Yong-liang Zhao

Plasma microRNAs, miR-223, miR-21 and miR-218, as Novel Potential Biomarkers for Gastric Cancer Detection

Background MicroRNAs (miRNAs), endogenous small non-coding RNAs, are stably detected in human plasma. Early diagnosis of gastric cancer (GC) is very important to improve the therapy effect and prolong the survival of patients. We aimed to identify whether four miRNAs (miR-223, miR-21, miR-218 and miR-25) closely associated with the tumorigenesis or metastasis of GC can serve as novel potential biomarkers for GC detection. Methodology We initially measured the plasma levels of the four miRNAs in 10 GC patients and 10 healthy control subjects by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and then compared plasma miRNA results with the expressions in cancer tissues from eight GC patients. Finally, the presence of miR-223, miR-21 and miR-218 in the plasma was validated in 60 GC patients and 60 healthy control subjects, and the areas under the receiver operating characteristic (ROC) curves of these miRNAs were analyzed. Results We found that the plasma levels of miR-223 (P<0.001) and miR-21 (P<0.001) were significantly higher in GC patients than in healthy controls, while miR-218 (P<0.001) was significantly lower. The ROC analyses yielded the AUC values of 0.9089 for miR-223, 0.7944 for miR-21 and 0.7432 for miR-218, and combined ROC analysis revealed the highest AUC value of 0.9531 in discriminating GC patients from healthy controls. Moreover, the plasma levels of miR-223 (P<0.001) and miR-21 (P = 0.003) were significantly higher in GC patients with stage I than in healthy controls. Furthermore, the plasma levels of miR-223 were significantly higher in GC patients with helicobacter pylori (Hp) infection than those without (P = 0.014), and significantly higher in healthy control subjects with Hp infection than those without (P = 0.016). Conclusions Plasma miR-223, miR-21 and miR-218 are novel potential biomarkers for GC detection.

Increased intratumoral IL-22-producing CD4 + T cells and Th22 cells correlate with gastric cancer progression and predict poor patient survival

IL-22-producing CD4+ T cells (IL-22+CD4+ T cells) and Th22 cells (IL-22+IL-17−IFN-γ−CD4+ T cells) represent newly discovered T-cell subsets, but their nature, regulation, and clinical relevance in gastric cancer (GC) are presently unknown. In our study, the frequency of IL-22+CD4+ T cells in tumor tissues from 76 GC patients was significantly higher than that in tumor-draining lymph nodes, non-tumor, and peritumoral tissues. Most intratumoral IL-22+CD4+ T cells co-expressed IL-17 and IFN-γ and showed a memory phenotype. Locally enriched IL-22+CD4+ T cells positively correlated with increased CD14+ monocytes and IL-6 and IL-23 detection ex vivo, and in vitro IL-6 and IL-23 induced the polarization of IL-22+CD4+ T cells in a dose-dependent manner and the polarized IL-22+CD4+ T cells co-expressed of IL-17 and IFN-γ. Moreover, IL-22+CD4+ T-cell subsets (IL-22+IL-17+CD4+, IL-22+IL-17−CD4+, IL-22+IFN-γ+CD4+, IL-22+IFN-γ−CD4+, and IL-22+IL-17+IFN-γ+CD4+ T cells), and Th22 cells were also increased in tumors. Furthermore, higher intratumoral IL-22+CD4+ T-cell percentage and Th22-cell percentage were found in patients with tumor-node-metastasis stage advanced and predicted reduced overall survival. In conclusion, our data indicate that IL-22+CD4+ T cells and Th22 cells are likely important in establishing the tumor microenvironment for GC; increased intratumoral IL-22+CD4+ T cells and Th22 cells are associated with tumor progression and predict poorer patient survival, suggesting that tumor-infiltrating IL-22+CD4+ T cells and Th22 cells may be suitable therapeutic targets in patients with GC.

