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Supplementary Figure S3. Identification of cytokines that are upregulated in AGS and U937 coculture. A, conditioned media of AGS monoculture and coculture of AGS and U937 cells was collected and subjected to cytokine arrays. B, gastric cancer cells were treated with recombinant proteins of CCL2, CCL5, IL-8, and CXCL10 for 24 hours, and the lysates were collected for detection of DNMT1 using Western blotting.

Epigenetic repression of the tumor suppressor gelsolin (GSN) is frequently observed in cancers. Chronic inflammation can promote tumor progression via aberrant DNA methylation. In this study, we investigated the role of tumor-associated macrophages (TAMs) in DNA methylation of the GSN gene during gastric cancer progression. Immunofluorescence staining of 121 gastric cancer tissues showed aberrant localization of GSN and DNA methyltransferase 1 (DNMT1) and juxtaposition of DNMT1 and M2 TAMs. Decreased GSN protein and mRNA expression and increased DNA methylation in the GSN promoter were observed in gastric cancer cell lines and clinical specimens. To examine the effect of TAMs on DNA methylation in gastric cancer cells, we performed in vitro coculture assays and found increased DNMT1 expression but decreased GSN expression in gastric cancer cells after coculture with U937 cells. Knockdown of DNMT1 expression in gastric cancer cells could abort U937 coculture-mediated GSN downregulation. Meanwhile, CCL5 was the main chemokine upregulated in coculture medium. Treatment with CCL5 could induce DNMT1 expression in gastric cancer cells via STAT3 signaling. Inhibiting DNMT1 activity with procainamide, inhibiting DNA methylation with 5-AZA, or inhibiting CCL5/CCR5 signaling with maraviroc reduced tumor growth in vivo. In conclusion, upregulation of DNMT1 by CCL5/CCR5/STAT3 signaling is critical for TAM-mediated GSN silencing in gastric cancer. This study identified potential targets for gastric cancer therapy. Cancer Immunol Res; 5(10); 885–97. ©2017 AACR.

Table S1. Expression of GSN in 121 gastric tumors according to tumor stages

Supplementary Materials and Methods

Table S4. Expression of CD68 in 121 gastric tumors according to tumor stages

Table S2. Expression of DNMT1 in 121 gastric tumors according to tumor stages

Table S3. Expression of CD204 in 121 gastric tumors according to tumor stages

Primers used for ChIP experiments

List of primers used in real-time PCR experiments

Purpose:KIT mutations, the most prevalent genetic event in gastrointestinal stromal tumors (GIST), are associated with malignant features and poor prognosis. Aggressive GISTs possess a high propensity to spread to the liver. This study aimed to explore the role of KIT mutations in GIST liver metastasis.Experimental Design: A total of 170 GISTs were used to determine the association between KIT mutations and liver metastasis. Immunohistochemistry was performed to assess the correlation of KIT mutations with CXCR4 and ETV1 expression. Genetic and pharmacologic methods were used to study the regulation of CXCR4 and ETV1 by KIT mutations.Results: Codons 557 and 558 in KIT exon 11 were deletion hot spots in GISTs. KIT exon 11 deletions involving codons 557–558 were highly associated with liver metastasis. Overexpression of mutant KIT with exon 11 codons 557–558 deletion (KIT Δ557–558) increased GIST cell motility and liver metastasis. Mechanistically, overexpression of KIT Δ557–558 in GIST cells increased ETV1 and CXCR4 expression. CXCR4 knockdown counteracted KIT Δ557–558–mediated cell migration. Moreover, KIT Δ557–558–induced CXCR4 expression could be abolished by silencing ETV1. The chromatin immunoprecipitation assay showed that ETV1 directly bound to the CXCR4 promoter. After ERK inhibitor PD325901 treatment, the upregulation of ETV1 by KIT Δ557–558 was prevented. In addition, KIT exon 11 codons 557–558 deletion enhanced CXCL12-mediated GIST cell migration and invasion.Conclusions:KIT exon 11 557–558 deletion upregulates CXCR4 through increased binding of ETV1 to the CXCR4 promoter in GIST cells, which thus promotes liver metastasis. These findings highlighted the potential therapeutic targets for metastatic GISTs. Clin Cancer Res; 22(14); 3477–87. ©2016 AACR.

