Patient-derived Tissue Research Articles

Loss of TARBP2 Drives the Progression of Hepatocellular Carcinoma via miR-145-SERPINE1 Axis.

The clinical outcomes of hepatocellular carcinoma (HCC) remain dismal. Elucidating the molecular mechanisms for the progression of aggressive HCC holds the promise for developing novel intervention strategies. The transactivation response element RNA-binding protein (TRBP/TARBP2), a key component of microRNA (miRNA) processing and maturation machinery has been shown to play conflicting roles in tumor development and progression. We sought to investigate the expression of TARBP2 in HCC using well-characterized HCC cell lines, patient-derived tissues and blood samples. Additionally, the potential prognostic and diagnostic value of TARBP2 in HCC were analyzed using Kaplan-Meier plots and ROC curve. Cell counting kit‐8 (CCK‐8), wound healing and transwell assays examined the ability of TARBP2 to induce cell proliferation, migration, and invasion in HCC cell lines. RNA sequencing was applied to identify the downstream elements of TARBP2. The interaction of potential targets of TARBP2, miR‐145 and serpin family E member 1 (SERPINE1), was assessed using luciferase reporter assay. TARBP2 expression was down-regulated in HCC cell lines relative to normal hepatocyte cells, with a similar pattern further confirmed in tissue and blood samples. Notably, the loss of TARBP2 was demonstrated to promote proliferation, migration, and invasion in HCC cell lines. Interestingly, the reduction of TARBP2 was shown to result in the upregulation of SERPINE1, also known as plasminogen activator inhibitor (PAI-1), which is a vital gene of the HIF-1 signaling pathway. Knockdown of SERPINE1 rescued the TARBP2-lost phenotype. Moreover, TARBP2 depletion induced the upregulation of SERPINE1 through reducing the processing of miR-145, which directly targets SERPINE1. Finally, overexpression of miR-145 repressed SERPINE1 and rescued the functions in sh-TARBP2 HCC cells. Our findings underscore a linear TARBP2-miR-145-SERPINE1 pathway that drives HCC progression, with the potential as a novel intervention target for aggressive HCC.

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium's cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling. Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

PR11-364P22.2/ATF3 protein interaction mediates IL-1β-induced catabolic effects in cartilage tissue and chondrocytes.

Osteoarthritis (OA) is a degenerative joint disease which lacks effective medical treatment due to ill‐defined molecular mechanisms underlying the pathology. Inflammation is a key factor that induces and aggravates OA. Therefore, the current study aims to explore roles of the dysregulated long non‐coding RNAs in the pro‐inflammatory cytokine IL‐1β‐mediated catabolic effects in cartilage tissue and chondrocytes. We identified RP11‐364P22.2 as dysregulated in OA patient‐derived cartilage tissues and highly responsive to IL‐1β stimulus. RNA pull‐down coupled with mass spectrometry demonstrated that RP11‐364P22.2 physically binds to activating transcription factor 3 (ATF3) and thus increases the protein stability and facilitates its nuclear translocation. Loss‐ and gain‐of‐function assays indicated that the interaction between RP11‐364P22.2 and ATF3 is indispensable for the detrimental effects of IL‐1β including growth inhibition, apoptosis induction as well as degradation of the key chondrocyte structural proteins of type II collage and Aggrecan and synthesis of the extracellular matrix‐degrading enzyme MMP13 in chondrocytes. In vivo, depletion of the RP11‐364P22.2 effector ATF3 drastically prevented OA development in the rats with surgical destabilization of the medial meniscus (DMM). These results highlight the important roles of lncRNAs in the pathogenesis of OA and indicate the RP11‐364P22.2/ATF3 regulatory axis as a potential therapeutic target of inflammation‐induced OA.

Antitumor Effects in Gas Plasma-Treated Patient-Derived Microtissues—An Adjuvant Therapy for Ulcerating Breast Cancer?

