Crosstalk between Cancer Cells and Cancer-Associated Fibroblasts Mediated by TGF-ß1-IGFBP7 Signaling Promotes the Progression of Infiltrative Gastric Cancer.

Patients with infiltrative-type gastric cancer (GC) (Ming's classification) have a poor prognosis due to more metastasis and recurrence. Cancer-associated fibroblasts (CAFs) in infiltrative-type extracellular matrix (ECM) have specific characteristics compared with those of expansive types with respect to metastasis, but the mechanism is still unclear. Based on our proteomics data, TCGA data analysis, and immunohistochemical staining results, significantly higher expression of IGFBP7 was observed in GC, especially in the infiltrative type, and was associated with a poor prognosis. Combining single-cell transcriptome data from GEO and multiple immunofluorescence staining on tissue showed that the differential expression of IGFBP7 mainly originated from myofibroblastic CAFs, the subgroup with higher expression of PDGFRB and α-SMA. After treating primary normal fibroblasts (NFs) with conditional medium or recombined protein, it was demonstrated that XGC-1-derived TGF-ß1 upregulated the expression of IGFBP7 in the cells and its secretion via the P-Smad2/3 pathway and mediated its activation with higher FAP, PDGFRB, and α-SMA expression. Then, either conditional medium from CAFs with IGFBP7 overexpression or recombined IGFBP7 protein promoted the migration, invasion, colony formation, and sphere growth ability of XGC-1 and MGC-803, respectively. Moreover, IGFBP7 induced EMT in XGC-1. Therefore, our study clarified that in the tumor microenvironment, tumor-cell-derived TGF-ß1 induces the appearance of the IGFBP7+ CAF subgroup, and its higher IGFBP7 extracellular secretion level accelerates the progression of tumors.

Apatinib remodels the immunosuppressive tumor ecosystem of gastric cancer enhancing anti-PD-1 immunotherapy.

Apatinib has been shown to clinically enhance anti-PD-1 immunotherapy for advanced gastric cancer (GC). However, the complexity of GC immunosuppression remains a challenge for precision immunotherapy. Here, we profile the transcriptomes of 34,182 single cells from GC patient-derived xenografts of humanized mouse models treated with vehicle, nivolumab, or nivolumab plus apatinib. Notably, excessive expression of CXCL5 in the CellCycle malignant epithelium, induced by anti-PD-1 immunotherapy and blocked by combined apatinib treatment, is found to be a key driver of tumor-associated neutrophil (TAN) recruitment in the tumor microenvironment through the CXCL5/CXCR2 axis. We further show that the protumor TAN signature is associated with anti-PD-1 immunotherapy-related progressive disease and poor cancer prognosis. Molecular and functional analyses in cell-derived xenograft models confirm the positive in vivo therapeutic effect of targeting the CXCL5/CXCR2 axis during anti-PD-1 immunotherapy. Altogether, our study elucidates the GC immunosuppressive landscape in anti-PD-1 immunotherapy and highlights potential targets for overcoming checkpoint immunotherapy resistance.

Dioscin potentiates the antitumor effect of suicide gene therapy in melanoma by gap junction intercellular communication-mediated antigen cross-presentation.

Dioscin (Dio), steroid saponin, exists in several medicinal herbs with potent anticancer efficacy. This study aimed to explore the effect of Dio on the immune-related modulation and synergistic therapeutic effects of the herpes simplex virus thymidine kinase/ganciclovir (HSV-Tk/GCV) suicide gene therapy system in murine melanoma, thereby providing a research basis to improve the potential immunomodulatory mechanism underlying combination therapy. Using both in vitro and in vivo experiments, we confirmed the immunocidal effect of Dio-potentiated suicide gene therapy on melanoma. The results showed that Dio upregulated connexin 43 (Cx43) expression and improved gap junction intercellular communication (GJIC) in B16 cells while increasing the cross-presentation of antigens by dendritic cells (DCs), eventually promoting the activation and antitumor immune killing effects of CD8+ T lymphocytes. In contrast, inhibition or blockade of the GJIC function (overexpression of mutant Cx43 tumor cells/Gap26) partially reversed the potentiating effect. The significant synergistic effect of Dio on HSV-Tk/GCV suicide gene therapy was further investigated in a B16 xenograft mouse model. The increased number and activation ratio of CD8+ T lymphocytes and the levels of Gzms-B, IFN-γ, and TNF-α in mice reconfirmed the potential modulatory effects of Dio on the immune system. Taken together, Dio targets Cx43 to enhance GJIC function, improve the antigens cross-presentation of DCs, and activate the antitumor immune effect of CD8+ T lymphocytes, thereby providing insights into the potential immunomodulatory mechanism underlying combination therapy.