KIT and PDGFRα mutation types in primary GISTs with respect to liver metastasis

List of antibodies

Rich in protein and adjacent to pancreatic cancer cells, pancreatic juice has been considered an attractive source to discover biomarkers for pancreatic ductal adenocarcinoma (PDAC). Here, we identified a novel biomarker, TBC1D23 in pancreatic juice of PDAC patients. High TBC1D23 expression was also observed in tumor tissues and sera, associated with lymphatic metastasis and shorter survival. TBC1D23 was strongly expressed in PDAC cell lines, and knocking down TBC1D23 reduced cell proliferation and migration in vitro and suppressed xenograft tumor growth and peritoneal metastasis in vivo. DNA microarray analysis identified the down-regulation of VEGF-C in TBC1D23 knockdown cells compared to control. Indeed, depletion of TBC1D23 decreased VEGF-C expression and secretion. Treatment with conditioned medium from PANC-1 cells promoted lymphangiogenesis in lymphatic endothelial cells, while this effect was counteracted by the VEGFR3 inhibitor MAZ51 or knockdown of TBC1D23, indicating that VEGF-C is a target gene of TBC1D23. Mechanistically, TBC1D23 could increase EGFR membrane distribution, enhance EGFR recycling, but inhibit EGFR endocytic degradation, suggesting that TBC1D23 up-regulates VEGF-C by maintaining EGFR capacity to transduce signals. This study clearly demonstrates the oncogenic role of TBC1D23 and EGFR/VEGF-C as its downstream signaling pathway for lymphatic metastasis.

Background: M2 tumor-associated macrophages can promote pancreatic cancer progression. Under ROS, cancer cells derived IL-4 can skew M2 with uncertain mechanism. In this study, we tried to elucidate the mechanism and design gene therapy to treat pancreatic cancer by inhibiting M2 macrophages. Methods: We used CRISPR/Cas9 to knock out CYBB that encodes NOX2 in macrophages and adopted CYBB-/- B6 mice. Co-culture assay and syngeneic orthotopic pancreatic cancer mice models were used to study the mechanism in skewing M2 and tumor growth. The decoy oligodeoxynucleotides were designed to inhibit M2 formation and control the tumor growth. Results: Co-culture with pancreatic cancer cells induced M2 polarization at 72 h, while CYBB knockout prevented it. In the syngeneic orthotopic model, M2 macrophages count was decreased and tumor formation was inhibited in Cybb-/- mice. Under secretion of ROS in activated macrophages, cancer cell derived IL-4 can increase CD204 expression on macrophages. Chromatin immunoprecipitation study showed a binding site of STAT6 and HDAC2 in the promotor region of Cybb gene. Using epigenetic inhibitors or knocking down STAT6 and HDAC2 could block the M2 skewing with reducing the IL-10 expression and keep on M1 macrophages with increasing the IL-12 expression. Gene therapy using STAT6-CYBB decoy oligodeoxynucleotides to interrupt STAT6/HDAC2 binding to CYBB promoter could inhibit M2 formation and reduce tumor growth significantly.Conclusion: Gene therapy using STAT6-CYBB decoy ODN can inhibit M2 formation to treat pancreatic cancer.

Pancreatic cancer (PC) has the highest frequency of developing cancer cachexia (CC)–sarcopenia (SC) syndrome, which negatively influences patients’ outcome, quality of life, and tolerance/response to treatments. However, the clinical impacts of CC, SC, and their associated factors on outcomes for advanced PC has yet to be fully investigated. A total of 232 patients were enrolled in this study for the retrospective review of their clinical information and the measurement of skeletal muscle areas at the third lumber vertebra by computed tomography scan to identify CC or SC. The association and concurrent occurrence of clinicopathological features in each patient, prevalence rates, and prognosis with the CC or SC were calculated. CC and SC were observed in 83.6% (n = 194) and 49.1% (n = 114) of PC patients, respectively. Low hemoglobin levels more often occurred in CC patients than in non-CC patients (p = 0.014). Older age (p = 0.000), female gender (p = 0.024), low body mass index (BMI) values (p = 0.004), low hemoglobin levels (p = 0.036), and low albumin levels (p = 0.001) were more often found in SC patients than in non-SC patients. Univariate and multivariate analyses showed that CC was an independent poor prognostic factor of overall survival (OS) and progression-free survival for all patients, the chemotherapy (C/T) subgroup, and the high BMI subgroup. Meanwhile, SC was an independent predictor of poor OS for the subgroups of C/T or high BMI but not for all patients. These findings reveal the clinical differences for CC and SC and provide useful information for predicting the prognosis of advanced PC patients and conducting personalized medicine.