Despite global research and continuous improvement in therapy, cancer remains a challenging disease globally, substantiating the need for new treatment avenues. Medical gas plasma technology has emerged as a promising approach in oncology in the last years. Several investigations have provided evidence of an antitumor action in vitro and in vivo, including our recent work on plasma-mediated reduction of breast cancer in mice. However, studies of gas plasma exposure on patient-derived tumors with their distinct microenvironment (TME) are scarce. To this end, we here investigated patient-derived breast cancer tissue after gas plasma-treated ex vivo. The tissues were disjoint to pieces smaller than 100 µm, embedded in collagen, and incubated for several days. The viability of the breast cancer tissue clusters and their outgrowth into their gel microenvironment declined with plasma treatment. This was associated with caspase 3-dependent apoptotic cell death, paralleled by an increased expression of the anti-metastatic adhesion molecule epithelial (E)-cadherin. Multiplex chemokine/cytokine analysis revealed a marked decline in the release of the interleukins 6 and 8 (IL-6, IL-8) and monocyte-chemoattractant-protein 1 (MCP) known to promote a cancer-promoting milieu in the TME. In summary, we provide here, for the first time, evidence of a beneficial activity of gas plasma exposure on human patient-derived breast cancer tissue.

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis.

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues

Prolonged detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and recurrence of PCR-positive tests have been widely reported in patients after recovery from COVID-19, but some of these patients do not appear to shed infectious virus. We investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the DNA of human cells in culture and that transcription of the integrated sequences might account for some of the positive PCR tests seen in patients. In support of this hypothesis, we found that DNA copies of SARS-CoV-2 sequences can be integrated into the genome of infected human cells. We found target site duplications flanking the viral sequences and consensus LINE1 endonuclease recognition sequences at the integration sites, consistent with a LINE1 retrotransposon-mediated, target-primed reverse transcription and retroposition mechanism. We also found, in some patient-derived tissues, evidence suggesting that a large fraction of the viral sequences is transcribed from integrated DNA copies of viral sequences, generating viral-host chimeric transcripts. The integration and transcription of viral sequences may thus contribute to the detection of viral RNA by PCR in patients after infection and clinical recovery. Because we have detected only subgenomic sequences derived mainly from the 3' end of the viral genome integrated into the DNA of the host cell, infectious virus cannot be produced from the integrated subgenomic SARS-CoV-2 sequences.

A data-independent acquisition-based global phosphoproteomics system enables deep profiling

Phosphoproteomics can provide insights into cellular signaling dynamics. To achieve deep and robust quantitative phosphoproteomics profiling for minute amounts of sample, we here develop a global phosphoproteomics strategy based on data-independent acquisition (DIA) mass spectrometry and hybrid spectral libraries derived from data-dependent acquisition (DDA) and DIA data. Benchmarking the method using 166 synthetic phosphopeptides shows high sensitivity (<0.1 ng), accurate site localization and reproducible quantification (~5% median coefficient of variation). As a proof-of-concept, we use lung cancer cell lines and patient-derived tissue to construct a hybrid phosphoproteome spectral library covering 159,524 phosphopeptides (88,107 phosphosites). Based on this library, our single-shot streamlined DIA workflow quantifies 36,350 phosphosites (19,755 class 1) in cell line samples within two hours. Application to drug-resistant cells and patient-derived lung cancer tissues delineates site-specific phosphorylation events associated with resistance and tumor progression, showing that our workflow enables the characterization of phosphorylation signaling with deep coverage, high sensitivity and low between-run missing values.

TIMP1 Triggers Neutrophil Extracellular Trap Formation in Pancreatic Cancer.