Oral Vaccination With Recombinant Pichia pastoris Expressing Iridovirus Major Capsid Protein Elicits Protective Immunity in Largemouth Bass (Micropterus salmoides).

Largemouth bass iridovirus (LMBV) can cause high mortality and lead to heavy economic loss in the cultivation of largemouth bass, but there was no effective treatment. Here, the present study constructed a recombinant Pichia pastoris expressing LMBV major capsid protein (MCPD). The recombinant GS115-pW317-MCPD was then used to immunize largemouth bass via oral administration, and mucosal immune response mediated by immunoglobulins (Igs) was measured after oral immunization. Serum antibody levels were measured by ELISA, neutralizing antibody titers were determined by serum neutralization test (SNT), antigen presentation-related gene expressions were detected by RT-PCR, and the histopathological characteristics of immunized fish were assessed after challenging with 0.1 ml 107.19 TCID50/ml LMBV. The relative percentage survival (RPS) was also determined. Our results showed that the serum antibody titers of immunized fish were significantly higher than that of control groups (P < 0.05). IgT and IgM expressions in gut were increased significantly after vaccination with GS115-pW317-MCPD; however, much stronger response in gut was observed as compared with gill. The expression levels of major histocompatibility complex (MHC) II, CD8, and T-cell receptor (TCR) were significantly elevated in GS115-pW317-MCPD group (P < 0.05), while CD4 and MHC I transcription levels remained unchanged after oral immunization (P > 0.05). The RPS of fish orally immunized with 1.0 × 108 CFU/g GS115-pW317-MCPD was reached up to 41.6% after challenge with 0.1 ml 109.46 TCID50/ml LMBV. Moreover, orally immunizing with GS115-pW317-MCPD can relieve the pathological damage caused by LMBV. Therefore, GS115-pW317-MCPD showed a promising potential against LMBV.

Multi-Transcriptomic Analysis Reveals the Heterogeneity and Tumor-Promoting Role of SPP1/CD44-Mediated Intratumoral Crosstalk in Gastric Cancer.

GC is a fatal disease with high heterogeneity and invasiveness. Recently, SPP1 has been reported to be involved in the tumor progression of multiple human cancers; however, the role of SPP1 in GC heterogeneity and whether it is associated with the invasiveness and mortality of GC remain unclear. Here, we combined multiple RNA sequencing approaches to evaluate the impact of SPP1 on GC. Through bulk RNA sequencing (bulk RNA-seq) and immunohistochemistry (IHC), we found that SPP1 was highly expressed in GC, and high levels of SPP1 were associated with macrophage infiltration, an advanced tumor stage, and higher mortality for advanced GC patients. Furthermore, through simultaneous single-cell and spatial analysis, we demonstrated that SPP1+ macrophages are tumor-specific macrophages unique to cancer and enriched in the deep layer of GC tissue. Cell-cell communication analysis revealed that SPP1/CD44 interactions between SPP1+ macrophages and their localized tumor epithelial cells could activate downstream target genes in epithelial cells to promote dynamic changes in intratumor heterogeneity. Moreover, these activated genes were found to be closely associated with poor clinical GC outcomes and with cancer-related pathways that promote GC progression, as shown by survival analysis and enrichment analysis, respectively. Collectively, our study reveals that tumor-specific SPP1+ macrophages drive the architecture of intratumor heterogeneity to evolve with tumor progression and that SPP1 may serve as a prognostic marker for advanced GC patients, as well as a potential therapeutic target for GC.

