating an adaptive immune resistance mechanism may exist. The group of patients infiltrated with lower density CD3+ T cells also without PDL1 expression in tumor cells predict the worst outcome in the subgroup of different PTNM stage, which may suggest an inactive immune status. These results highlights the need to assess both PDL1 expression in all tumor context and the characterization of the GC immune microenvironment.

The contingent governance theory based on state-contingent ownership has exerted an important impact on economic academia, and its application in the field of company law is also of significant value. The lack of internal restraint mechanism and public supervision of limited liability companies makes it feasible to implement contingent governance. Under the mechanism of contingent governance, creditors of a limited liability company may intervene in its governance. The right of intervention is mainly reflected in three aspects, i.e., (1) mandatory debt-for-equity swap, (2) obligation of the actual controller to creditors when the limited liability company is on the edge of insolvency, and (3) creditor’s right of objection with respect to the decision of corporate substantial business.

The subcellular localization of mulberry type III polyketide synthases. (A) The control vector pLGNL-EGFP, (B) pLGNL-MnPKS1-EGFP, (C) pLGNL-MnPKS2-EGFP, (D) pLGNL-MnCHS2-EGFP, (E) pLGNL-MnCHS6-EGFP, (G) pLGNL-MnSTS7-EGFP, and (F) pLGNL-MnSTS8-EGFP were transiently expressed in tobacco epidermal cells. Column (a), bright filed; column (b), EGFP fluorescence; and column (c), merged fluorescence. (PDF 3312 kb)

The phylogenetic tree used to calculate phylogenetic dN/dS ratio for multispecies set. (PDF 1102 kb)

In vivo characterization of MnPKS1 and MnPKS2 by coexpression with 4-coumaroyl-CoA ligase in Escherichia coli. Mn4CL: E. coli-expressed Mn4CL. Mn4CL+MnPKS1: E. coli-coexpressed Mn4CL and MnPKS1. Mn4CL+MnPKS2: E. coli-coexpressed Mn4CL and MnPKS2. (A) 4-Coumaric acid (1) and naringenin (2) were used as standard compounds. Selected ion chromatograms generated during liquid chromatography-electrospray ionization mass spectrometry analyses of the compounds are provided in the small panel on the right: 4-coumaric acid, m/z = 163.1; naringenin, m/z = 271.1. (B) Resveratrol (3) was used as the standard compound. A selected ion chromatogram of the compound is provided in the small panel on the right: resveratrol, m/z = 227.1. (PDF 941 kb)

Type III polyketide synthase genes in the M. notabilis genome. Genes have been named based on scaffold locations. ORF length: open reading frame length confirmed by clone analysis. *: genes without expression data. Table S2: Estimation of the mean Ks values for the genes flanking mulberry type III PKS genes. The mean Ks value was calculated for each homologous pair of protein-coding genes between genomic fragments containing type III PKS genes if microsynteny was detected. Table S3: Parameter estimates of codon-substitution evolutionary models for three MnCHS-like genes with undetectable expression. The likelihood ratio tests are as follows: Branch model, one ratio model 0 vs two ratio model 2; Clade model C, M2a_rel vs CmC; Branch site model, model A null (ω is fixed as 1) vs model A, ω = d N /d S . b = background; f = foreground; o=other; p0, p1, and p2 = proportion of sites with d N /d S ratios = ω0, ω1, and ω2, respectively; lnL = ln of the likelihood; P-value = P values of the likelihood ratio tests. Table S4: Type I and type II functional divergence between MnSTS and MnCHS families as well as in MnSTS subfamilies. Table S5: Primers used for cloning mulberry type III PKS and 4CL genes. Four genes labelled with asterisk, including MnSTS2, MnSTS3, MnSTS8 and MnSTS9 shared the forward (from initiation codon ‘ATG’) and reverse (end with stop codon ‘TAG’) primers, while other primers matching to variable regions have been used to distinguish them. Table S6: Sequences of primers used for quantitative reverse transcription PCR. (DOCX 36 kb)

General flavonoid and stilbene biosynthetic pathways in mulberry. The enzymes shown in these pathways are as follows: PAL, phenylalanine ammonia-lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-cumaroyl CoA-lyase; CHS, chalcone synthase; STS, stilbene synthase. (PDF 783 kb)

Comparison of mulberry type III polyketide synthase amino acid sequences. Conserved sites are shaded. The asterisk indicates the site contains a conserved Thr197 in CHS, but a Gly in PKS1 and PKS2. Purple arrow: positive selection and type II divergence sites; red arrow: positive selection sites; green arrow: type I divergence sites; yellow arrow: type II divergence sites. Red box: catalytic triad; blue box: co-enzyme A binding sites; yellow box: important residues for functional diversity. (PDF 2015 kb)

Coexpression of M. notabilis 4CL and type III PKS in E. coli. The results were detected in a SDS-PAGE gel (12%). (A): soluble protein fraction; (B): insoluble protein fraction; Lane 1: pCold-4CL; Lane 2: pCold-4CL+pET28a-MnPKS1; Lane 3: pCold-4CL+pET28a-MnPKS2; Lane 4: pCold-4CL+pET28a-MnCHS2; Lane 5: pCold-4CL+pET28a-MnCHS6; Lane 6: pCold-4CL+pET28a-MnSTS7; Lane 7: pCold-4CL+pET28a-MnSTS8; Lane 8: pCold-4CL control (no IPTG); Lane 9: pCold-4CL+pET28a-MnPKSs control (no IPTG); Lane 10: pCold+pET28a empty plasmids control. Marker: molecular weight standard. (PDF 1187 kb)

Background Type III polyketide synthases are important for the biosynthesis of flavonoids and various plant polyphenols. Mulberry plants have abundant polyphenols, but very little is known about the mulberry type III polyketide synthase genes. An analysis of these genes may provide new targets for genetic improvement to increase relevant secondary metabolites and enhance the plant tolerance to biotic and abiotic stresses. Results Eighteen genes encoding type III polyketide synthases were identified, including six chalcone synthases (CHS), ten stilbene synthases (STS), and two polyketide synthases (PKS). Functional characterization of four genes representing most of the MnCHS and MnSTS genes by coexpression with 4-Coumaroyl-CoA ligase in Escherichia coli indicated that their products were able to catalyze p-coumaroyl-CoA and malonyl-CoA to generate naringenin and resveratrol, respectively. Microsynteny analysis within mulberry indicated that segmental and tandem duplication events contributed to the expansion of the MnCHS family, while tandem duplications were mainly responsible for the generation of the MnSTS genes. Combining the evolution and expression analysis results of the mulberry type III PKS genes indicated that MnCHS and MnSTS genes evolved mainly under purifying selection to maintain their original functions, but transcriptional subfunctionalization occurred during long-term species evolution. Moreover, mulberry leaves can rapidly accumulated oxyresveratrol after UV-C irradiation, suggesting that resveratrol was converted to oxyresveratrol. Conclusions Characterizing the functions and evolution of mulberry type III PKS genes is crucial for advancing our understanding of these genes and providing the basis for further studies on the biosynthesis of relevant secondary metabolites in mulberry plants. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2843-7) contains supplementary material, which is available to authorized users.