Semaphorins (SEMAs) are axon guidance factors that participate in axonal connections and nerve system development. However, the functional roles of SEMAs in tumorigenesis are still largely uncovered. By using in silico data analysis, we found that SEMA6C was downregulated in pancreatic cancer, and its reduction was correlated with worse survival rates. RNA sequencing revealed that cell cycle-related genes, especially cyclin D1, were significantly altered after blockage of SEMA6C by neutralizing antibodies or ectopic expressions of SEMA6C. Mechanistic investigation demonstrated that SEMA6C acts as a tumor suppressor in pancreatic cancer by inhibiting the AKT/GSK3 signaling axis, resulting in a decrease in cyclin D1 expression and cellular proliferation. The enhancement of cyclin D1 expression and cyclin-dependent kinase activation in SEMA6C-low cancer created a druggable target of CDK4/6 inhibitors. We also elucidated the mechanism underlying SEMA6C downregulation in pancreatic cancer and demonstrated a novel regulatory role of miR-124-3p in suppressing SEMA6C. This study provides new insights of SEMA6C-mediated anti-cancer action and suggests the treatment of SEMA6C-downregulated cancer by CDK4/6 inhibitors.

Background & Aims Limited data are available for tumor immune microenvironment (TIME) in Epstein-Barr virus (EBV)-associated lymphoepithelioma-like cholangiocarcinoma (EBV-LELCC), a rare subtype of intrahepatic cholangiocarcinoma (IHCC). We aimed to investigate TIME features in EBV-LELCC and the correlation between the components of TIME and the clinical outcomes. Methods Tumor tissues from Five EBV-LELCC cases confirmed through EBER in situ hybridization and five stage-matched conventional IHCC (non-EBV IHCC) cases were collected. These samples were used to evaluate genetic alterations, TIME composition, and PD-L1 expression through ion AmpliSeq comprehensive cancer panel, PanCancer immune profiling panel, immunohistochemistry, and immunofluorescence staining. The correlation between clinical outcomes and TIME components was analyzed in the two EBV-LELCC cases receiving anti-PD-1 treatment.Results The genetic mutations identified in EBV-LELCC were BARD1, CD19, CD79B, EPHA5, KDM5A, MUC6, MUC16, PTEN, RECQL4, TET1, and TNFAIP3. PD-L1 was highly expressed in tumor-infiltrating immune cells, especially the T cells and macrophages. The TIME of EBV-LELCC displayed abundant immune cell infiltration with a stronger adaptive immune response. Increased Th1 cells, NK CD56dim cells, and M1 macrophages, decreased M2 macrophages, exhausted CD8 T cell infiltration, and increased T cell activation signatures in TIME were associated with longer survival. Two patients with metastatic EBV-LELCC had good disease control after anti-PD-1 antibody treatment. A significantly larger TIME component made EBV-LELCCs more sensitive to immune checkpoint blockade (ICB). Conclusion A better understanding of the composition of TIME in EBV-LELCC is critical for predicting the clinical outcomes of ICB treatment.

Posttranscriptional modifications in RNA have been considered to contribute to disease pathogenesis and tumor progression. NOL1/NOP2/Sun domain family member 2 (NSUN2) is an RNA methyltransferase that promotes tumor progression in several cancers. Pancreatic cancer relapse inevitably occurs even in cases where primary tumors have been successfully treated. Associations of cancer progression due to reprogramming of the cancer methyl-metabolome and the cancer genome have been noted, but the effect of base modifications, namely 5-methylcytosine (m