Identification of a Tumor Microenvironment-relevant Gene set-based Prognostic Signature and Related Therapy Targets in Gastric Cancer.

Rationale: The prognosis of gastric cancer (GC) patients is poor, and there is limited therapeutic efficacy due to genetic heterogeneity and difficulty in early-stage screening. Here, we developed and validated an individualized gene set-based prognostic signature for gastric cancer (GPSGC) and further explored survival-related regulatory mechanisms as well as therapeutic targets in GC. Methods: By implementing machine learning, a prognostic model was established based on gastric cancer gene expression datasets from 1699 patients from five independent cohorts with reported full clinical annotations. Analysis of the tumor microenvironment, including stromal and immune subcomponents, cell types, panimmune gene sets, and immunomodulatory genes, was carried out in 834 GC patients from three independent cohorts to explore regulatory survival mechanisms and therapeutic targets related to the GPSGC. To prove the stability and reliability of the GPSGC model and therapeutic targets, multiplex fluorescent immunohistochemistry was conducted with tissue microarrays representing 186 GC patients. Based on multivariate Cox analysis, a nomogram that integrated the GPSGC and other clinical risk factors was constructed with two training cohorts and was verified by two validation cohorts. Results: Through machine learning, we obtained an optimal risk assessment model, the GPSGC, which showed higher accuracy in predicting survival than individual prognostic factors. The impact of the GPSGC score on poor survival of GC patients was probably correlated with the remodeling of stromal components in the tumor microenvironment. Specifically, TGFß and angiogenesis-related gene sets were significantly associated with the GPSGC risk score and poor outcome. Immunomodulatory gene analysis combined with experimental verification further revealed that TGFß1 and VEGFB may be developed as potential therapeutic targets of GC patients with poor prognosis according to the GPSGC. Furthermore, we developed a nomogram based on the GPSGC and other clinical variables to predict the 3-year and 5-year overall survival for GC patients, which showed improved prognostic accuracy than clinical characteristics only. Conclusion: As a tumor microenvironment-relevant gene set-based prognostic signature, the GPSGC model provides an effective approach to evaluate GC patient survival outcomes and may prolong overall survival by enabling the selection of individualized targeted therapy.

Inhibition of Bcl6b promotes gastric cancer by amplifying inflammation in mice.

BACKGROUND: Chronic gastritis has been demonstrated to be a key cause of gastric cancer (GC), and control of gastric inflammation is regarded as an effective treatment for the clinical prevention of gastric carcinogenesis. However, there remains an unmet need to identify the dominant regulators of gastric oncogenesis-associated inflammation in vivo. METHODS: The mouse model for the study of inflammation-associated GC was induced by Benzo[a]pyrene (BaP) intragastric administration in Bcl6b-/- and wildtype mice on a C57BL/6 background. 5-Aza-2'-deoxycytidine (5-Aza), the demethylation drug, was intraperitoneally injected to restore Bcl6b expression. Human GC tissue array was used to analyse patient survival based on BCL6B and CD3 protein expression. RESULTS: Bcl6b was gradually downregulated by its own promoter hypermethylation in parallel to an increasing inflammatory response during the progression of BaP-induced gastric carcinogenesis in mice. Moreover, knockout of Bcl6b dramatically worsened the severity of gastric cancer and aggravated the inflammatory response in the BaP-induced mice GC model. Re-activation of Bcl6b by 5-Aza impeded inflammatory amplification and BaP-induced GC development, prolonging survival time in wildtype mice, whereas no notable curative effect occurred in Bcl6b-/- mice with 5-Aza treatment. Finally, significant negative correlations were detected between the mRNA levels of BCL6B and inflammatory cytokines in human GC tissues; patients harbouring BCL6B-negetive and severe-inflammation GC tumours were found to exhibit the shortest survival time. CONCLUSIONS: Epigenetic inactivation of Bcl6b promotes gastric cancer through amplification of the gastric inflammatory response in vivo and offers a new approach for GC treatment and regenerative medicine.

Tumor endothelial cell-derived cadherin-2 promotes angiogenesis and has prognostic significance for lung adenocarcinoma.

