Gastric Organoids Research Articles

Abstract 1730: Unraveling the oncogenic potential of KDM2A with gastric organoids

Abstract Background: Gastric adenocarcinoma (GAC), prevalent and often lethal, imposes a serious global health challenge. Therefore, it is critical to identify novel oncogenes (drivers) that can be therapeutically targeted. Of epigenetic modifications, histone lysine methylation is a hallmark of epigenetic and transcriptional regulation of gene expression. Therefore, investigating the involvement of histone methylation modifiers in GAC is paramount to gaining a comprehensive understanding of the disease but also to discover novel targets. Our analysis of the GAC genome indicates that these epigenetic modifiers are amplified in GAC. Objective and Hypothesis: The overall objective is to define the oncogenic role of KDM2A in GAC pathogenesis and progression. The central hypothesis is that KDM2A promotes GAC by upregulating the expression of oncogenic proteins via the activation of epigenomic signatures/pathways.KDM2A (FBXL11) is a histone lysine demethylase that demethylates H3K36me2 and represses target genes involved in various biological processes, including cell proliferation, DNA damage repair, and stem cell maintenance. Previously, we reported that KDM2A was necessary for the tumorigenic and metastatic capabilities of non-small lung cancer cells through transcriptionally repressing the expression of DUSP3 and HDAC3, which led to activation of ERK signaling and upregulation of cell cycle-associated genes. Results: Our analysis of the GAC genome indicates that KDM2A is one of the top demethylases frequently amplified and overexpressed (14%). In addition, KDM2A expression was significantly higher in GAC samples than in normal tissues, and high expression of KDM2A was associated with poor survival in GAC patients. To examine the effect of KDM2A knockdown on characteristics of GAC, we used gastric organoids generated from KDM2Aflox/flox mouse stomach. We expressed wild-type (Flag-KDM2A) and catalytic mutant (Flag-KDM2A-mut) in these organoids. Ectopic expression of KDM2A wild-type, but not mutant, increased the proliferation of organoids. We showed that KDM2A knockdown decreased the proliferation and invasion of two GAC cells, GA0518 and AGS. In addition, we determine the effect of overexpression of KDM2A on cell proliferation by comparing the control organoids to KDM2A overexpressed using IHC staining of Ki-67 (a proliferation marker) and other activated oncogenic signaling PI3KCA/mTOR/AKT, EGFR. We showed that GA5018 cells were very sensitive to the KDM2A inhibitor (daminozide) alone and in combination with erlotinib (EGFRi inhibitor) and RAD001 (RAD1 mTOR inhibitor). Significance and Innovation: In contrast to advances in cellular signaling pathways in GAC pathogenesis, how GAC development is epigenetically regulated remains largely unknown. Our results from using organoids indicate that KDM2A is an oncoprotein. Citation Format: Shilpa S. Dhar, Calena Brown, Constantin Zod, Jaffer A. Ajani, Min Gyu Lee. Unraveling the oncogenic potential of KDM2A with gastric organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1730.

Abstract 229: Molecular-genomic characterization of metastatic gastric cancer organoids and establishment of organoid-immune cell co-culture model