Increased Circulating Th22 and Th17 Cells are Associated with Tumor Progression and Patient Survival in Human Gastric Cancer

Although Th22 and Th17 cells have been reported to play critical roles during autoimmunity and inflammation, information on their role in cancer-immunity is limited. In this study, we investigated clinical relevance of circulating Th22 and Th17 cells in patients with gastric cancer (GC). Using multi-color flow cytometry and PMA stimulation, we determined the levels of Th22, Th17 and Th1 cells in the peripheral blood of 32 GC patients and 19 healthy donors, and evaluated their correlations with tumor stage and overall survival. Compared with healthy donors, the frequencies of circulating CD4+IL-22+ T cells, CD4+IL-17+ T cells, Th22 (CD4+IL-22+IL-17-INF-γ−) cells, Th17 (CD4+IL-17+INF-γ−) cells were increased in patients with GC, but there was no significant differences in the frequencies of CD4+IFN-γ+ T cells and Th1 (CD4+IL-17−INF-γ+) cells. Th22 cells showed positive correlation with Th17 cells and CD4+IL-17+ T cells in patients with GC. Furthermore, the frequencies of Th22 and Th17 cells were significantly higher in stage III–IV GC patients versus stage I–II and correlated with patients’ overall survival. These data suggest that circulating Th22 cells as well as Th17 cells are increased in the peripheral blood of GC patients with tumor progression, and that these cells may be promising novel clinical markers for GC.

Increased tumor-infiltrating CD8+Foxp3+ T lymphocytes are associated with tumor progression in human gastric cancer

BackgroundCD8+Foxp3+ T lymphocytes have been detected in tumors. However, the distribution, phenotypic features, and regulation of these cells in gastric cancer remain unknown.MethodsThe levels of CD8+Foxp3+ T lymphocytes in the peripheral blood, tumor-draining lymph nodes, non-tumor tissues, and tumor tissues of patients with gastric cancer were detected by flow cytometry. Foxp3 induction in CD8+Foxp3− T cells was investigated in vitro. The suppressive function of CD8+Foxp3+ T lymphocytes was analyzed by their effect on CD4+ T-cell proliferation and IFN-γ production. The percentages of CD8+Foxp3+ T lymphocytes were evaluated for the association with tumor stage.ResultsThe frequency of CD8+Foxp3+ T lymphocytes in tumor tissues was significantly higher than that in non-tumor tissues, and similar results were also observed in tumor-draining lymph nodes compared with peripheral blood. Most intratumoral CD8+Foxp3+ T lymphocytes were activated effector cells (CD45RA−CD27−). TGF-β1 levels were positively correlated with the frequency of CD8+Foxp3+ T lymphocytes in tumor tissues, and in vitro TGF-β1 could induce the generation of CD8+Foxp3+ T lymphocytes in a dose-dependent manner. Furthermore, intratumoral CD8+Foxp3+ T lymphocytes suppressed the proliferation and IFN-γ production of CD4+ T cells. Finally, intratumoral CD8+Foxp3+ T lymphocytes were significantly increased with tumor progression in terms of tumor-node-metastasis (TNM) stage.ConclusionsOur data have shown that increased intratumoral CD8+Foxp3+ T lymphocytes are associated with tumor stage and potentially influence CD4+ T-cell functions, which may provide insights for developing novel immunotherapy protocols against gastric cancer.

Tumour-activated neutrophils in gastric cancer foster immune suppression and disease progression through GM-CSF-PD-L1 pathway

