Gastric Cancer Organoids Research Articles

Precision Medicine for Gastric Cancer: Current State of Organoid Drug Testing

Gastric cancer (GC) presents a significant health challenge and ranks as the fifth most common cancer in the world. Unfortunately, most patients with GC exhaust standard care treatment options due to late diagnosis and tumour heterogeneity that leads to drug resistance, resulting in poor survival outcomes. Potentially, this situation can be improved by personalising treatment choice. Organoids are an emerging cell model system that recapitulates tumour heterogeneity and drug responses. Coupled with genomic analysis, organoid culture can be used to guide personalised medicine. The GC organoid field, however, lacks standardised methodologies for assessing organoid drug sensitivities. Comparing results across different GC organoid studies and correlating organoid drug responses with patient outcomes is challenging. Hence, we aim to summarise the methodologies used in GC organoid drug testing and correlation with clinical outcomes and discuss design considerations and limitations to enhance the robustness of such studies in the future.

Circ_0008315 promotes tumorigenesis and cisplatin resistance and acts as a nanotherapeutic target in gastric cancer

IntroductionCisplatin-based chemotherapy is one of the fundamental therapeutic modalities for gastric cancer (GC). Chemoresistance to cisplatin is a great clinical challenge, and its underlying mechanisms remain poorly understood. Circular RNAs (circRNAs) are involved in the pathophysiology of multiple human malignancies.MethodsHigh-throughput sequencing was performed to determine the differentially expressed profile of circRNA in GC tissues and cisplatin-resistant GC cells. Quantitative real-time polymerase chain reaction and Fluorescence in situ hybridization was utilized to confirm the dysregulation of circ_0008315 in GC tissues. To evaluate the prognostic significance of circ_0008315 in GC, we used Kaplan-Meier plot. The self-renewal ability of drug-resistant GC cell was verified through tumor sphere formation assay. GC organoids were constructed to simulate the tumor microenvironment and verified the function of circ_0008315 in cisplatin resistance of gastric cancer. In vivo evaluation was conducted using patient-derived xenograft models. Dual-luciferase reporter gene, RNA immunoprecipitation and miRNA pull-down assays were employed to investigate the molecular mechanisms of circ_0008315 in GC.ResultsWe revealed that a novel circRNA hsa_circ_0008315 was upregulated in GC and cisplatin-resistant GC cells. Elevated circ_0008315 was also observed in cisplatin-resistant GC organoid model. High circ_0008315 expression predicted unfavorable survival outcome in GC patients. Downregulation of circ_0008315 expression inhibited proliferation, mobility, and epithelial-mesenchymal transition of GC cells in vitro and in vivo. Reducing circ_0008315 expression in cisplatin-resistant GC organoid model reversed cisplatin resistance. Mechanistically, circ_0008315 modulated the stem cell properties of GC through the miR-3666/CPEB4 signaling pathway, thereby promoting cisplatin resistance and GC malignant progression. Furthermore, we developed PLGA-PEG nanoparticles targeting circ_0008315, and the nanoparticles could effectively inhibit GC proliferation and cisplatin resistance.ConclusionCirc_0008315 exacerbates GC progression and cisplatin resistance, and can be used as a prognostic predictor. Circ_0008315 may function as a promising nanotherapeutic target for GC treatment.Graphical

Establishment of patient-derived organoids and a characterization based drug discovery platform for treatment of gastric cancer