Background: Chitinase 3-like-1 (CHI3L1) is a secretion glycoprotein associated with the immunosuppressive tumor microenvironment (TME). The secretory mode of CHI3L1 makes it a promising target for cancer treatment. We have previously reported that Rab37 small GTPase mediates secretion of IL-6 in macrophages to promote cancer progression, whereas the roles of Rab37 in the intracellular trafficking and exocytosis of CHI3L1 are unclear. Methods: We examined the concentration of CHI3L1 in the culture medium of splenocytes and bone marrow derived macrophages (BMDMs) from wild-type or Rab37 knockout mice, and macrophage or T cell lines expressing wild type, active GTP-bound or inactive GDP-bound Rab37. Vesicle isolation, total internal reflection fluorescence microscopy, and real-time confocal microscopy were conducted. We developed polyclonal neutralizing-CHI3L1 antibodies (nCHI3L1 Abs) to validate the therapeutic efficacy in orthotopic lung, pancreas and colon cancer allograft models. Multiplex fluorescence immunohistochemistry was performed to detect the protein level of Rab37 and CHI3L1, and localization of the tumor-infiltrating immune cells in allografts from mice or tumor specimens from cancer patients. Results: We demonstrate a novel secretion mode of CHI3L1 mediated by the small GTPase Rab37 in T cells and macrophages. Rab37 mediated CHI3L1 intracellular vesicle trafficking and exocytosis in a GTP-dependent manner, which is abolished in the splenocytes and BMDMs from Rab37 knockout mice and attenuated in macrophage or T cell lines expressing the inactive Rab37. The secreted CHI3L1 activated AKT, ß-catenin and NF-κB signal pathways in cancer cells and macrophages to foster a protumor TME characterized by activating M2 macrophages and increasing the population of regulatory T cells. Our developed nCHI3L1 Abs showed the dual properties of reducing tumor growth/metastases and eliciting an immunostimulatory TME in syngeneic orthotopic lung, pancreas and colon tumor models. Clinically, high plasma level or intratumoral expression of CHI3L1 correlated with poor survival in 161 lung cancer, 155 pancreatic cancer and 180 colon cancer patients. Conclusions: These results provide the first evidence that Rab37 mediates CHI3L1 secretion in immune cells and highlight nCHI3L1 Abs that can simultaneously target both cancer cells and tumor microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and treatment‐resistant malignancy. The lack of pathway‐informed biomarkers hampers the development of rational diagnostics or therapies. Recently, the protein abnormal spindle‐like microcephaly‐associated (ASPM) was identified as a novel Wnt and stemness regulator in PDAC, while the pathogenic roles of its protein isoforms remain unclarified. We developed novel isoform‐specific antibodies and genetic knockdown (KD) of putative ASPM isoforms, whereby we uncovered that the levels of ASPM isoform 1 (iI) and ASPM‐iII are variably upregulated in PDAC cells. ASPM isoforms show remarkably different subcellular locations; specifically, ASPM‐iI is exclusively localized to the cortical cytoplasm of PDAC cells, while ASPM‐iII is predominantly expressed in cell nuclei. Mechanistically, ASPM‐iI co‐localizes with disheveled‐2 and active β‐catenin as well as the stemness marker aldehyde dehydrogenase‐1 (ALDH‐1), and its expression is indispensable for the Wnt activity, stemness, and the tumorigenicity of PDAC cells. By contrast, ASPM‐iII selectively regulates the expression level of cyclin E and cell cycle progression in PDAC cells. The expression of ASPM‐iI and ASPM‐iII displays considerable intratumoral heterogeneity in PDAC tissues and only that of ASPM‐iI was prognostically significant; it outperformed ALDH‐1 staining and clinico‐pathological variables in a multivariant analysis. Collectively, the distinct expression patterns and biological functions of ASPM isoforms may illuminate novel molecular mechanisms and prognosticators in PDAC and may pave the way for the development of therapies targeting this novel oncoprotein. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Background: The dense fibrotic stroma enveloping pancreatic tumors is a major cause of drug resistance. Pancreatic stellate cells (PSCs) in the stroma can be activated to induce intra-tumor fibrosis and worsen patient survival; however, the molecular basics for the regulation of PSC activation remains unclear. Methods: The in vitro coculture system was used to study cancer cell-PSC interactions. Atomic force microscopy was used to measure the stiffness of tumor tissues and coculture gels. Cytokine arrays, qPCR, and Western blotting were performed to identify the potential factors involved in PSC activation and to elucidate underlying pathways. Results: PSC activation characterized by α-SMA expression was associated with increased pancreatic tumor stiffness and poor prognosis. Coculture with cancer cells induced PSC activation, which increased organotypic coculture gel stiffness and cancer cell invasion. Cancer cells-derived PAI-1 identified from coculture medium could activate PSCs, consistent with pancreatic cancer tissue microarray analysis showing a strong positive correlation between PAI-1 and α-SMA expression. Suppression by knocking down PAI-1 in cancer cells demonstrated the requirement of PAI-1 for coculture-induced PSC activation and gel stiffness. PAI-1 could be upregulated by KRAS in pancreatic cancer cells through ERK. In PSCs, inhibition of LRP-1, ERK, and c-JUN neutralized the effect of PAI-1, suggesting the contribution of LRP-1/ERK/c-JUN signaling. Furthermore, activated PSCs might exacerbate malignant behavior of cancer cells via IL-8 because suppression of IL-8 signaling reduced pancreatic tumor growth and fibrosis in vivo. Conclusions: KRAS-mutant pancreatic cancer cells can activate PSCs through PAI-1/LRP-1 signaling to promote fibrosis and cancer progression.

Due to the increasing incidence of malignant gliomas, particularly glioblastoma multiforme (GBM), a simple and reliable GBM diagnosis is needed to screen early the death-threaten patients. This study aimed to identify a protein that can be used to discriminate GBM from low-grade astrocytoma and elucidate further that it has a functional role during malignant glioma progressions. To identify proteins that display low or no expression in low-grade astrocytoma but elevated levels in GBM, glycoprotein fibronectin (FN) was particularly examined according to the mining of the Human Protein Atlas. Web-based open megadata minings revealed that FN was mainly mutated in the cBio Cancer Genomic Portal but dominantly overexpressed in the ONCOMINE (a cancer microarray database and integrated data-mining platform) in distinct tumor types. Furthermore, numerous different cancer patients with high FN indeed exhibited a poor prognosis in the PrognoScan mining, indicating that FN involves in tumor malignancy. To investigate further the significance of FN expression in glioma progression, tumor specimens from five malignant gliomas with recurrences that received at least two surgeries were enrolled and examined. The immunohistochemical staining showed that FN expression indeed determined the distinct progressions of malignant gliomas. Furthermore, the expression of vimentin (VIM), a mesenchymal protein that is strongly expressed in malignant cancers, was similar to the FN pattern. Moreover, the level of epithelial–mesenchymal transition (EMT) inducer transforming growth factor-beta (TGF-β) was almost recapitulated with the FN expression. Together, this study identifies a protein FN that can be used to diagnose GBM from low-grade astrocytoma, moreover, its expression functionally determines the malignant glioma progressions via TGF-β-induced EMT pathway.