Tumor-derived protein tissue inhibitor of metalloproteinases-1 (TIMP1) correlates with poor prognosis in many cancers, including highly lethal pancreatic ductal adenocarcinoma (PDAC). The noncanonical signaling activity of TIMP1 is emerging as one basis for its contribution to cancer progression. However, TIMP1-triggered progression-related biological processes are largely unknown. Formation of neutrophil extracellular traps (NET) in the tumor microenvironment is known to drive progression of PDAC, but factors or molecular mechanisms initiating NET formation in PDAC remain elusive. In this study, gene-set enrichment analysis of a human PDAC proteome dataset revealed that TIMP1 protein expression most prominently correlates with neutrophil activation in patient-derived tumor tissues. TIMP1 directly triggered formation of NETs in primary human neutrophils, which was dependent on the interaction of TIMP1 with its receptor CD63 and subsequent ERK signaling. In genetically engineered PDAC-bearing mice, TIMP1 significantly contributed to NET formation in tumors, and abrogation of TIMP1 or NETs prolonged survival. In patient-derived PDAC tumors, NETs predominantly colocalized with areas of elevated TIMP1 expression. Furthermore, TIMP1 plasma levels correlated with DNA-bound myeloperoxidase, a NET marker, in the blood of patients with PDAC. A combination of plasma levels of TIMP1 and NETs with the clinically established marker CA19-9 allowed improved identification of prognostically distinct PDAC patient subgroups. These observations may have a broader impact, because elevated systemic levels of TIMP1 are associated with the progression of a wide range of neutrophil-involved inflammatory diseases. SIGNIFICANCE: These findings highlight the prognostic relevance of TIMP1 and neutrophil extracellular traps in highly lethal pancreatic cancer, where a noncanonical TIMP1/CD63/ERK signaling axis induces NET formation. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/13/3568/F1.large.jpg.

Club Cell Functions As A Wnt‐responsible Progenitor For Tissue Repair In COPD

Introduction Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide that is characterized by progressive loss of alveolar tissue. Endogenous lung stem cells and tissue repair pathways are thought be dysfunctional and their reactivation is a promising therapeutic approach for COPD. Wnt/β-catenin signaling is critical for stem cell function and is reduced in human COPD lungs. We have previously demonstrated that therapeutic Wnt/β-catenin initiated tissue repair in COPD patient-derived lung tissue ex vivo. Recently, we identified an epithelial progenitor population, which were activated by Wnt/β-catenin signaling to form alveolar organoids. Thus, we hypothesize that club cells represent a novel Wnt-responsive epithelial progenitor for tissue repair in COPD. Methods Single cell RNA sequencing (scRNA seq), organoid assay, fate tracing studies, flow cytometry, immunofluorescence, FACS sorting, porcine pancreatic elastase (PPE) induced mouse COPD model. Results First, using scRNA seq, we identified club cells as the major progenitor cells in the Wnt-responsive epithelial progenitor cell population. Further, we perform fate tracing studies using a Scgb1a1Cre-ERT;Rosa-Tomato;TCF/Lef:H2B-GFP mouse line combined with lung organoid assays to further demonstrate that activation of the Wnt/β-catenin signaling using the GSK3β inhibitor CHIR9920 increased the number of alveolar organoids formed by club cells. Finally, in a PPE-induced mouse COPD model, we identified an impaired organoid forming capacity of club cells, which could be restored by activation of the Wnt/β-catenin signaling. Conclusion Our study reveals a novel mechanism of club cell activation by Wnt/β-catenin signaling in COPD.

Co-Targeting PIM Kinase and PI3K/mTOR in NSCLC.