BackgroundGastric cancer (GC) encompasses many different histological and molecular subtypes. It is a major driver of cancer mortality because of poor survival and limited treatment options. Personalised medicine in the form of patient-derived organoids (PDOs) represents a promising approach for improving therapeutic outcomes. The goal of this study was to overcome the limitations of current models by ameliorating organoid cultivation.MethodsOrganoids derived from cancer tissue were evaluated by haematoxylin and eosin staining, immunohistochemistry, mRNA, and whole-exome sequencing. Three representative chemotherapy drugs, 5-fluorouracil, docetaxel, and oxaliplatin, were compared for their efficacy against different subtypes of gastric organoids by ATP assay and apoptosis staining. In addition, drug sensitivity screening results from two publicly available databases, the Genomics of Drug Sensitivity in Cancer and Cancer Cell Line Encyclopaedia, were pooled and applied to organoid lines. Once key targeting genes were confirmed, chemotherapy was used in combination with poly (ADP ribose) polymerase (PARP)-targeted therapy.ResultsWe successfully constructed GC PDOs surgically resected from GC patient tissue. PDOs closely reflected the histopathological and genomic features of the corresponding primary tumours. Whole-exosome sequencing and mRNA analysis revealed that changes to the original tumour genome were maintained during long-term culture. The drugs caused divergent responses in intestinal, poorly differentiated intestinal, and diffuse gastric cancer organoids, which were confirmed in organoid lines. Poorly differentiated intestinal GC patients benefited from a combination of 5-fluorouracil and veliparib.ConclusionThe present study demonstrates that combining chemotherapy with PARP targeting may improve the treatment of chemotherapy-resistant tumours.

Drug-induced senescence by aurora kinase inhibitors attenuates innate immune response of macrophages on gastric cancer organoids

Diffuse-type gastric cancer (DGC) is a subtype of gastric cancer with aggressiveness and poor prognosis. It is of great significance to find sensitive drugs for DGC. In the current study, a total of 20 patient-derived organoids (PDOs) were analyzed for screening the therapeutic efficacy of small molecule kinases inhibitors on gastric cancers, especially the therapeutic difference between intestinal-type gastric cancer (IGCs) and DGCs. The IGCs are sensitive to multiple kinases inhibitors, while DGCs are resistant to most of these kinases inhibitors. It was found that DGCs showed drug-induced senescent phenotype after treatment by aurora kinases inhibitors (AURKi) Barasertib-HQPA and Danusertib. The cell diameter of cancer cells are increased with stronger staining of senescence-associated β-galactosidase (SA-β-GAL), and characteristic appearance of multinucleated giant cells. The senescent cancer cells secrete large amounts of chemokine MCP-1/CCL2, which recruit and induce macrophage to M2-type polarization in PDOs of DGC (DPDOs)-macrophage co-culture system. The up-regulation of local MCP-1/CCL2 can interact with MCP-1/CCL2 receptor (CCR2) expressed on macrophages and suppress their innate immunity to cancer cells. Overall, the special response of DGC to AURKi suggests that clinicians should select a sequential therapy with senescent cell clearance after AURKi treatment for DGC.

OLFM4 promotes the progression of intestinal metaplasia through activation of the MYH9/GSK3β/β-catenin pathway

BackgroundIntestinal metaplasia (IM) is classified into complete intestinal metaplasia (CIM) and incomplete intestinal metaplasia (IIM). Patients diagnosed with IIM face an elevated susceptibility to the development of gastric cancer, underscoring the critical need for early screening measures. In addition to the complexities associated with diagnosis, the exact mechanisms driving the progression of gastric cancer in IIM patients remain poorly understood. OLFM4 is overexpressed in several types of tumors, including colorectal, gastric, pancreatic, and ovarian cancers, and its expression has been associated with tumor progression.MethodsIn this study, we used pathological sections from two clinical centers, biopsies of IM tissues, precancerous lesions of gastric cancer (PLGC) cell models, animal models, and organoids to explore the role of OLFM4 in IIM.ResultsOur results show that OLFM4 expression is highly increased in IIM, with superior diagnostic accuracy of IIM when compared to CDX2 and MUC2. OLFM4, along with MYH9, was overexpressed in IM organoids and PLGC animal models. Furthermore, OLFM4, in combination with Myosin heavy chain 9 (MYH9), accelerated the ubiquitination of GSK3β and resulted in increased β-catenin levels through the Wnt signaling pathway, promoting the proliferation and invasion abilities of PLGC cells.ConclusionsOLFM4 represents a novel biomarker for IIM and could be utilized as an important auxiliary means to delimit the key population for early gastric cancer screening. Finally, our study identifies cell signaling pathways involved in the progression of IM.

Success rate of current human-derived gastric cancer organoids establishment and influencing factors: A systematic review and meta-analysis.