Background Cancer subtype switching, which involves unclear cancer cell origin, cell fate decision, and transdifferentiation of cells within a confined tumor microenvironment, remains a major problem in pancreatic cancer (PDA). Results By analyzing PDA subtypes in The Cancer Genome Atlas, we identified that epigenetic silencing of apoptosis-associated tyrosine kinase (AATK) inversely was correlated with mRNA expression and was enriched in the quasi-mesenchymal cancer subtype. By comparing early mouse pancreatic lesions, the non-invasive regions showed AATK co-expression in cells with acinar-to-ductal metaplasia, nuclear VAV1 localization, and cell cycle suppression; but the invasive lesions conversely revealed diminished AATK expression in those with poorly differentiated histology, cytosolic VAV1 localization, and co-expression of p63 and HNF1α. Transiently activated AATK initiates acinar differentiation into a ductal cell fate to establish apical-basal polarization in acinar-to-ductal metaplasia. Silenced AATK and ectopically expressed p63 and HNF1α allow the proliferation of ductal PanINs in mice. Conclusion Epigenetic silencing of AATK regulates the cellular transdifferentiation, proliferation, and cell cycle progression in converting PDA-subtypes.

Background: Exon 11 deletion of c-KIT is the common mutation site with the worst prognosis of gastrointestinal stromal tumors (GISTs). Autophagy has shown to have both protumor and antitumor functions and protect GIST cells from imatinib (IM)-induced apoptosis. In this study, we aimed to explore whether c-KIT exon 11 deletions of GISTs drive refractory to IM treatment through regulation of autophagy.Methods: 251 GISTs were included in this study to determine the association of c-KIT exon 11 deletions with prognosis and IM resistance. Immunohistochemistry, immunofluorescence, Western blotting were used to assess the correlation between c-KIT exon 11 deletions and autophagy and the underlying mechanism. We also studied the antitumor effect of IM in combination with the autophagy inhibitor chloroquine (CQ) in GIST62 cell derived xenograft mice model.Results: GISTs with c-KIT exon 11 deletions had worse survival and IM-resistant phenotype and exhibited higher autophagy levels as compared with GISTs harboring wild type or other c-KIT mutations. In addition, LC3-II expression strongly increased in GIST cell lines carrying c-KIT exon 11 deletions. Treated with autophagy inhibitors or knockdown of the autophagy-related gene ATG5 effectively inhibit GIST cells viability. Co-treatment with IM and CQ significantly suppressed tumor growth as compared with either agent alone, suggesting that autophagy protect GIST cells from IM-induced cell death both in vitro and in vivo. Furthermore, c-KIT exon 11 deletions can induce autophagy by activating SRC and ULK1. SRC inhibitor can block this action.Conclusion: Our study clearly demonstrates that KIT exon 11 deletions induce ULK1-SRC related autophagy is one of reasons for imatinib resistance clinically. Combination with autophagy inhibitor shows significant promise in treatment for imatinib refractory GISTs.

Additional file 1: Figure S1. The TCGA cohort patients formed clusters of: (1) benign (n=36), (2) early development and differentiation (n=86), (3) transition (n=36), and (4) QM-PDA (n=20) based on differential gene expression patterns.

Additional file 6: Table S1. Survival analysis of VAV1 targeted the cell cycle pathway gene network and cytokeratin 6B expression in a combined analysis of 1207 pancreatic cancer samples from 10 studies included in the cBioPortal [61, 62].

Additional file 7: Table S2. Details of reagents and materials. #, not available; *, in the TSA IHC Kit a higher dilution factor was chosen for optimized signal to background ratio.

Additional file 2: Figure S2. Collection of AATK expression in ADM in KC mice. Insert, higher magnification of Fig. 7a, region 1.

Additional file 3: Figure S3. miR-338-3p in situ hybridization in adjacent normal and tumoral sections of pancreatic cancer. U6 snRNA expression was used as positive control.

Additional file 4: Figure S4. The individual features of human pancreatic cancer and adjacent normal tissue sections.