Simple SummaryPIM kinases interact with major oncogenic players, including the PI3K/Akt pathway, and provide an escape mechanism leading to drug resistance. This study examined PIM kinase expression in NSCLC and the potential of PIM1 as a prognostic marker. The effect on cell signaling of novel preclinical PI3K/mTOR/PIM kinase inhibitor IBL-301 was compared to PI3K/mTOR inhibition in vitro and ex vivo. PI3K-mTOR inhibitor sensitive (H1975P) and resistant (H1975GR) cells were compared for altered IL6/STAT3 pathway expression and sensitivity to IBL-301. All three PIM kinases are expressed in NSCLC and PIM1 is a marker of poor prognosis. IBL-301 inhibited c-Myc, the PI3K-Akt and JAK/STAT pathways in vitro and in NSCLC tumor tissue explants. IBL-301 also inhibited secreted pro-inflammatory cytokine MCP-1. PIM kinases were activated in H1975GR cells which were more sensitive to IBL-301 than H1975P cells. A miRNA signature of PI3K-mTOR resistance was validated. Co-targeting PIM kinase and PI3K-mTOR warrants further clinical investigation.PIM kinases are constitutively active proto-oncogenic serine/threonine kinases that play a role in cell cycle progression, metabolism, inflammation and drug resistance. PIM kinases interact with and stabilize p53, c-Myc and parallel signaling pathway PI3K/Akt. This study evaluated PIM kinase expression in NSCLC and in response to PI3K/mTOR inhibition. It investigated a novel preclinical PI3K/mTOR/PIM inhibitor (IBL-301) in vitro and in patient-derived NSCLC tumor tissues. Western blot analysis confirmed PIM1, PIM2 and PIM3 are expressed in NSCLC cell lines and PIM1 is a marker of poor prognosis in patients with NSCLC. IBL-301 decreased PIM1, c-Myc, pBAD and p4EBP1 (Thr37/46) and peIF4B (S406) protein levels in-vitro and MAP kinase, PI3K-Akt and JAK/STAT pathways in tumor tissue explants. IBL-301 significantly decreased secreted pro-inflammatory cytokine MCP-1. Altered mRNA expression, including activated PIM kinase and c-Myc, was identified in Apitolisib resistant cells (H1975GR) by an IL-6/STAT3 pathway array and validated by Western blot. H1975GR cells were more sensitive to IBL-301 than parent cells. A miRNA array identified a dysregulated miRNA signature of PI3K/mTOR drug resistance consisting of regulators of PIM kinase and c-Myc (miR17-5p, miR19b-3p, miR20a-5p, miR15b-5p, miR203a, miR-206). Our data provides a rationale for co-targeting PIM kinase and PI3K-mTOR to improve therapeutic response in NSCLC.

Identification of novel targets of miR-622 in hepatocellular carcinoma reveals common regulation of cooperating genes and outlines the oncogenic role of zinc finger CCHC-type containing 11

The poor prognosis of advanced hepatocellular carcinoma (HCC) is driven by diverse features including dysregulated microRNAs inducing drug resistance and stemness. Lin-28 homolog A (LIN28A) and its partner zinc finger CCHC-type containing 11 (ZCCHC11) cooperate in binding, oligouridylation and subsequent degradation of tumorsuppressive let-7 precursor microRNAs. Functionally, activation of LIN28A was recently shown to promote stemness and chemoresistance in HCC. However, the expression and regulation of LIN28A in HCC had been unclear. Moreover, the expression, regulation and function of ZCCHC11 in liver cancer remained elusive.In contrast to "one-microRNA-one-target" interactions, we identified common binding sites for miR-622 in both LIN28A and ZCCHC11, suggesting miR-622 to function as a superior pathway regulator. Applying comprehensive microRNA database screening, human hepatocytes and HCC cell lines, patient-derived tissue samples as well as "The Cancer Genome Atlas" (TCGA) patient cohorts, we demonstrated that loss of tumorsuppressive miR-622 mediates derepression and overexpression of LIN28A in HCC. Moreover, the cooperator of LIN28A, ZCCHC11, was newly identified as a prognostic and therapeutic target of miR-622 in liver cancer.Together, identification of novel miR-622 target genes revealed common regulation of cooperating genes and outlines the previously unknown oncogenic role of ZCCHC11 in liver cancer.