Human-derived gastric cancer organoids (GCOs) are widely used in gastric cancer research; however, the culture success rate is generally low. To explore the potential influencing factors, and the literature on successful culture rates of GCOs was reviewed using meta-analysis. PubMed, Web of Science, and EMBASE were searched for studies. Two trained researchers selected the studies and extracted data. STATA 17.0 software was used for meta-analysis of the incidence of each outcome event. The adjusted Methodological Index for Non-Randomized Studies scale was used to assess the quality of the included studies. Funnel plots and Egger's test were used to detect publication bias. Subgroup analyses were conducted for sex, tissue source, histological classification, and the pathological tumor-node-metastasis (pTNM) cancer staging system. Eight studies with a pooled success rate of 66.6% were included. GCOs derived from women and men had success rates of 67% and 46.7%, respectively. GCOs from surgery or biopsy/endoscopic submucosal dissection showed success rates of 70.9% and 53.7%, respectively. GCOs of poorly-differentiated, moderately-differentiated and signet-ring cell cancer showed success rates of 64.6%, 31%, and 32.7%, respectively. GCOs with pTNM stages I-II and III-IV showed success rates of 38.3% and 65.2%, respectively. Y-27632 and non-Y-27632 use showed success rates of 58.2% and 70%, respectively. GCOs generated with collagenase were more successful than those constructed with Liberase TH and TrypLE (72.1% vs 71%, respectively). EDTA digestion showed a 50% lower success rate than other methods (P = 0.04). GCO establishment rate is low and varies by sex, tissue source, histological type, and pTNM stage. Omitting Y-27632, and using Liberase TH, TrypLE, or collagenase yields greater success than EDTA.

Enhanced LRP8 expression induced by Helicobacter pylori drives gastric cancer progression by facilitating β-Catenin nuclear translocation

IntroductionHelicobacter pylori (H. pylori) infection has been associated with gastric carcinogenesis. However, the precise involvement of LRP8, the low-density lipoprotein receptor-related protein 8, in H. pylori pathogenesis and gastric cancer (GC) remains poorly understood. ObjectivesTo investigate the potential role of LRP8 in H. pylori infection and gastric carcinogenesis. MethodsThree-dimensional human-derived gastric organoids (hGO) and gastric cancer organoids (hGCO) were synthesized from the tissues obtained from human donors. In this work, multi-omics combined with in vivo and in vitro studies were conducted to investigate the potential involvement of LRP8 in H. pylori-induced GC. ResultsWe found that H. pylori infection significantly upregulated the expression of LRP8 in human GC tissues, cells, organoids, and mouse gastric mucous. In particular, LRP8 exhibited a distinct enrichment in cancer stem cells (CSC). Functionally, silencing of LRP8 affected the formation and proliferation of tumor spheroids, while increased expression of LRP8 was associated with increased proliferation and stemness of GC cells and organoids. Mechanistically, LRP8 promotes the binding of E-cadherin to β-catenin, thereby promoting nuclear translocation and transcriptional activity of β-catenin. Furthermore, LRP8 interacts with the cytotoxin-associated gene A (CagA) to form the CagA/LRP8/β-catenin complex. This complex further amplifies H. pylori-induced β-catenin nuclear translocation, leading to increased transcription of inflammatory factors and CSC markers. Clinical analysis demonstrated that abnormal overexpression of LRP8 is correlated with a poor prognosis and resistance to 5-Fluorouracil in patients with GC. ConclusionOur findings provide valuable information on the molecular intricacies of H. pylori-induced gastric carcinogenesis, offering potential therapeutic targets and prognostic markers for GC.

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.

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.

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.

Abstract C157: Targeting PIK3CA and CDKN2A alterations in Esophageal Adenocarcinoma with Alpelisib and Palbociclib

