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

SummaryLung cancer is an extremely heterogeneous disease, and its treatment remains one of the most challenging tasks in medicine. Few existing laboratory lung cancer models can faithfully recapitulate the diversity of the disease and predict therapy response. Here, we establish 12 patient-derived organoids from the most common lung cancer subtype, lung adenocarcinoma (LADC). Extensive gene and histopathology profiling show that the tumor organoids retain the histological architectures, genomic landscapes, and gene expression profiles of their parental tumors. Patient-derived lung cancer organoids are amenable for biomarker identification and high-throughput drug screening in vitro. This study should enable the generation of patient-derived lung cancer organoid lines, which can be used to further the understanding of lung cancer pathophysiology and to assess drug response in personalized medicine.

ABSTRACTIntroduction: Gastric cancer is among the most common cancers worldwide. Despite declining incidences, the prognosis remains dismal in Western countries and is better in Asian countries with national cancer screening programs. Complete endoscopic or surgical resection of the primary tumor with or without lymphadenectomy offers the only chance of cure in the early stage of the disease. Survival of more locally advanced gastric cancers was improved by the introduction of perioperative, adjuvant and palliative chemotherapy. However, the identification and usage of novel predictive and diagnostic targets is urgently needed.Areas covered: Recent comprehensive molecular profiling of gastric cancer proposed four molecular subtypes, i.e. Epstein-Barr virus-associated, microsatellite instable, chromosomal instable and genomically stable carcinomas. The new molecular classification will spur clinical trials exploring novel targeted therapeutics. This review summarizes recent advancements of the molecular classification, and based on that, putative pitfalls for the development of tissue-based companion diagnostics, i.e. prevalence of actionable targets and therapeutic efficacy, tumor heterogeneity and tumor evolution, impact of ethnicity on gastric cancer biology, and standards of care in the East and West.Expert commentary: The overall low prevalence of actionable targets and tumor heterogeneity are the two main obstacles of precision medicine for gastric cancer.

Metastasis is a major factor leading to an unfavorable prognosis in gastric cancer (GC). However, factors driving GC metastasis are not fully understood. Single-cell transcriptome analysis was done on three primary GC samples, one adjacent nontumor sample, and six GC metastasis samples (GSE163558) to clarify cellular composition characteristics, differential genes, and screen genes related to epithelial-mesenchymal transition (EMT). Effects of GPX3 on GC growth and metastasis were assessed through in vitro cell experiments, a GC liver metastasis model, a GC organoid model, and an organoid xenograft nude mouse model. The primary tumor samples showed a higher proportion of epithelial cells, and analysis revealed a significant reduction in GPX3 levels in GC metastasis samples within the subpopulation of epithelial cells undergoing EMT. Cell experiments demonstrated low expression of GPX3 in GC cells, and overexpression of GPX3 inhibited cell migration, invasion, and EMT in GC cells. Further validation in a nude mouse liver metastasis model confirmed the repressive role of GPX3 in GC metastasis. Additionally, GPX3 could inhibit the growth of patient-derived GC organoids and impede tumor growth and metastasis in an organoid xenograft nude mouse model. This study, based on single-cell transcriptome analysis, revealed the potential inhibitory factor GPX3 in metastatic GC and validated its effects on GC growth and metastasis using GC cells and organoids in vitro and in vivo experiments. These findings offer insights into understanding GC heterogeneity and targeting GPX3 in GC therapeutic strategies.

To establish an in vitro study model of gastric cancer, gastric cancer organoid culture system, by biopsy under gastroscope, and to explore and analyze the related factors affecting the success rate of culture, to provide a better in vitro model for the study of gastric cancer. Twenty-six patients with advanced gastric cancer were collected. Organoids were cultured by biopsy under gastroscope. Paraffin sections were made and HE staining was used to compare the consistency of gastroscopic pathological morphology and organoids. To explore the influencing factors of cultivating gastric cancer organoids in combination with clinical data. A total of 26 cases were collected by gastroscopy, and 12 cases of gastric cancer organoids were successfully cultured after identification, with a success rate of about 48%. Its histopathological morphology was highly consistent with that of gastric cancer. According to the pathological type, 21 cases were poorly differentiated adenocarcinoma and 12 cases were successful. Four cases of signet ring cell carcinoma failed. According to the location of the lesion, the success rate of sampling and culture of gastric antrum was significantly lower, which may be related to antral edema and anatomical characteristics of gastric antrum. Some of the failed cases are related to the quality of sampling, technique and contamination of tissue cells. We have successfully established gastric cancer organoids through endoscopic biopsy, and analyzed the factors affecting the success rate of culture from various angles, to improve and enhance the organoid culture technology and provide a better platform for tumor research.