Translating complexity and heterogeneity of pancreatic tumor: 3D in vitro to in vivo models

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive type of cancer with an overall survival rate of less than 7-8%, emphasizing the need for novel effective therapeutics against PDAC. However only a fraction of therapeutics which seemed promising in the laboratory environment will eventually reach the clinic. One of the main reasons behind this low success rate is the complex tumor microenvironment (TME) of PDAC, a highly fibrotic and dense stroma surrounding tumor cells, which supports tumor progression as well as increases the resistance against the treatment. In particular, the growing understanding of the PDAC TME points out a different challenge in the development of efficient therapeutics - a lack of biologically relevant in vitro and in vivo models that resemble the complexity and heterogeneity of PDAC observed in patients. The purpose and scope of this review is to provide an overview of the recent developments in different in vitro and in vivo models, which aim to recapitulate the complexity of PDAC in a laboratory environment, as well to describe how 3D in vitro models can be integrated into drug development pipelines that are already including sophisticated in vivo models. Hereby a special focus will be given on the complexity of in vivo models and the challenges in vitro models face to reach the same levels of complexity in a controllable manner. First, a brief introduction of novel developments in two dimensional (2D) models and ex vivo models is provided. Next, recent developments in three dimensional (3D) in vitro models are described ranging from spheroids, organoids, scaffold models, bioprinted models to organ-on-chip models including a discussion on advantages and limitations for each model. Furthermore, we will provide a detailed overview on the current PDAC in vivo models including chemically-induced models, syngeneic and xenogeneic models, highlighting hetero- and orthotopic, patient-derived tissues (PDX) models, and genetically engineered mouse models. Finally, we will provide a discussion on overall limitations of both, in vitro and in vivo models, and discuss necessary steps to overcome these limitations to reach an efficient drug development pipeline, as well as discuss possibilities to include novel in silico models in the process.

Anti-Tumor Effects of MAPK-Dependent Tumor-Selective Oncolytic Vaccinia Virus Armed with CD/UPRT against Pancreatic Ductal Adenocarcinoma in Mice

Engineered vaccinia virus serves as an oncolytic virus for cancer virotherapy. We evaluated the oncolytic characteristics of VGF- and O1-deleted recombinant mitogen-activated protein kinase (MAPK)-dependent vaccinia virus (MDRVV). We found that compared with viruses with the deletion of either gene alone, MDRVV is more attenuated in normal cells and can replicate in cancer cells that exhibit constitutive ERK1/2 activation in the MAPK pathway. We armed MDRVV with a bifunctional fusion gene encoding cytosine deaminase and uracil phosphoribosyltransferase (CD/UPRT), which converts 5-fluorocytosine (5-FC) into chemotherapeutic agents, and evaluated its oncolytic activity alone or in combination with 5-FC in human pancreatic cancer cell lines, tumor mouse models of peritoneal dissemination and liver metastasis, and ex vivo-infected live pancreatic cancer patient-derived tissues. CD/UPRT-armed MDRVV alone could efficiently eliminate pancreatic cancers, and its antitumor effects were partially enhanced in combination with 5-FC in vitro and in vivo. Moreover, the replication of MDRVV was detected in tumor cells of patient-derived, surgically resected tissues, which showed enlarged nuclei and high expression of pERK1/2 and Ki-67, and not in stromal cells. Our findings suggest that systemic injections of CD/UPRT-armed MDRVV alone or in combination with 5-FC are promising therapeutic strategies for pancreatic ductal adenocarcinoma.

Role of Annexin A1 in Squamous Cell Lung Cancer Progression.

Lung cancer remains the primary cause of cancer-related death worldwide, and its molecular mechanisms of tumor progression need further characterization to improve the clinical management of affected patients. The role of Annexin A1 (ANXA1) in tumorigenesis and cancer progression in general and especially in lung cancer remains to be controversial and seems to be highly tissue specific and inconsistent among tumor initiation, progression, and metastasis. In the current study, we investigated ANXA1 expression in 81 squamous cell lung cancer (SQCLC), 86 pulmonary adenocarcinoma (AC), and 30 small cell lung cancer (SCLC) patient-derived tissue samples and its prognostic impact on patient's survival. Mechanistically, we analyzed the impact of ANXA1 expression on proliferation and migration of SQCLC cell lines using CRISPR-Cas9 and mammalian overexpression vectors. Strong expression of ANXA1 was significantly correlated to longer overall survival only in SQCLC patients (P = 0.019). Overexpression of ANXA1 promoted proliferation in SQCLC cell lines but suppressed their migration, while knockout of ANXA1 promoted cell migration and suppressed proliferation. In conclusion, ANXA1 expression might elongate patients' survival by inhibiting tumor cell migration and subsequent metastasis.