Epigenetic inhibitors have shown anticancer effects. Combination chemotherapy with epigenetic inhibitors has shown high effectiveness in gastric cancer clinical trials, but severe side effect and local progression are the causes of treatment failure. Therefore, we sought to develop an acidity-sensitive drug delivery system to release drugs locally to diminish unfavorable outcome of gastric cancer. In this study, we showed that, as compared with single agents, combination treatment with the demethylating agent 5'-aza-2'-deoxycytidine and HDAC inhibitors Trichostatin A or LBH589 decreased cell survival, blocked cell cycle by reducing number of S-phase cells and expression of cyclins, increased cell apoptosis by inducing expression of Bim and cleaved Caspase 3, and reexpressed tumor suppressor genes more effectively in MGCC3I cells. As a carrier, reconstituted apolipoprotein B lipoparticles (rABLs) could release drugs in acidic environments. Orally administrated embedded drugs not only showed inhibitory effects on gastric tumor growth in a syngeneic orthotopic mouse model, but also reduced the hepatic and renal toxicity. In conclusion, we have established rABL-based nanoparticles embedded epigenetic inhibitors for local treatment of gastric cancer, which have good therapeutic effects but do not cause severe side effects.

Stroke and brain cancer are two distinct diseases. However, the relationship between both diseases has rarely been examined. This study investigated the longitudinal risk for developing brain cancer in stroke patients. To study this, we first reviewed the malignant gliomas previously with or without stroke using brain magnetic resonance imaging (MRI) images and the past histories. Two ischemic stroke patients before the malignant glioma were identified and belonged to the glioblastoma mutiforme (GBM). Particularly, both GBM specimens displayed strong hypoxia-inducible factor 1α (HIF-1α) expression in immunohistochemical (IHC) staining. To elucidate the significance of this relationship, we then used a nationwide population-based cohort in Taiwan to investigate the risk for the incidence of brain cancer in patients previously with or without stroke. The incidence of all tumors in the stroke group was lower than that in the control group with an adjusted hazard ratio (HR) of 0.79 (95% confidence interval [CI]: 0.74-0.84) in both gender and age older than 60 years. But the stroke patients had higher risk of developing only brain cancer with an adjusted HR of 3.09 (95% CI: 1.80-5.30), and otherwise had lower risk of developing head and neck, digestive, respiratory, bone and skin, as well as other tumors, all with p

Cancer immunotherapy targeting immune checkpoints has exhibited promising clinical outcomes in many cancers, but it offers only limited benefits for pancreatic cancer (PC). Cancer stem cells (CSCs), a minor subpopulation of cancer cells, play important roles in tumor initiation, progression, and drug resistance. Accumulating evidence suggests that CSCs employ immunosuppressive effect to evade the immune recognition. However, clinical implications of the associations among CD8+ T cells infiltration, programmed death receptor ligand-1 (PD-L1) expression, and CSCs existence are poorly understood in PC. Immunostaining and quantitative analysis were performed to assess CD8+ T cells infiltration, PD-L1 expression, and their relationship with CD44+/CD133+ CSCs and disease progression in PC. CD8+ T cells infiltration was associated with better survival while PD-L1 expression was correlated with PC recurrence. Both the low CD8+ T cells infiltration/high PD-L1 expression group and the high CD8+ T cells infiltration/high PD-L1 expression group show high levels of CD44+/CD133+ CSCs, but patients with low CD8+ T cells infiltration/high PD-L1 expression had worse survival and higher recurrence risk than those with high CD8+ T cells infiltration/high PD-L1 expression. Moreover, CD8+ T cells infiltration could reduce unfavorable prognostic effect of high co-expression of PD-L1 and CD44/CD133. Our study highlights an interaction among CD8+ T cells infiltration, PD-L1 expression, and CD44+/CD133+ CSCs existence, which contributes to PC progression and immune evasion.

Cancer cachexia (CC), characterized by body weight loss and sarcopenia, contributes to over 20% of all cancer-related death. Approximately 80% of pancreatic cancer (PC) patients develop CC during disease progression. Pro-inflammatory cytokines, including interleukin (IL)-1&beta, IL-6, IL-8, and tumor necrosis factor (TNF)-&alpha, have been correlated with CC, however, its prognostic significance remains unclear. In this study, serum levels of the CC-related cytokines were determined in normal donors and PC patients. IL-8 expression was assessed in PC tissue microarrays. The correlation of levels of each cytokine with disease progression, weight loss, and sarcopenia was calculated. The relationships among the baseline variables, CC, and IL-8 expression with disease progression were examined using univariate and multivariate analyses. Of these mentioned cytokines, only serum IL-8 level was elevated in the locally advanced group (n = 55) compared with the normal (n = 17) and resected groups (n = 55). Serum IL-8 level was positively correlated with CC status, weight loss, sarcopenia, but was negatively correlated with total psoas area (TPA). IL-8 expression in tissue samples was also positively associated with weight loss. Furthermore, serum IL-8 level was an independent predictor of survival. In conclusion, elevated serum IL-8 level significantly correlates with CC and sarcopenia and can be used as a prognostic indicator in PC.