Background: The prognosis of gastric cancer (GC) is improving due to the development of chemotherapy, however, intrinsic or acquired resistance to anti-cancer drugs is still a major clinical challenge. Oxaliplatin (L-OHP) is one of the anti-cancer drugs used as first-line treatment for GC. As with other anti-cancer drugs, although acquired resistance to L-OHP is a major problem, no biomarkers are clinically available for L-OHP response. Recently, cancer stem cells (CSCs) were shown to be involved in the acquisition of drug resistance. Hence, the major goal is to discover novel predictive biomarkers and overcome L-OHP resistance in GC in relation to CSCs. Methods: We used the organoid model; a culture method that allows 3D in vitro culturing of tissue-like structures and is presumed to contain many stem/progenitor cells. We established three independent L-OHP-resistant gastric cancer organoids (GCOs) by adding increasing doses of L-OHP in the culture medium over time. Gene expression profiles of the pairs of parental and resistant organoids were evaluated using microarray analysis. We analyzed the microarray data of L-OHP-resistant GCOs with our previous study from 5-fluorouracil (5-FU)-resistant GCOs (GSE154127). We validated the upregulated genes in the L-OHP-resistant GCOs by qRT-PCR. To confirm the use of upregulated genes as a novel biomarker, we immunohistochemically evaluated the expression levels in GC tissue samples. Furthermore, we analyzed the effects of knockdown using both GC cell lines and GCOs. Results: Through the comparison of the data, we detected myoferlin (MYOF) to be a candidate gene responsible only for L-OHP resistance. qRT-PCR results confirmed the results of the microarray and found that high expression of MYOF correlated significantly with the IC50 of L-OHP in GCOs. Immunohistochemistry analysis for MYOF using 132 GC cases showed that high expression of MYOF was significantly associated with poor prognosis, T grade, N grade, and lymphatic invasion, and showed MYOF to be an independent prognostic indicator, especially in the GC patients treated with platinum-based chemotherapy. The knockdown or inhibition of MYOF by WJ460 in GC cell line and GCOs enhanced L-OHP sensitivity while it reduced cell growth, spheroid/organoid formation, migration, invasion, and in vivo tumorigenesis. Conclusion: We highlight that MYOF is highly involved in L-OHP resistance and tumor progression in GC. Our results indicate that MYOF could be a promising biomarker in GC to predict L-OHP sensitivity and tumor progression and a potential candidate gene as a therapeutic target. Citation Format: Kenji Harada, Naoya Sakamoto, Shoichi Ukai, Tsuyoshi Takashima, Ryota Maruyama, Daiki Taniyama, Kazuaki Tanabe, Hideki Ohdan, Wataru Yasui. Identification of MYOF as a novel biomarker by using oxaliplatin-resistant gastric cancer organoid model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6186.