Abstract Our study investigates the efficacy of targeted therapies for Esophageal Adenocarcinoma (EAC) tumors harboring an activating PIK3CA mutation and inactivating CDKN2A mutation. We tested the small molecule inhibitors alpelisib, which targets the Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit (PIK3CA), and palbociclib, which targets Cyclin D kinase 4/6 (CDK4/6), in EAC in vitro organoid and in vivo mouse models. EAC and Gastric cancer (GC) organoid models were treated with single agent alpelisib or palbociclib on a log scale to determine their IC50 values. An EAC organoid harboring activating PIK3CA E545K and inactivating CDKN2A D74A mutations demonstrated more significant growth inhibition when treated with each agent alone versus standard of care chemotherapy. Subsequent experiments involved combination treatment of our EAC-mutant organoid with alpelisib and palbociclib as well as a representative GC organoid and EAC organoids lacking these specific mutations as controls. We confirmed PIK3CA downstream target engagement after alpelisib treatment by qPCR, western blot analysis and immunofluorescence analysis. Once in vitro efficacy was confirmed, EAC-mutant organoids were transplanted into mice by either subcutaneous or orthotopic injections and treated with chemotherapy, alpelisib and palbociclib alone or in combination. Our data demonstrate that a PIK3CA/CDKN2A mutated patient-derived EAC organoid is sensitive to alpelisib and palbociclib in a dose dependent manner, and analysis of PIK3CA downstream targets by western and qPCR was consistent with growth inhibition of treated organoids. These findings offer support for the use of alpelisib and palbociclib in the treatment of EAC patients with PIK3CA/CDKN2A mutations. Citation Format: Naryan Rustgi, Thomas Ryan, Jin Cho, Ryan Moy, Sam Yoon, Sandra Ryeom, Changhwan Yoon. Targeting PIK3CA and CDKN2A alterations in Esophageal Adenocarcinoma with Alpelisib and Palbociclib [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C157.

Ailanthone synergizes with PARP1 inhibitor in tumour growth inhibition through crosstalk of DNA repair pathways in gastric cancer.

In our previous research, we proved that ailanthone (AIL) inhibits the growth of gastric cancer (GC) cells and causes apoptosis by inhibiting P23. However, we still find some GC organoids are insensitive to AIL. We have done some sequencing analysis and found that the insensitive strains are highly expressed in PARP1. In this study, we investigated whether AIL can enhance the anti-tumour effect of PARPi in GC. CCK8 and spheroid colony formation assay were used to measure anti-tumour effects. SynergyFinder software was used to calculate the synergy score of the drug combination and flow cytometry was used to detect apoptosis. Western blot, IHC, IF tests were used to measure protein expression. Finally, nude mouse xenograft models were used to verify the invitro mechanisms. High expression of PARP1 was found to be the cause of drug insensitivity. When AIL is paired with a PARP1 inhibitor, olaparib (OLP), drug sensitivity improves. We discovered that this combination functions by blocking off HSP90-BRCA1 interaction and inhibiting the activity of PARP1, thus in turn inhibiting the homologous recombination deficiency and base excision repair pathway to finally achieve synthetic lethality through increased sensitivity. Moreover, P23 can regulate BRCA1 in GC invitro. This study proves that the inhibitory effect of AIL on BRCA1 allowed even cancer cells with normal BRCA1 function to be sensitive to PARP inhibitors when it is simultaneously administered with OLP. The results greatly expanded the scope of the application of PARPi.

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.

Wnt/β-catenin pathway is a key signaling pathway to trastuzumab resistance in gastric cancer cells

BackgroundTrastuzumab is the only approved target agent for the first-line treatment of human epidermal growth factor receptor-2 (HER-2) positive gastric cancer; however, trastuzumab resistance is a major problem in clinical practice. To comprehend the mechanism of trastuzumab resistance, we focused on the Wnt/β-catenin signaling pathway and its influence on the phenotypes and behavior of trastuzumab-resistant gastric cancer cells.MethodsTrastuzumab-resistant NCI-N87R cells were established in vitro from the human gastric cancer cell line NCI-N87 by dose-escalating repeated trastuzumab treatment. We investigated the phenotypes of NCI-N87R cells, including Wnt signaling pathway activity. Gastric cancer organoid cells were incubated with complete medium and Wnt3a-depletion medium, and their resistance to trastuzumab was compared.ResultsNCI-N87R exhibited stemness and epithelial-mesenchymal transition (EMT)-like phenotypes, along with decreased levels of the epithelial marker E-cadherin and increased levels of the mesenchymal markers Vimentin and Snail along with an increased Wnt signaling pathway activity. When gastric cancer cells were incubated in Wnt3a-conditioned medium. Wnt signaling pathway activity and resistance to trastuzumab increased. Gastric cancer patient-derived organoids incubated in Wnt3a-depletion medium were more susceptible to dose-dependent inhibition of cell viability by trastuzumab than those incubated in complete medium.ConclusionsTrastuzumab-resistant gastric cancer cells exhibited EMT-like phenotype, and trastuzumab resistance was promoted by the Wnt/β-catenin signaling pathway. The Wnt/β-catenin pathway is a key signaling pathway for trastuzumab resistance in gastric cancer cells.