Abstract Advanced gastric cancer (AGC) showed marked molecular heterogeneity with aggressive behavior and treatment resistance, especially AGC with ascites from peritoneal metastasis. Tumor organoids are valuable in vitro model for mimicking organ characteristics and are used for developing therapeutic strategies. Therefore, we aimed to understand the characteristics of organoids established from AGC patient-derived fluids. Also, we are in progress of setting up an organoid-immune cell co-culture model for drug screening of immune checkpoint inhibitors (ICI) for AGC. All established organoids from peritoneal and pleural fluid of AGC patients were authenticated through short tandem repeat (STR) profiling, comparing with their matched PBMCs. We compared the morphological characteristics using Operetta High Content Imaging (Perkin Elmer). Also, the in house targeted sequencing and RNA sequencing data were obtained from the genome database of the Song-Dang Institute for Cancer Research and it was verified using molecular methods. Then, we co-cultured organoids with healthy donor PBMCs that were pre-activated using anti-human CD3/CD28 Dynabeads (Gibco). 3D spheroids, rather than single cells, and the PBMCs were co-cultured in matrigel (Corning Inc.) for more than 3 days. The overall success rate of the organoid establishment was 53.8% (78/145), with different sources of peritoneal (54%, 61/113) and pleural fluids (53.1%, 17/32). The organoid success rate was various as in EBV positive (66.7%, 2/3) and dMMR (0%, 0/4). Based on the analysis of 91 patients eligible for histopathology, the success rate of HER2-negative (57.5%, 46/80) is higher than that of HER2-positive (36.4%, 4/11). The organoids were observed in a variety of morphologies categorized in 3 groups such as grape-like, compact and glandular structure. Based on genomic data of 31 organoids, we could subgrouping for TCGA subtypes and EMT subtype. RTKs were amplified in 10 organoids, including the amplification of ERBB2 (n=2), MET (n=4) and FGFR (n=4). And KRAS alterations including amplification (n=2) and G12A/D (n=2) mutation were also detected. Then, we established the condition for organoid-immune co-culture model with PBMCs with Dynabead activation for 3-10 days. We confirmed that higher dose of PBMCs killed the tumor cells with reduction of organoid size and partial disintegration. We established the proper PBMC dose and characterization to maintain the organoids co-culture for further drug (ICI) sensitivity testing. In conclusion, we evaluated phenotypic and molecular characteristics of gastric cancer organoids established from malignant peritoneal and pleural effusion. The established organoid-immune cell co-culture models can be utilized to evaluate a variety of drug screenings, including ICI and various combinations, helping in the selection of drugs for individual patient. Citation Format: Han Byeol Mun, juin Park, Jinsoo Jang, Jingmin Che, Tae Soo Kim, Woo Sun Kwon, Hyun Cheol Chung, Sun Young Rha. Molecular-genomic characterization of metastatic gastric cancer organoids and establishment of organoid-immune cell co-culture model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 229.

Abstract 7281: Targeting the YAP/TAZ-TEAD pathway with TEAD inhibitors synergizes with chemotherapy and blocks diffuse gastric cancer progression

Abstract Introduction: Diffuse gastric cancer (DGC) accounts for one third of gastric cancers and is associated with poor differentiation, discohesive growth, chemoresistance, and worse survival compared to intestinal-type tumors. DGC is typically genomically stable, and there are no approved DGC-targeted therapies. Recent studies have identified a potential tumor-promoting role for Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif (YAP/TAZ), which interact with TEAD transcription factors to regulate gene expression. Therefore, we evaluated the activity and mechanism of TEAD inhibitors in DGC preclinical models. Methods: Gastric cancer patient-derived organoids (PDOs) and cell lines were treated with TEAD inhibitors (K-975, VT103, VT104, VT107) and/or 5-fluorouracil (5-FU) for in vitro assays evaluating cell proliferation or invasion. Additional experiments were conducted to test the in vivo activity of TEAD inhibitors or shRNA-mediated knockdown of YAP in subcutaneous xenograft, orthotopic xenograft, and lung metastasis models using a genetically engineered mouse model of gastric cancer driven by oncogenic KrasG12D and loss of Trp53/Cdh1. Results: TEAD inhibitors K-975, VT103, VT104, and VT107 reduced proliferation, colony formation, and invasion in DGC cell lines and PDOs. We observed no effect of TEAD inhibitors on the viability of normal gastric organoids. Combining the TEAD1-selective inhibitor VT103 with 5-FU chemotherapy resulted in further reduction of DGC PDO viability relative to monotherapy, including in a PDO derived from a patient with locally advanced DGC that demonstrated intrinsic resistance to neoadjuvant chemotherapy. In flank tumors and orthotopic models, inhibition of the YAP/TAZ-TEAD pathway using YAP shRNA and VT103, either alone or in combination with 5-FU, abrogated primary tumor formation, with the strongest inhibition observed with combination treatment. Loss of YAP or VT103 treatment also significantly reduced lung metastasis after tail vein injection of mouse gastric cancer cells. We hypothesized that TEAD inhibition blocks an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Consistent with this hypothesis, TEAD1 inhibition with VT103 or YAP knockdown reduced expression of the CSC marker CD44 in vitro and decreased expression of CD44 and the EMT marker Slug in tumor xenografts. Conclusions: The YAP/TAZ-TEAD pathway is an important driver of gastric cancer growth and metastasis, particularly in DGC where it may promote EMT and cancer stemness. TEAD inhibition reduces gastric cancer growth and invasion in vitro and in vivo in preclinical models and demonstrates synergistic activity with 5-FU chemotherapy. These results suggest a potential role for evaluating the efficacy of TEAD inhibitors in patients with DGC. Citation Format: Anastasiia Bulakhova, Jin Sun Cho, Jack T. Rifkin, Jaewon Kim, Thomas J. Ryan, Tracy T. Tang, Ryan H. Moy. Targeting the YAP/TAZ-TEAD pathway with TEAD inhibitors synergizes with chemotherapy and blocks diffuse gastric cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7281.