BACKGROUND The interactions between cancer stem cells (CSCs) and tumor-associated macrophages (TAMs) can promote tumor progression, maintain the CSCs population, and reduce therapeutic effects. The objective of this study was to investigate the coexpression of CSCs and TAMs and its clinical significance in pancreatic ductal adenocarcinoma (PDAC). METHODS Ninety-six patients with PDAC were included in this study. Tissue microarrays were constructed for immunostaining of the CSCs markers CD44 and CD133 and the TAMs marker CD204. Correlations between the expression of CSCs and TAMs markers and clinicopathologic characteristics or disease progression were analyzed. RESULTS Expression levels of CD44/CD133 and CD204 were significantly higher in tumor tissues than in normal tissues (P

Background & Aims Many patients with pancreatic ductal adenocarcinoma (PDAC) develop recurrent or metastatic diseases after surgery, so it is important to identify those most likely to benefit from aggressive therapy. Disruption of tissue microarchitecture is an early step in pancreatic tumorigenesis and a parameter used in pathology grading of glandular tumors. We investigated whether changes in gene expression during pancreatic epithelial morphogenesis were associated with outcomes of patients with PDAC after surgery. Methods We generated architectures of human pancreatic duct epithelial cells in a 3-dimensional basement membrane matrix. We identified gene expression profiles of the cells during different stages of tubular morphogenesis (tubulogenesis) and of PANC-1 cells during spheroid formation. Differential expression of genes was confirmed by immunoblot analysis. We compared the gene expression profile associated with pancreatic epithelial tubulogenesis with that of PDAC samples from 27 patients, as well as with their outcomes after surgery. Results We identified a gene expression profile associated with tubulogenesis that resembled the profile of human pancreatic tissue with differentiated morphology and exocrine function. Patients with PDACs with this profile fared well after surgery. Based on this profile, we established a 6−28 gene tubulogenesis-specific signature that accurately determined the prognosis of independent cohorts of patients with PDAC (total n = 128; accuracy = 81.2%−95.0%). One gene, ASPM , was down-regulated during tubulogenesis but up-regulated in human PDAC cell lines and tumor samples; up-regulation correlated with patient outcomes (Cox regression P = .0028). Bioinformatic, genetic, biochemical, functional, and clinical correlative studies showed that ASPM promotes aggressiveness of PDAC by maintaining Wnt-β-catenin signaling and stem cell features of PDAC cells. Conclusions We identified a gene expression profile associated with pancreatic epithelial tubulogenesis and a tissue architecture−specific signature of PDAC cells that is associated with patient outcomes after surgery.

Epigenetic repression of the tumor suppressor gelsolin (GSN) is frequently observed in cancers. Chronic inflammation can promote tumor progression via aberrant DNA methylation. In this study, we investigated the role of tumor-associated macrophages (TAMs) in DNA methylation of the GSN gene during gastric cancer progression. Immunofluorescence staining of 121 gastric cancer tissues showed aberrant localization of GSN and DNA methyltransferase 1 (DNMT1) and juxtaposition of DNMT1 and M2 TAMs. Decreased GSN protein and mRNA expression and increased DNA methylation in the GSN promoter were observed in gastric cancer cell lines and clinical specimens. To examine the effect of TAMs on DNA methylation in gastric cancer cells, we performed in vitro coculture assays and found increased DNMT1 expression but decreased GSN expression in gastric cancer cells after coculture with U937 cells. Knockdown of DNMT1 expression in gastric cancer cells could abort U937 coculture-mediated GSN downregulation. Meanwhile, CCL5 was the main chemokine upregulated in coculture medium. Treatment with CCL5 could induce DNMT1 expression in gastric cancer cells via STAT3 signaling. Inhibiting DNMT1 activity with procainamide, inhibiting DNA methylation with 5-AZA, or inhibiting CCL5/CCR5 signaling with maraviroc reduced tumor growth in vivo. In conclusion, upregulation of DNMT1 by CCL5/CCR5/STAT3 signaling is critical for TAM-mediated GSN silencing in gastric cancer. This study identified potential targets for gastric cancer therapy. Cancer Immunol Res; 5(10); 885–97. ©2017 AACR.