The ATR inhibitor VE-821 increases the sensitivity of gastric cancer cells to cisplatin

BackgroundChemoresistance is a common event after cancer chemotherapy, including gastric cancer (GC). Cisplatin has been reported to induce the DNA damage response (DDR), thus leading to chemoresistance. VE-821, a specific inhibitor of ATR, has been proven to suppress a variety of solid malignancies effectively. Our study aimed to explore the effect of VE-821 on enhancing the chemical sensitivity to cisplatin and clarify the potential molecular mechanisms. MethodsCell viability and apoptosis of MKN-45 and AGS were measured by CCK8 and flow cytometry assay respectively. Western blotting was used to detect the expression of target proteins. TCGA database was used to analyze the correlation between the ATR expression with the prognosis of GC patients. The viability of GC organoids was detected by Cell Titer Glo (CTG) through luminescence. ResultsCisplatin inhibited the proliferation and induced apoptosis of GC cells with a relatively high IC50 value, and increased the phosphorylation levels of ATR-CHK1 and H2AX. VE-821 achieved the same effects but by downregulating the phosphorylation levels of the ATR-CHK1 pathway. Besides, higher ATR expression in GC tissues was positively correlated with higher pathological stage in GC patients. Interestingly, ATR inhibition reversed cisplatin-induced STAT3 activation and enhanced H2AX levels. Moreover, VE-821 significantly sensitized GC cells to cisplatin, and these two drugs had synergistic effects in GC cell lines, organoids, and in vivo. ConclusionOur results suggested VE-821 sensitized GC cells to cisplatin via reversing DDR activation. And VE-821 treatment may be a promising therapeutic strategy for GC patients with cisplatin resistance.

Creation of EGD-Derived Gastric Cancer Organoids to Predict Treatment Responses.

Gastric adenocarcinoma (GAd) is the third leading cause of cancer-related deaths worldwide. Most patients require perioperative chemotherapy, yet methods to accurately predict responses to therapy are lacking. Thus, patients may be unnecessarily exposed to considerable toxicities. Here, we present a novel methodology using patient-derived organoids (PDOs) that rapidly and accurately predicts the chemotherapy efficacy for GAd patients. Methods:Endoscopic GAd biopsies were obtained from 19 patients, shipped overnight, and PDOs were developed within 24 h. Drug sensitivity testing was performed on PDO single-cells with current standard-of-care systemic GAd regimens and cell viability was measured. Whole exome sequencing was used to confirm the consistency of tumor-related gene mutations and copy number alterations between primary tumors, PDOs, and PDO single-cells. Results:Overall, 15 of 19 biopsies (79%) were appropriate for PDO creation and single-cell expansion within 24 h of specimen collection and overnight shipment. With our PDO single-cell technique, PDOs (53%) were successfully developed. Subsequently, two PDO lines were subjected to drug sensitivity testing within 12 days from initial biopsy procurement. Drug sensitivity assays revealed unique treatment response profiles for combination drug regimens in both of the two unique PDOs, which corresponded with the clinical response. Conclusions:The successful creation of PDOs within 24 h of endoscopic biopsy and rapid drug testing within 2 weeks demonstrate the feasibility of our novel approach for future applications in clinical decision making. This proof of concept sets the foundation for future clinical trials using PDOs to predict clinical responses to GAd therapies.