Abstract 3382: Unveiling gastric premalignancy: A comprehensive study of tumor initiation and evolution using organoid models

Abstract Tumor initiation and evolution is still a poorly characterized process. Organoid models recapitulate three-dimensional tissue structure, differentiation, and disease pathogenesis. These models can help to better characterize pre-neoplastic processes and tumor evolution, with implications for early cancer detection and personalized therapeutic strategies for patients with gastric cancer (GC). In our research, we performed whole exome sequencing (WES) on 24 GC patient tumors, uncovering distinct mutation frequencies in well-known tumor suppressor genes -APC, TP53, and ARID1A (18.75%, 37.5%, 43.75%, respectively) -compared to The Cancer Genome Atlas (TCGA) PanCancer Altas Cohort (n=440) (13.48%, 50.56%, 22.47% respectively). To gain a deeper understanding of how these mutated genes promote gastric premalignancy, we generated multiple isogenic gastric organoid lines with mutant APC (AT-dmut) or ARID1A (ArT-dmut) on a TP53 mutant background as well as TP53 mutant-only lines (T-mut) using our established CRISPR/Cas9 genome-editing platform and two HLA patient-derived normal gastric organoid lines (HL-PDOs). After confirmation of functional gene knockouts, we focused on elucidating carcinogenic changes via a two-pronged approach: (1) a comparative analysis of growth and morphology between these mutant organoid models and the parental normal gastric organoids and (2) an in-depth examination of mutational and transcriptional changes in our edited AT-mut, ArT-mut and T-mut organoid models. We cultured each edited organoid line for up to 350 days and collected histological and molecular data at multiple time points. Preliminary analysis shows an increased growth rate of all edited cell lines compared to parental normal lines. The ArT-mut and AT-mut cell lines exhibit distinct morphological and 3D structural alterations, while T-mut cell lines do not. We expect to find additional mutation gains in these cells’ lines compared to the parental normal gastric organoids that promote gastric phenotype with the greatest gain of alternations at the 350-day timepoint. These additional mutations and transcriptional changes will help us gain insights into the pathways altered in gastric cancer initiation and early progression that may be specific to HLAs. Our overall goal is to enhance our understanding of the molecular mechanisms underlying gastric premalignancy among HLAs, ultimately contributing to advancements in early detection and tailored therapeutic interventions for HLA patients with GC and advancing cancer health equity. Citation Format: Alexa Morales Arana, Nicole B. Halmai, Hongyong Zhang, Paul Lott, Ana Estrada, University of California Minority Patient-DerivedXenograft Development and Trial (UCaMP) Center, Luis Carvajal-Carmona. Unveiling gastric premalignancy: A comprehensive study of tumor initiation and evolution using organoid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3382.

Bibliometrics analysis based on the Web of Science: Current trends and perspective of gastric organoid during 2010-2023.