Objective Neutrophils are prominent components of solid tumours and exhibit distinct phenotypes in different tumour microenvironments. However, the nature, regulation, function and clinical relevance of neutrophils in human gastric cancer (GC) are presently unknown. Design Flow cytometry analyses were performed to examine levels and phenotype of neutrophils in samples from 105 patients with GC. Kaplan-Meier plots for overall survival were performed using the log-rank test. Neutrophils and T cells were isolated, stimulated and/or cultured for in vitro and in vivo regulation and function assays. Results Patients with GC showed a significantly higher neutrophil infiltration in tumours. These tumour-infiltrating neutrophils showed an activated CD54+ phenotype and expressed high level immunosuppressive molecule programmed death-ligand 1 (PD-L1). Neutrophils activated by tumours prolonged their lifespan and strongly expressed PD-L1 proteins with similar phenotype to their status in GC, and significant correlations were found between the levels of PD-L1 and CD54 on tumour-infiltrating neutrophils. Moreover, these PD-L1+ neutrophils in tumours were associated with disease progression and reduced GC patient survival. Tumour-derived GM-CSF activated neutrophils and induced neutrophil PD-L1 expression via Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) signalling pathway. The activated PD-L1+ neutrophils effectively suppressed normal T-cell immunity in vitro and contributed to the growth and progression of human GC in vivo; the effect could be reversed by blocking PD-L1 on these neutrophils. Conclusions Our results illuminate a novel mechanism of PD-L1 expression on tumour-activated neutrophils in GC, and also provide functional evidence for these novel GM-CSF-PD-L1 pathways to prevent, and to treat this immune tolerance feature of GC.

Aberrant expression of Cx43 is associated with the peritoneal metastasis of gastric cancer and Cx43-mediated gap junction enhances gastric cancer cell diapedesis from peritoneal mesothelium.

The process of peritoneal metastasis involves the diapedesis of intra-abdominal exfoliated gastric cancer cells through the mesothelial cell monolayers; however, the related molecular mechanisms for this process are still unclear. Heterocellular gap-junctional intercellular communication (GJIC) between gastric cancer cells and mesothelial cells may play an active role during diapedesis. In this study we detected the expression of connexin 43 (Cx43) in primary gastric cancer tissues, intra-abdominal exfoliated cancer cells, and matched metastatic peritoneal tissues. We found that the expression of Cx43 in primary gastric cancer tissues was significantly decreased; the intra-abdominal exfoliated cancer cells and matched metastatic peritoneal tissues exhibited increasing expression compared with primary gastric cancer tissues. BGC-823 and SGC-7901 human gastric cancer cells were engineered to express Cx43 or Cx43T154A (a mutant protein that only couples gap junctions but provides no intercellular communication) and were co-cultured with human peritoneal mesothelial cells (HPMCs). Heterocellular GJIC and diapedesis through HPMC monolayers on matrigel-coated coverslips were investigated. We found that BGC-823 and SGC-7901 gastric cancer cells expressing Cx43 formed functional heterocellular gap junctions with HPMC monolayers within one hour. A significant increase in diapedesis was observed in engineered Cx43-expressing cells compared with Cx43T154A and control group cells, which suggested that the observed upregulation of diapedesis in Cx43-expressing cells required heterocellular GJIC. Further study revealed that the gastric cancer cells transmigrated through the intercellular space between the mesothelial cells via a paracellular route. Our results suggest that the abnormal expression of Cx43 plays an essential role in peritoneal metastasis and that Cx43-mediated heterocellular GJIC between gastric cancer cells and mesothelial cells may be an important regulatory step during metastasis. Finally, we observed that the diapedesis of exfoliated gastric cancer cells through mesothelial barriers is a viable route of paracellular migration.

Cumulative summation analysis of learning curve for robot-assisted gastrectomy in gastric cancer

The purpose of this study was to determine the learning curve for robot‐assisted gastrectomy using the Cumulative Summation (CUSUM) technique.