PHGDH preserves one-carbon cycle to confer metabolic plasticity in chemoresistant gastric cancer during nutrient stress

Molecular classification of gastric cancer (GC) identified a subgroup of patients showing chemoresistance and poor prognosis, termed SEM (Stem-like/Epithelial-to-mesenchymal transition/Mesenchymal) type in this study. Here, we show that SEM-type GC exhibits a distinct metabolic profile characterized by high glutaminase (GLS) levels. Unexpectedly, SEM-type GC cells are resistant to glutaminolysis inhibition. We show that under glutamine starvation, SEM-type GC cells up-regulate the 3 phosphoglycerate dehydrogenase (PHGDH)-mediated mitochondrial folate cycle pathway to produce NADPH as a reactive oxygen species scavenger for survival. This metabolic plasticity is associated with globally open chromatin structure in SEM-type GC cells, with ATF4/CEBPB identified as transcriptional drivers of the PHGDH-driven salvage pathway. Single-nucleus transcriptome analysis of patient-derived SEM-type GC organoids revealed intratumoral heterogeneity, with stemness-high subpopulations displaying high GLS expression, a resistance to GLS inhibition, and ATF4/CEBPB activation. Notably, coinhibition of GLS and PHGDH successfully eliminated stemness-high cancer cells. Together, these results provide insight into the metabolic plasticity of aggressive GC cells and suggest a treatment strategy for chemoresistant GC patients.

Abstract 1193: Sensory innervation directly promotes gastric cancer and modulates the tumor microenvironment

Abstract Tumor innervation contributes directly to cancer progression, but there appears to be organ-specific variation in the type of innervation. While our previous studies showed cholinergic signaling promoted gastric cancer and vagal denervation inhibited gastric cancer, it is noteworthy that sensory nerves comprise 80% of the vagal axons. To investigate the role of sensory innervation in gastric carcinogenesis, we performed retrograde neural tracing and identified CGRP+ peptidergic neurons in nodose ganglia and dorsal root ganglia as primary gastric sensory neurons. While VAChT+ parasympathetic nerves are the main nerves innervating the normal stomach, CGRP+ sensory nerves show the greatest degree of expansion in mouse gastric tumors. Indeed, mouse gastric tumors show upregulation of NGF, a growth factor essential for sensory nerve survival, and gastric NGF overexpression in an NGF knock-in mouse also resulted in increased sensory innervation. Inhibition of NGF/Trk signaling by Entrectinib markedly attenuates NGF-dependent sensory nerve expansion. In addition to overall increased axonogenesis of sensory nerves, mouse models of gastric cancer showed upregulation of αCGRP peptide in gastric tumors and Calca mRNA in gastric sensory neurons, indicating increased activation of sensory neurons during gastric carcinogenesis. To study the effect of such activation, sensory neurons were then directly activated by treatment of Trpv1-Cre;hM3Dq mice with clozapine-N-oxide (CNO), which led to marked gastric hyperplasia with increased proliferation. Interestingly, in an acetic acid-induced ulcer model, activation of sensory neurons reduced the ulcer size and strongly promoted gland regeneration. Activation of sensory neurons, in a syngeneic, orthotopic model of gastric cancer in the Trpv1-Cre;hM3Dq mice, led to a significant increase in tumor volume, Ki-67+ cancer cells, CD31+ vessels and M2 macrophage polarization, as well as decreased CD8+ T cells. Analysis of available single-cell transcriptome data from mouse and human stomach showed that Calcrl/Ramp1 (CGRP receptors) are expressed in gastric epithelial cells and most stromal cells, while the expression levels of Tacr1 (receptor for Substance P) and Sstr1-5 (receptors for Somatostatin) were much lower. Importantly, some Calcrl/Ramp1+ cells are proliferative cells with strong Ki-67 expression. Our data show that Calcrl is also overexpressed in mouse gastric tumors, while TCGA data reveals significant upregulation of CALCRL in human gastric cancers and that high expression of CALCRL is correlated with poor overall survival. Finally, we established a co-culture model using DRGs and gastric cancer organoids, and found that sensory nerves can generate synaptic-like terminals on cancer organoids. In conclusion, our data indicate that sensory innervation directly promotes gastric cancer growth and also modulates the tumor microenvironment. Citation Format: Xiaofei Zhi, Feijing Wu, Jin Qian, Yosuke Ochiai, Guodong Lian, Ermanno Malagola, Timothy C. Wang. Sensory innervation directly promotes gastric cancer and modulates the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1193.