Three-dimensional organoid culture systems have been established as a robust tool for elucidating mechanisms and performing drug efficacy testing. The use of gastric organoid models holds significant promise for advancing personalized medicine research. However, a comprehensive bibliometric review of this bur-geoning field has not yet been published. To analyze and understand the development, impact, and direction of gastric organoid research using bibliometric methods using data from the Web of Science Core Collection (WoSCC) database. This analysis encompassed literature pertaining to gastric organoids published between 2010 and 2023, as indexed in the WoSCC. CiteSpace and VOSviewer were used to depict network maps illustrating collaborations among authors, institutions and keywords related to gastric organoid. Citation, co-citation, and burst analysis methodologies were applied to assess the impact and progress of research. A total of 656 relevant studies were evaluated. The majority of research was published in gastroenterology-focused journals. Globally, Yana Zavros, Hans Clevers, James M Wells, Sina Bartfeld, and Chen Zheng were the 5 most productive authors, while Hans Clevers, Huch Meritxell, Johan H van Es, Marc Van de Wetering, and Sato Toshiro were the foremost influential scientists in this area. Institutions from the University Medical Center Utrecht, Netherlands Institute for Developmental Biology (Utrecht), and University of Cincinnati (Cincinnati, OH, United States) made the most significant contributions. Currently, gastric organoids are used mainly in studies investigating gastric cancer (GC), Helicobacter pylori-infective gastritis, with a focus on the mechanisms of GC, and drug screening tests. Key focus areas of research using gastric organoids include unraveling disease mechanisms and enhancing drug screening techniques. Major contributions from renowned academic institutions highlight this field's dynamic growth.

Mechanism of action of CDH1 gene on gastric organoid growth and E-cadherin expression in mice

Mechanism of action of CDH1 gene on gastric organoid growth and E-cadherin expression in mice

E-cadherin loss drives diffuse-type gastric tumorigenesis via EZH2-mediated reprogramming.

Diffuse-type gastric adenocarcinoma (DGAC) is a deadly cancer often diagnosed late and resistant to treatment. While hereditary DGAC is linked to CDH1 mutations, the role of CDH1/E-cadherin inactivation in sporadic DGAC tumorigenesis remains elusive. We discovered CDH1 inactivation in a subset of DGAC patient tumors. Analyzing single-cell transcriptomes in malignant ascites, we identified two DGAC subtypes: DGAC1 (CDH1 loss) and DGAC2 (lacking immune response). DGAC1 displayed distinct molecular signatures, activated DGAC-related pathways, and an abundance of exhausted T cells in ascites. Genetically engineered murine gastric organoids showed that Cdh1 knock-out (KO), KrasG12D, Trp53 KO (EKP) accelerates tumorigenesis with immune evasion compared with KrasG12D, Trp53 KO (KP). We also identified EZH2 as a key mediator promoting CDH1 loss-associated DGAC tumorigenesis. These findings highlight DGAC's molecular diversity and potential for personalized treatment in CDH1-inactivated patients.