Patient-derived organoids model cervical tissue dynamics and viral oncogenesis in cervical cancer.

Cervical cancer is a common gynecological malignancy often caused by high-risk human papillomavirus. There is a paucity of human-derived culture systems to study the cervical epithelium and the cancers derived thereof. Here we describe a long-term culturing protocol for ecto- and endocervical epithelia that generates 3D organoids that stably recapitulate the two tissues of origin. As evidenced for HSV-1, organoid-based cervical models may serve to study sexually transmitted infections. Starting from Pap brush material, a small biobank of tumoroids derived from affected individuals was established that retained the causative human papillomavirus (HPV) genomes. One of these uniquely carried the poorly characterized HPV30 subtype, implying a potential role in carcinogenesis. The tumoroids displayed differential responses to common chemotherapeutic agents and grew as xenografts in mice. This study describes an experimental platform for cervical (cancer) research and for future personalized medicine approaches.

A GATA6-centred gene regulatory network involving HNFs and ΔNp63 controls plasticity and immune escape in pancreatic cancer

ObjectiveMolecular taxonomy of tumours is the foundation of personalised medicine and is becoming of paramount importance for therapeutic purposes. Four transcriptomics-based classification systems of pancreatic ductal adenocarcinoma (PDAC) exist, which...

Identification of prognostic genes and tumor-infiltrating immune cells in the tumor microenvironment of esophageal squamous cell carcinoma and esophageal adenocarcinoma.

BackgroundEsophageal cancer (EC) is a highly aggressive malignancy that is classified as esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Infiltrating stromal/immune cells, a major component of the tumor immune microenvironment (TIME), have prognostic significance in various cancers.MethodsIn this study we investigated genes and immune factors in the tumor microenvironment (TME) of ESCC and EAC that can serve as prognostic biomarkers. Stromal and immune scores were calculated using the Estimation of Stromal and Immune Cells in Malignant Tumor Tissues Using Expression Data (ESTIMATE) algorithm based on gene expression profiles of patient-derived tumor tissues in The Cancer Genome Atlas database. The correlation between ESTIMATE scores and survival rates in EC were analyzed. A comparison of high and low stromal/immune score groups revealed multiple differentially expressed genes (DEGs) as candidate prognostic genes; their role in immune-related biological processes was evaluated by functional and protein-protein interaction (PPI) network analyses, and the genes were validated using Gene Expression Omnibus datasets. Additionally, 22 tumor-infiltrating immune cell (TIIC) subsets were analyzed using the CIBERSORT algorithm.ResultsMedian stromal score was higher whereas immune score was lower in ESCC than in EAC (both P<0.01). Stromal score was lower in female as compared to male ESCC patients (P<0.05), and was significantly correlated with T stage (P<0.05). In EAC, median immune score was higher in female as compared to male patients (P<0.05) and was correlated with tumor-node-metastasis stage (P<0.05). The identified DEGs were mainly involved in lymphocyte (especially T-lymphocyte) activation and carbohydrate binding. Moreover, the levels of infiltrating resting-stage dendritic cells, CD8+ T cells, naïve B cells, activated mast cells, and resting memory CD4+ T cells were significantly correlated with EC prognosis (P<0.05).ConclusionsThe immune microenvironment of ESCC and EAC are quite different. We have found genes with prognostic value in multiple tumor databases.