Purpose: KIT mutations, the most prevalent genetic event in gastrointestinal stromal tumors (GIST), are associated with malignant features and poor prognosis. Aggressive GISTs possess a high propensity to spread to the liver. This study aimed to explore the role of KIT mutations in GIST liver metastasis. Experimental Design: A total of 170 GISTs were used to determine the association between KIT mutations and liver metastasis. Immunohistochemistry was performed to assess the correlation of KIT mutations with CXCR4 and ETV1 expression. Genetic and pharmacologic methods were used to study the regulation of CXCR4 and ETV1 by KIT mutations. Results: Codons 557 and 558 in KIT exon 11 were deletion hot spots in GISTs. KIT exon 11 deletions involving codons 557–558 were highly associated with liver metastasis. Overexpression of mutant KIT with exon 11 codons 557–558 deletion (KIT Δ557–558) increased GIST cell motility and liver metastasis. Mechanistically, overexpression of KIT Δ557–558 in GIST cells increased ETV1 and CXCR4 expression. CXCR4 knockdown counteracted KIT Δ557–558–mediated cell migration. Moreover, KIT Δ557–558–induced CXCR4 expression could be abolished by silencing ETV1. The chromatin immunoprecipitation assay showed that ETV1 directly bound to the CXCR4 promoter. After ERK inhibitor PD325901 treatment, the upregulation of ETV1 by KIT Δ557–558 was prevented. In addition, KIT exon 11 codons 557–558 deletion enhanced CXCL12-mediated GIST cell migration and invasion. Conclusions: KIT exon 11 557–558 deletion upregulates CXCR4 through increased binding of ETV1 to the CXCR4 promoter in GIST cells, which thus promotes liver metastasis. These findings highlighted the potential therapeutic targets for metastatic GISTs. Clin Cancer Res; 22(14); 3477–87. ©2016 AACR.

Supplementary Materials and Methods.(DOCX 17Â kb)

Background: Acute pancreatitis (AP), a sudden inflammation of the pancreas, can cause severe complications and high mortality despite treatment. The use of mesenchymal stem cells (MSCs) for the treatment of AP has attracted significant attention in novel treatment strategy; however, the mode of action of spleen-derived MSCs (sp-MSCs) in AP remains unknown. Method: MSCs isolated from mouse spleen (msp-MSCs) were used to investigate the effects in animal models of cerulean-induced acute pancreatitis (CAP) and pancreatic ischemia injury (PII). Results: Msp-MSCs had multipotent differentiation capacities and immunoregulatory functions. A greater number of Qtracker-labeled msp-MSCs were detected in pancreas of mice with CAP than of control mice. Infused msp-MSCs reduced serum levels of amylase, lipase, and myeloperoxidase, and pancreatic edema, necrosis level, expression of inflammation cytokines, and CD3+T cell infiltration. In PII model, infused msp-MSCs promoted cell growth and thus improved pancreatic dysfunction. Conclusion: Msp-MSCs exert protective effects on CAP- and PII-induced pancreatic injury and might be developed as a potential therapeutic agent for pancreatitis treatment.

List of antibodies used in this study. (DOCX 20Â kb)

Interleukin (IL)-20, a proinflammatory cytokine of the IL-10 family, is involved in acute and chronic renal failure. The aim of this study was to elucidate the role of IL-20 during diabetic nephropathy development. We found that IL-20 and its receptor IL-20R1 were upregulated in the kidneys of mice and rats with STZ-induced diabetes. In vitro, IL-20 induced MMP-9, MCP-1, TGF-β1 and VEGF expression in podocytes. IL-20 was upregulated by hydrogen peroxide, high-dose glucose and TGF-β1. In addition, IL-20 induced apoptosis in podocytes by activating caspase-8. In STZ-induced early diabetic nephropathy, IL-20R1-deficient mice had lower blood glucose and serum BUN levels and a smaller glomerular area than did wild-type controls. Anti-IL-20 monoclonal antibody (7E) treatment reduced blood glucose and the glomerular area and improved renal functions in mice in the early stage of STZ-induced diabetic nephropathy. ELISA showed that the serum IL-20 level was higher in patients with diabetes mellitus than in healthy controls. The findings of this study suggest that IL-20 induces cell apoptosis of podocytes and plays a role in the pathogenesis of early diabetic nephropathy. Serious kidney damage often found in type 1 and type 2 diabetes has been linked to the natural signaling molecule interleukin-20 (IL-20). Ming-Shi Chang and colleagues at the National Cheng Kung University in Taiwan found increased levels of both IL-20 and its receptor molecule in mice and rats with chemically induced diabetes. They also found that IL-20 induced changes in gene activity, leading to cell death in cultured specialized kidney cells known as podocytes. Patients with diabetes were also shown to have increased IL-20 levels. Treating diabetic mice with an antibody that bound to and interfered with the activity of IL-20 improved kidney function and reduced biochemical indicators of diabetes. Understanding the mechanism of IL-20 involvement in this common complication of diabetes could help devise treatments to prevent or limit the damage.