A162 DEFINING INNATE IMMUNE CELL MECHANISMS UPON HELICOBACTER PYLORI MEDIATED TUMORIGENESIS

Abstract Aims Hypothesis: H. pylori interacts with eosinophils to inhibit degranulation, alter eosinophil gene expression and promote gastric tumorigenesis Aim 1: Elucidate the mechanisms underlying the interactions between eosinophils and H. pylori. Aim 2: Determine the role of eosinophils during H. pylori mediated gastric tumorigenesis. Methods Histological and immunofluorescent staining. Hemotoxilin and eosin staining is important to analyze gastric gland structure and parietal cell loss. Immunofluorescent staining is used to detect and localize specific antigens present in cells and cell membranes. Gastric organoids: Gastric organoids are an important tool for modelling dissease. Gastric organoids generated from adult stem cells from gastric glands depend on growth factors that mimic tissue regeneration. Bone marrow derived eosinophils: Bone marrow progenitor cells isolated from murine femur and tibia are easily converted into maturer eosinophils with the addition of cytokine interleukin-5. Results Addition of mouse derived eosinophils to gastric organoids significantly increases number of organoids. To understand the relationship between eosinophils and gastric epithelial cell (GEC) growth, I designed a co-culture experiment that involved incubating mature eosinophils with gastric glands in an extracellular matrix. These findings revealed a significant increase in the number of gastric organoids per well when eosinophils were present compared to the gastric organoids without eosinophils. This observation supports the hypothesis that eosinophils may play a crucial role in promoting gastric tumorigenesis. Exposure of mouse derived eosinophils to H. pylori significantly increases gastric organoid proliferation. To explore the impact of stimulating eosinophils prior to incubation with GECs I incubated the eosinophils with H. pylori. As expected, there was a significant increase in the number of organoids per well with the addition of uninfected eosinophils. Interestingly, when H. pylori incubated eosinophils were added to the gastric glands, there was a significant increase in the number of gastric organoids per well compared to uninfected eosinophils. Conclusions My preliminary findings have indicated that eosinophils can promote gastric epithelial stem cell proliferation. These results led me to ask how treatment of eosinophils with H. pylori subsequently impacts gastric organoid growth and gastric stem cell proliferation. Interestingly, I found H. pylori treated eosinophils increase growth and gastric stem cell proliferation. These results support my hypothesis as increased proliferation is considered a hallmark of tumorigenesis. There little known about resident and inflammatory GI eosinophils and the role of eosinophils during tumorigenesis in unknown. A better understanding of the role eosinophils play can uncover novel mechanisms to be targeted for therapeutic intervention. Funding Agencies CIHR

Establishment and Characterization of Patient-derived Gastric Organoids from Biopsies of Benign Gastric Body and Antral Epithelium.

Gastric patient-derived organoids (PDOs) offer a unique tool for studying gastric biology and pathology. Consequently, these PDOs find increasing use in a wide array of research applications. However, a shortage of published approaches exists for producing gastric PDOs from single-cell digests while maintaining a standardized initial cell seeding density. In this protocol, the emphasis is on the initiation of gastric organoids from isolated single cells and the provision of a method for passaging organoids through fragmentation. Importantly, the protocol demonstrates that a standardized approach to the initial cell seeding density consistently yields gastric organoids from benign biopsy tissue and allows for standardized quantification of organoid growth. Finally, evidence supports the novel observation that gastric PDOs display varying rates of formation and growth based on whether the organoids originate from biopsies of the body or antral regions of the stomach. Specifically, it is revealed that the use of antral biopsy tissue for organoid initiation results in a greater number of organoids formed and more rapid organoid growth over a 20-day period when compared to organoids generated from biopsies of the gastric body. The protocol described herein offers investigators a timely and reproducible method for successfully generating and working with gastric PDOs.

Gastric cancer patient-derived organoids model for the therapeutic drug screening

BackgroundGastric cancer (GC) is a highly heterogeneous disease featuring many various histological and molecular subtypes. Therefore, it is imperative to have well-characterized in vitro models for personalized treatment development. Gastric cancer patient-derived organoids (PDOs), re-capitulating in vivo conditions, exhibit high clinical efficacy in predicting drug sensitivity to facilitate the development of cancer precision medicine. MethodsPDOs were established from surgically resected GC tumor tissues. Histological and molecular characterization of PDOs and primary tissues were performed via IHC and sequencing analysis. We also conducted drug sensitivity tests using PDO cultures with five chemotherapeutic drugs and twenty-two targeted drugs. ResultsWe have successfully constructed a PDOs biobank that included EBV+, intestinal/CIN, diffuse/GS, mixed and Her2+ GC subtypes, and these PDOs captured the pathological and genetic characteristics of corresponding tumors and exhibited different sensitivities to the tested agents. In a clinical case study, we performed an additional drug sensitivity test for a patient who reached an advanced progressive stage after surgery. We discovered that the combination of napabucasin and COTI-2 exhibited a stronger synergistic effect than either drug alone. ConclusionPDOs maintained the histological and genetic characteristics of original cancer tissues. PDOs biobank opens up new perspectives for studying cancer cell biology and personalized medicine as a preclinical study platform.