FOXO3 is a latent tumor suppressor for FOXO3-positive and cytoplasmic-type gastric cancer cells

FOXO3 is a member of the FOXO transcription factors thought to play a tumor-suppressor role in gastrointestinal cancer, while tumor-promoting function of FOXO3 has also been reported. These results suggest a context-dependent function of FOXO3 in tumor development. However, the relationship between the FOXO3 expression pattern and its role in tumorigenesis has not been elucidated. We examined the FOXO3 expression in 65 human primary gastric cancer and patient-derived xenograft tissues by immunohistochemistry and identified three subtypes according to subcellular localization: FOXO3-nuclear accumulated (FOXO3-Nuc), FOXO3-nuclear/cytoplasmic or cytoplasmic distributed (FOXO3-Cyt), and FOXO3-negative. In the FOXO3-Cyt gastric cancer cells, the expression of the constitutive active mutant FOXO3 (Act-ER FOXO3) induced the nuclear accumulation of FOXO3 and significantly suppressed colony formation and proliferation. The inhibition of the PI3K-AKT pathway by inhibitor treatment also suppressed the proliferation of FOXO3-Cyt gastric cancer cells, which was associated with the nuclear accumulation of endogenous FOXO3. Furthermore, the expression of Act-ER FOXO3 by an endogenous promoter significantly suppressed gastric tumorigenesis in Gan mice, a model of gastric cancer. Finally, treatment of FOXO3-Cyt human gastric cancer-derived organoids with an AKT inhibitor significantly suppressed the survival and proliferation. These results indicate that FOXO3 is a latent tumor suppressor for FOXO3-Cyt-type gastric cancer cells and that activation of the PI3K-AKT pathway protects this type of gastric cancer cell from FOXO3-mediated growth suppression via constitutive nuclear export. Thus, the inhibition of the PI3K-AKT pathway and nuclear translocation of endogenous FOXO3 may have therapeutic applications in the treatment of FOXO3-positive and cytoplasmic-type gastric cancer.

Somatic GNAQ R183Q mutation is located within the sclera and episclera in patients with Sturge-Weber syndrome

AimsTo determine the correspondence between GNAQ R183Q (c.548G＞A) mutation in abnormal scleral tissue of patients with Sturge-Weber syndrome (SWS) secondary glaucoma and explore the role of GNAQ R183Q in glaucoma...

Role of TGF-β in pancreatic ductal adenocarcinoma progression and PD-L1 expression.

The transforming growth factor-beta (TGF-β) pathway plays a paradoxical, context-dependent role in pancreatic ductal adenocarcinoma (PDAC): a tumor-suppressive role in non-metastatic PDAC and a tumor-promotive role in metastatic PDAC. We hypothesize that non-SMAD-TGF-β signaling induces PDAC progression. We investigated the expression of non-SMAD-TGF-β signaling proteins (pMAPK14, PD-L1, pAkt and c-Myc) in patient-derived tissues, cell lines and an immunocompetent mouse model. Experimental models were complemented by comparing the signaling proteins in PDAC specimens from patients with various survival intervals. We manipulated models with TGF-β, gemcitabine (DNA synthesis inhibitor), galunisertib (TGF-β receptor inhibitor) and MK-2206 (Akt inhibitor) to investigate their effects on NF-κB, β-catenin, c-Myc and PD-L1 expression. PD-L1 expression was also investigated in cancer cells and tumor associated macrophages (TAMs) in a mouse model. We found that tumors from patients with aggressive PDAC had higher levels of the non-SMAD-TGF-β signaling proteins pMAPK14, PD-L1, pAkt and c-Myc. In PDAC cells with high baseline β-catenin expression, TGF-β increased β-catenin expression while gemcitabine increased PD-L1 expression. Gemcitabine plus galunisertib decreased c-Myc and NF-κB expression, but induced PD-L1 expression in some cancer models. In mice, gemcitabine plus galunisertib treatment decreased metastases (p=0.018), whereas galunisertib increased PD-L1 expression (p<0.0001). In the mice, liver metastases contained more TAMs compared to the primary pancreatic tumors (p=0.001), and TGF-β increased TAM PD-L1 expression (p<0.05). In PDAC, the non-SMAD-TGF-β signaling pathway leads to more aggressive phenotypes, TAM-induced immunosuppression and PD-L1 expression. The divergent effects of TGF-β ligand versus receptor inhibition in tumor cells versus TAMs may explain the TGF-β paradox. Further evaluation of each mechanism is expected to lead to the development of targeted therapies.