In lung cancer, antiangiogenic strategies targeting tumor-derived endothelial cells (TECs) afford a survival advantage, but the characteristics of TECs have not been comprehensively elucidated. Herein, high-purity (> 98%) TECs were obtained, and these cells retained expression of EC markers and exhibited high viability. ITRAQ-2DLC-MS/MS was performed to profile the proteome and the heterogeneity of ECs. Only 31 of 1820 identified proteins were differentially expressed between adenocarcinoma (ADC)- and squamous cell carcinoma (SCC)-derived TECs (TEC-A and TEC-S, respectively), and cadherin-2 (CDH2) was the most significantly upregulated protein in TEC-A samples. Positive immunostaining for CDH2 (score > 3) was significantly more frequent in the endothelium of ADC tissues than in that of SCC tissues. Loss- or gain-of-function analysis showed that CDH2 significantly promoted in vitro and in vivo angiogenesis and sensitivity to the antagonist exherin. The MAPK/ERK and MAPK/JNK signaling pathways may play crucial roles in CDH2-induced HIF-1α/VEGF-mediated angiogenesis. Moreover, high CDH2 expression in TECs was significantly associated with tumor stage, visceral pleural metastasis, and decreased overall survival in patients with ADC but not SCC. Together, these data indicate the importance of CDH2 in angiogenesis and highlight its potential both for antiangiogenic therapy and as a candidate prognostic marker for ADC.

Regulatory network of circRNA-miRNA-mRNA contributes to the histological classification and disease progression in gastric cancer.

BACKGROUND: Little has been known about the role of non-coding RNA regulatory network in the patterns of growth and invasiveness of gastric cancer (GC) development. METHODS: MicroRNAs (miRNAs) microarray was used to screen differential miRNA expression profiles in Ming's classification. The significant differential expressions of representative miRNAs and their interacting circular RNA (circRNA) were confirmed in GC cell line and 63 pairs of GC samples. Then, a circRNA/miRNA network was constructed by bioinformatics approaches to identify molecular pathways. Finally, we explored the clinical value of the common targets in the pathway by using receiver operating characteristic curve and survival analysis. RESULTS: Significantly differential expressed miRNAs were found in two pathological types of GC. Both of miR-124 and miR-29b were consistently down-regulated in GC. CircHIPK3 could play a negative regulatory role on miR-124/miR-29b expression and associated with T stage and Ming's classification in GC. The bioinformatics analyses showed that targets expression of circHIPK3-miR-124/miR-29b axes in cancer-related pathways was able to predict the status of GC and associated with individual survival time. CONCLUSIONS: The targets of circHIPK3-miR-124/miR-29b axes involved in the progression of GC. CircHIPK3 could take part in the proliferation process of GC cell and may be potential biomarker in histological classification of GC.

Tumor-originated exosomal lncUEGC1 as a circulating biomarker for early-stage gastric cancer.

Conventional tumor markers for non-invasive diagnosis of gastric cancer (GC) exhibit insufficient sensitivity and specificity to facilitate detection of early gastric cancer (EGC). We aimed to identify EGC-specific exosomal lncRNA biomarkers that are highly sensitive and stable for the non-invasive diagnosis of EGC. Hence, in the present study, exosomes from the plasma of five healthy individuals and ten stage I GC patients and from culture media of four human primary stomach epithelial cells and four gastric cancer cells (GCCs) were isolated. Exosomal RNA profiling was performed using RNA sequencing to identify EGC-specific exosomal lncRNAs. A total of 79 and 285 exosomal RNAs were expressed at significantly higher levels in stage I GC patients and GCCs, respectively, than that in normal controls. Through combinational analysis of the RNA sequencing results, we found two EGC-specific exosomal lncRNAs, lncUEGC1 and lncUEGC2, which were further confirmed to be remarkably up-regulated in exosomes derived from EGC patients and GCCs. Furthermore, stability testing demonstrates that almost all the plasma lncUEGC1 was encapsulated within exosomes and thus protected from RNase degradation. The diagnostic accuracy of exosomal lncUEGC1 was evaluated, and lncUEGC1 exhibited AUC values of 0.8760 and 0.8406 in discriminating EGC patients from healthy individuals and those with premalignant chronic atrophic gastritis, respectively, which was higher than the diagnostic accuracy of carcinoembryonic antigen. Consequently, exosomal lncUEGC1 may be promising in the development of highly sensitive, stable, and non-invasive biomarkers for EGC diagnosis.