Oncogenic Fatty Acid Metabolism Rewires Energy Supply Chain in Gastric Carcinogenesis

Background & AimsGastric carcinogenesis develops within a sequential carcinogenic cascade from pre-cancerous metaplasia to dysplasia and adenocarcinoma, and oncogenic gene activation can drive the process. Metabolic reprogramming is considered a key mechanism for cancer cell growth and proliferation. However, it remains unclear how metabolic changes contribute to the progression of metaplasia to dysplasia. We have examined metabolic dynamics during gastric carcinogenesis using a novel mouse model that induces Kras activation in zymogen-secreting chief cells. MethodsWe generated a Gif-rtTA;TetO-Cre;KrasG12D (GCK) mouse model which continuously induces active Kras expression in chief cells following doxycycline treatment. Histological examination and imaging mass spectrometry were performed in the GCK mouse stomachs at 2 to 14 weeks after doxycycline treatment. Mouse and human gastric organoids were used for metabolic enzyme inhibitor treatment. The GCK mice were treated with a stearoyl-CoA desaturase (SCD) inhibitor to inhibit the fatty acid desaturation. Tissue microarrays were used to assess the SCD expression in human gastrointestinal cancers. ResultsThe GCK mice developed metaplasia and high-grade dysplasia within 4 months. Metabolic reprogramming from glycolysis to fatty acid metabolism occurred during metaplasia progression to dysplasia. Altered fatty acid desaturation through SCD produces a novel Eicosenoic acid, which fuels dysplastic cell hyperproliferation and survival. The SCD inhibitor killed both mouse and human dysplastic organoids and selectively targeted dysplastic cells in vivo. SCD was upregulated during carcinogenesis in human gastrointestinal cancers. ConclusionsActive Kras expression only in gastric chief cells drives the full spectrum of gastric carcinogenesis. Also, oncogenic metabolic rewiring is an essential adaptation for high energy demand in dysplastic cells.

RHOAL57V drives the development of diffuse gastric cancer through IGF1R-PAK1-YAP1 signaling.

Cancer-associated mutations in the guanosine triphosphatase (GTPase) RHOA are found at different locations from the mutational hotspots in the structurally and biochemically related RAS. Tyr42-to-Cys (Y42C) and Leu57-to-Val (L57V) substitutions are the two most prevalent RHOA mutations in diffuse gastric cancer (DGC). RHOAY42C exhibits a gain-of-function phenotype and is an oncogenic driver in DGC. Here, we determined how RHOAL57V promotes DGC growth. In mouse gastric organoids with deletion of Cdh1, which encodes the cell adhesion protein E-cadherin, the expression of RHOAL57V, but not of wild-type RHOA, induced an abnormal morphology similar to that of patient-derived DGC organoids. RHOAL57V also exhibited a gain-of-function phenotype and promoted F-actin stress fiber formation and cell migration. RHOAL57V retained interaction with effectors but exhibited impaired RHOA-intrinsic and GAP-catalyzed GTP hydrolysis, which favored formation of the active GTP-bound state. Introduction of missense mutations at KRAS residues analogous to Tyr42 and Leu57 in RHOA did not activate KRAS oncogenic potential, indicating distinct functional effects in otherwise highly related GTPases. Both RHOA mutants stimulated the transcriptional co-activator YAP1 through actin dynamics to promote DGC progression; however, RHOAL57V additionally did so by activating the kinases IGF1R and PAK1, distinct from the FAK-mediated mechanism induced by RHOAY42C. Our results reveal that RHOAL57V and RHOAY42C drive the development of DGC through distinct biochemical and signaling mechanisms.