HIF-1α induces the epithelial-mesenchymal transition in gastric cancer stem cells through the Snail pathway

Substantial evidence suggests that the epithelial-mesenchymal transition (EMT) phenotype is associated with the invasive characteristics of cancer stem cells (CSCs),which possess an EMT phenotype that may predominate in tumor invasion and metastasis. However, the mechanisms for the generation and regulation of these CSCs have not been clearly defined. As hypoxia and EMT-related factors may have important functions in EMT-like CSCs, the aim of this study was to investigate the effects of hypoxia on these cells. CSCs were established from the gastric cancer cell lines MGC-803 and SGC7901, and the relationship between hypoxia and EMT-like CSCs was investigated in gastric cancer. After hypoxia treatment, some gastric CSCs exhibited a marked increase in hypoxia-inducible factor-1α (HIF-1α)expression and increased migration and invasion capabilities compared with the normoxic control. These CSCs were defined by activation of the mesenchymal cell marker Vimentin and by inhibition of the epithelial cell marker E-cadherin. Our analyses also show that HIF-1α was responsible for activating EMT via increased expression of the transcription factor Snail in gastric CSCs. Moreover, inhibition of Snail by shRNA reduced HIF-1α-induced EMT in gastric CSCs. The results demonstrated that hypoxia-induced EMT-like CSCs rely on HIF-1αto activate Snail, which may result in recurrence and metastasis of gastric cancer.

Tumor-Associated Monocytes/Macrophages Impair NK-Cell Function via TGFβ1 in Human Gastric Cancer

Patients with gastric cancer had few NK cells infiltrating their tumors, a condition associated with tumor progression and poor survival. These intratumoral NK cells were functionally impaired, due to the presence of TGFβ1 derived from tumor-associated monocytes/macrophages. Natural killer (NK) cells are a major component of the host antitumor immune response in human cancer. However, the nature, functional regulation, and clinical relevance of NK cells in gastric cancer remain largely unknown. In this study, we showed that the percentages of NK cells in tumors were significantly decreased, and low percentages of tumor-infiltrating NK cells were positively correlated with poor survival and disease progression. Although the expression of activating and inhibitory receptors on NK cells was shown to be not different between tumor and nontumor tissues, NK cells in tumors had impaired effector functions, characterized by decreased IFNγ, TNFα, and Ki-67 expression. We found that tumor-infiltrating monocytes/macrophages were physically close to NK cells, and their percentages negatively correlated with IFNγ+ and TNFα+ NK-cell percentages. Ex vivo study showed that isolated tumor-associated monocytes/macrophages could impair NK-cell expression of IFNγ, TNFα, and Ki-67. Blockade of TGFβ1 attenuated such monocytes/macrophages-mediated impairment of NK-cell function. Our data suggest that human NK-cell function was impaired by tumor-associated monocytes/macrophages, and that restoring NK-cell function may be an important therapeutic strategy to prevent tumor immune escape in gastric cancer. Cancer Immunol Res; 5(3); 248–56. ©2017 AACR.

Interleukin 6 induces M2 macrophage differentiation by STAT3 activation that correlates with gastric cancer progression

Interleukin 6 (IL-6) was abundant in the tumor microenvironment and played potential roles in tumor progression. In our study, the expression of IL-6 in tumor tissues from 36 gastric cancer (GC) patients was significantly higher than in non-tumor tissues. Moreover, the number of CD163+CD206+ M2 macrophages that infiltrated in tumor tissues was significantly greater than those infiltrated in non-tumor tissues. The frequencies of M2 macrophages were positively correlated with the IL-6 expression in GC tumors. We also found that IL-6 could induce normal macrophages to differentiate into M2 macrophages with higher IL-10 and TGF-β expression, and lower IL-12 expression, via activating STAT3 phosphorylation. Accordingly, knocking down STAT3 using small interfering RNA decreased the expression of M2 macrophages-related cytokines (IL-10 and TGF-β). Furthermore, supernatants from IL-6-induced M2 macrophages promote GC cell proliferation and migration. Moreover, IL-6 production and CD163+CD206+ M2 macrophage infiltration in tumors were associated with disease progression and reduced GC patient survival. In conclusion, our data indicate that IL-6 induces M2 macrophage differentiation (IL-10highTGF-βhighIL-12p35low) by activating STAT3 phosphorylation, and the IL-6-induced M2 macrophages exert a pro-tumor function by promoting GC cell proliferation and migration.