Yes-associated protein/TEA domain family member and hepatocyte nuclear factor 4-alpha (HNF4α) repress reciprocally to regulate hepatocarcinogenesis in rats and mice.

Great progress has been achieved in the study of Hippo signaling in regulating tumorigenesis; however, the downstream molecular events that mediate this process have not been completely defined. Moreover, regulation of Hippo signaling during tumorigenesis in hepatocellular carcinoma (HCC) remains largely unknown. In the present study, we systematically investigated the relationship between Yes-associated protein/TEA domain family member (YAP-TEAD) and hepatocyte nuclear factor 4-alpha (HNF4α) in the hepatocarcinogenesis of HCC cells. Our results indicated that HNF4α expression was negatively regulated by YAP1 in HCC cells by a ubiquitin proteasome pathway. By contrast, HNF4α was found to directly associate with TEAD4 to compete with YAP1 for binding to TEAD4, thus inhibiting the transcriptional activity of YAP-TEAD and expression of their target genes. Moreover, overexpression of HNF4α was found to significantly compromise YAP-TEAD-induced HCC cell proliferation and stem cell expansion. Finally, we documented the regulatory mechanism between YAP-TEAD and HNF4α in rat and mouse tumor models, which confirmed our in vitro results. CONCLUSION: There is a double-negative feedback mechanism that controls TEAD-YAP and HNF4α expression in vitro and in vivo, thereby regulating cellular proliferation and differentiation. Given that YAP acts as a dominant oncogene in HCC and plays a crucial role in stem cell homeostasis and tissue regeneration, manipulating the interaction between YAP, TEADs, and HNF4α may provide a new approach for HCC treatment and regenerative medicine. (Hepatology 2017;65:1206-1221).

Interleukine-1beta and interleukine-6 levels in striatum and other brain structures after MPTP treatment: influence of behavioral lateralization.

MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induces diminution of the dopamine in nigrostriatal pathway and cognitive deficits in mice. MPTP treatment also increases pro-inflammatory cytokine production in substantia nigra and striatum. Since, pro-inflammatory cytokines influence striatal dopamine content and provoke cognitive impairments, the cognitive defects induced by MPTP may be partly due to brain cytokine induction in other structures than nigrostriatal pathway. Furthermore, behavioral lateralization, as assessed by paw preference, influences cytokine production at the periphery and in the central nervous system. Behavioral lateralization may thus influence brain cytokine levels after MPTP. In order to address these issues, mice selected for paw preference were injected with 25 mg/kg MPTP i.p. for five consecutive days after which striatal dopamine and DOPAC contents were measured by HPLC and IL-1beta and IL-6 quantified by ELISA in the striatum, cerebral cortex, hippocampus and hypothalamus. The results showed that MPTP treatment induced dramatic loss of DA in striatum, simultaneously, IL-6 levels decreased in the striatum and increased in hippocampus and hypothalamus, while IL-1beta levels decreased in the striatum, cerebral cortex and hippocampus. Interestingly, striatal dopamine turnover under basal conditions as well as striatal IL-1beta and IL-6 levels under basal conditions and after MPTP depended on behavioral lateralization. Left pawed mice showed a higher decrease in dopamine turnover and lower cytokine levels as compared to right pawed animals. Behavioral lateralization also influenced IL-6 hippocampal levels under basal conditions and IL-1beta cortical levels after MPTP. From these results, it can be concluded that MPTP-induced cognitive defects are accompanied by an alteration of pro-inflammatory cytokine levels in brain structures other than those involved in the nigrostriatal pathway. In addition, MPTP-induced dopamine decrease is influenced by behavioral lateralization, possibly through an effect on brain cytokine levels.