Establishment of a novel overlay culture method that enables immune response assessment using gastric cancer organoids

Organoid technology, a novel 3D cell culture system, can reproduce a patient’s cancer and may be a novel immunotherapy experimental model. However, currently no gastric cancer organoid (GCO) models in which the organoid and immune cells are in free contact and sufficiently react with each other exist. In this study, we aimed to create a coculture model in which immune cells can move freely and stay in contact with GCOs. We coated the bottom surface of the plate with Matrigel and adhered stem cells to the Matrigel surface, instead of completely embedding them in Matrigel to culture organoids. This method allowed GCOs to grow on the Matrigel surface while maintaining a three-dimensional structure and reproducing the characteristics of the patient’s cancer. We cocultured GCOs and immune cells. Using this model, immune cells could freely move and were in sufficient contact with the cultured GCOs. Our model allowed real-time observation of the immune response and tumor destruction with time. In addition, the GCO killing assay was assessed with natural killer cells from the same patient. This organoid culture model enabled repeated evaluation of the GCO killing assay with various immune cells in vitro. We established a new experimental model that allowed free movement of immune cells and sufficient contact with GCOs. Using this model, it may be possible to predict the effects of immune checkpoint inhibitors in vitro (using GCOs) before administering them to patients.

Bioprinted Organoids Platform with Tumor Vasculature for Implementing Precision Personalized Medicine Targeted Towards Gastric Cancer

AbstractAccurate prediction of treatment response for cancer patients is essential for overcoming intrinsic therapy resistance that results from genetic heterogeneity, varying tumor growth kinetics, and the complex tumor microenvironment. To achieve this goal, there is an urgent need for effective preclinical in vitro models that recapitulate the molecular–pathologic features and intricate ecology of native tumors for precision medicine. In this study, a vascularized organoid model (VOM) composed of patient‐derived gastric cancer organoids (PDOs), perfusable endothelium, and stomach decellularized extracellular matrix is presented that enables the prediction of clinical response to VEGFR2‐targeted therapy in gastric cancer patients. The results indicate that VOMs are dependent on the PDO molecular subtype. Moreover, VOMs accurately reproduce the clinically observed responses of patients treated with VEGFR2 inhibitor. Therefore, VOMs represent a valuable platform for providing clinical predictions for personalized testing and potential discovery of therapeutic drugs in various cancers that lack standardized regimens.

The influence of Helicobacter pylori, proton pump inhibitor, and obesity on the gastric microbiome in relation to gastric cancer development

Helicobacter pylori infection is still the main risk factor for the development of gastric cancer (GC). We explore the scientific evidence for the role of the gastric microbiome beyond Helicobacter pylori (H. pylori) in gastric carcinogenesis. The composition of the gastric microbiome in healthy individuals, in presence and absence of H. pylori infection, in proton pump inhibitor (PPI)-users, obese individuals, and GC patients was investigated. Possible mechanisms for microbial involvement, limitations of available research and options for future studies are provided.A common finding amongst studies was increased levels of Streptococcus, Prevotella, Neisseria, and Actinomyces in healthy individuals or those with H. pylori-negative gastritis. In PPI-users the risk for GC increases with the treatment duration, and the gastric microbiome shifts, with the most consistent increase in the genus Streptococcus. Similarly, in obese individuals, Streptococcus was the most abundant genus, with an increased risk for cardia GC. The genera Streptococcus, Lactobacillus and Prevotella were found to be more prominent in GC patients in multiple studies. Potential mechanisms of non-H. pylori microbiota contributing to GC are linked to lipopolysaccharide production, contribution to inflammatory pathways, and the formation of N-nitroso compounds and reactive oxygen species.In conclusion, the knowledge of the gastric microbiome in GC is mainly descriptive and based on sequencing of gastric mucosal samples. For a better mechanistic understanding of microbes in GC development, longitudinal cohorts including precancerous lesions, different regions in the stomach, and subtypes of GC, and gastric organoid models for diffuse and intestinal type GC should be employed.

Establishing an Infection Model Through Microinjection of Gastric Organoids with Pathogenic Bacteria

Establishing an Infection Model Through Microinjection of Gastric Organoids with Pathogenic Bacteria

Establishing an Infection Model Through Microinjection of Gastric Organoids with Pathogenic Bacteria

Establishing an Infection Model Through Microinjection of Gastric Organoids with Pathogenic Bacteria

Generation of human gastric assembloids from primary fetal organoids

PurposeUnderstanding human gastric epithelium homeostasis remains partial, motivating the exploration of innovative in vitro models. Recent literature showcases the potential of fetal stem cell-derived organoids in developmental and disease modelling and translational therapies. To scale the complexity of the model, we propose to generate assembloids, aiming to increase gastric maturation to provide new structural and functional insights.MethodsHuman fetal gastric organoids (fGOs) were expanded in 3D Matrigel cultures. Confluent organoid cultures were released from the Matrigel dome and resuspended in a collagen I hydrogel. Subsequently, the organoid mixture was seeded in a ring shape within a 24-well plate and allowed to gelate. The structure was lifted in the medium and cultured in floating conditions, allowing for organoid self-assembling into a gastric assembloid. After 10 days of maturation, the assembloids were characterized by immunostaining and RT-PCR, comparing different fetal developmental stages.ResultsSuccessful generation of human fetal gastric assembloids (fGAs) was achieved using spontaneous self-aggregation within the collagen I hydrogel. Immunostaining analysis of early and late fGAs showed the establishment of apico-basal cell polarity, secretion of gastric mucins, and the presence of chromogranin A in both samples. Transcriptional markers analysis revealed distinct disparities in markers associated with mature cell types between late and early fetal stages.ConclusionsfGOs can reliably be generated from human fetal samples. This pioneering assembloid approach paves the way for advancing our comprehension of human gastric epithelium homeostasis and its perturbation, offering a better in vitro platform for the study of gastric epithelial development and therapeutic translation.

Heterogeneity of Phase II Enzyme Ligands on Controlling the Progression of Human Gastric Cancer Organoids as Stem Cell Therapy Model.

Gastric cancer (GC) organoids are frequently used to examine cell proliferation and death as well as cancer development. Invasion/migration assay, xenotransplantation, and reactive oxygen species (ROS) production were used to examine the effects of antioxidant drugs, including perillaldehyde (PEA), cinnamaldehyde (CA), and sulforaphane (SFN), on GC. PEA and CA repressed the proliferation of human GC organoids, whereas SFN enhanced it. Caspase 3 activities were also repressed on treatment with PEA and CA. Furthermore, the tumor formation and invasive activities were repressed on treatment with PEA and CA, whereas they were enhanced on treatment with SFN. These results in three-dimensional (3D)-GC organoids showed the different cancer development of phase II enzyme ligands in 2D-GC cells. ROS production and the expression of TP53, nuclear factor erythroid 2-related factor (NRF2), and Jun dimerization protein 2 were also downregulated on treatment with PEA and CA, but not SFN. NRF2 knockdown reversed the effects of these antioxidant drugs on the invasive activities of the 3D-GC organoids. Moreover, ROS production was also inhibited by treatment with PEA and CA, but not SFN. Thus, NRF2 plays a key role in the differential effects of these antioxidant drugs on cancer progression in 3D-GC organoids. PEA and CA can potentially be new antitumorigenic therapeutics for GC.

Replicative stress in gastroesophageal cancer is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

Gastroesophageal adenocarcinoma (GEA) is an aggressive malignancy with chromosomal instability (CIN). To understand adaptive responses enabling DNA damage response (DDR) and CIN, we analyzed matched normal, premalignant, and malignant gastric lesions from human specimens and a carcinogen-induced mouse model, observing activation of replication stress, DDR, and p21 in neoplastic progression. In GEA cell lines, expression of DDR markers correlated with ploidy abnormalities, such as number of high-level focal amplifications and whole-genome duplication (WGD). Integrating TP53 status, ploidy abnormalities, and DDR markers into a compositive score helped predict GEA cell lines with enhanced sensitivity to Chk1/2 and Wee1 inhibition, either alone or combined with irinotecan (SN38). We demonstrate that Chk1/2 or Wee1 inhibition combined with SN38/irinotecan shows greater anti-tumor activity in human gastric cancer organoids and an invivo xenograft mouse model. These findings indicate that specific DDR biomarkers and ploidy abnormalities may predict premalignant progression and response to DDR pathway inhibitors.