Recent advances in metabolomics have deepened our understandings of the roles that specific modes of metabolism play in the maintenance of stem cells. Several pieces of evidence have demonstrated that cancer stem cells (CSCs) play a key role in the acquisition of drug resistance. Organoid is a novel 3D cell culture system through using the specific niche factors in a dish and is believed to harbor abundant stem cells; cancer organoid could possibly be useful for scrutinizing CSC biology. In this study, we successfully established 5-FU resistant gastric cancer organoids (GCOs) from patient-derived specimens, which were further confirmed by significant changes in the expression of molecules related to 5-FU metabolism. We then performed metabolome analysis using 3 pairs of 5-FU resistant/ parental GCOs and 10 pairs of non-neoplastic gastric organoid/ GCOs generated from the same patients. PCA plotting revealed that 5-FU resistant GCOs showed a distinct signature compared to those of their parental GCOs and 10 pairs of non-neoplastic gastric organoid/ GCOs. Among various significant changes in the metabolic pathways, we focused on the excessive reliance on the energy from TAC cycle in 5-FU resistant GCO because the difference was specifically seen in the comparison between 5-FU resistant and parental GCOs: not found in the comparison between 10 pairs of non-neoplastic gastric organoid and GCOs. We examined IC50 value for Tigecyclin and VLX600, both of which have an inhibitory effect on TCA cycle, by MTT assay using 5-FU resistant GCO and 2 gastric cancer cell lines (MKN-45 and MKN-74). Although there was almost no difference in IC50 values for Tigecyclin and VLX600 between 2 gastric cell lines and their 5-FU resistant cells, IC50 for both of TCA cycle inhibitors in 5-FU resistant GCO was significantly lower compared to that in parental GCO. Further studies are ongoing in order to verify the significance of dependency on TCA cycle, which could potentially lead to the development of new therapy for 5-FU resistant gastric cancer. Citation Format: Naoya Sakamoto, Kazuhito Naka, Shoichi Ukai, Ririno Honma, Daiki Taniyama, Tsuyoshi Takashima, Ryota Maruyama, Kazuaki Tanabe, Hideki Ohdan, Wataru Yasui. Metabolome in 5-FU resistant gastric cancer organoids [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4762.

Gastric cancer (GC) is a complex disease linked to a series of environmental factors and unhealthy lifestyle habits, and especially to genetic alterations. GC represents the second leading cause of cancer-related deaths worldwide. Its onset is subtle, and the majority of patients are diagnosed once the cancer is already advanced. In recent years, there have been innovations in the management of advanced GC including the introduction of new classifications based on its molecular characteristics. Thanks to new technologies such as next-generation sequencing and microarray, the Cancer Genome Atlas and Asian Cancer Research Group classifications have also paved the way for precision medicine in GC, making it possible to integrate diagnostic and therapeutic methods. Among the objectives of the subdivision of GC into subtypes is to select patients in whom molecular targeted drugs can achieve the best results; many lines of research have been initiated to this end. After phase III clinical trials, trastuzumab, anti-Erb-B2 receptor tyrosine kinase 2 (commonly known as ERBB2) and ramucirumab, anti-vascular endothelial growth factor receptor 2 (commonly known as VEGFR2) monoclonal antibodies, were approved and introduced into first- and second-line therapies for patients with advanced/metastatic GC. However, the heterogeneity of this neoplasia makes the practical application of such approaches difficult. Unfortunately, scientific progress has not been matched by progress in clinical practice in terms of significant improvements in prognosis. Survival continues to be low in contrast to the reduction in deaths from many common cancers such as colorectal, lung, breast, and prostate cancers. Although several target molecules have been identified on which targeted drugs can act and novel products have been introduced into experimental therapeutic protocols, the overall approach to treating advanced stage GC has not substantially changed. Currently, surgical resection with adjuvant or neoadjuvant radiotherapy and chemotherapy are the most effective treatments for this disease. Future research should not underestimate the heterogeneity of GC when developing diagnostic and therapeutic strategies aimed toward improving patient survival.

Construction and application of a genetically engineered mouse model of gastric adenocarcinoma.

BackgroundGastric cancer ranks as the fifth most common malignancy worldwide. Of note, this entity shows a rising incidence rate over the last decades. Surgical resection and adjuvant chemotherapy are the...

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.

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.

Precision medicine for gastric cancer (GC) is still an unsolved issue, because most available target drugs are not specifically designed for GC. Exploring novel signaling molecules with target value for GC is in urgent need. This study aimed to reveal that interleukin-2 receptor subunit gamma (IL2RG) is such a key molecule in human GC progression. GC tissues and paracancerous gastric tissues were collected from 7 patients (5 males and 2 females) during tumor radical excision surgery. These tissues were used to identify the differentially expressed genes (DEGs) with RNA-seq and serial bioinformatics analyses including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, gene expression profiling interactive analysis (GEPIA), and survival analysis. RT-qPCR and western blotting were performed to compare the mRNA and protein expression levels of IL2RG between GC tissues and adjacent normal gastric tissues as well as between GC cell line SGC-7901 and normal gastric epithelial cell line GES-1. Results showed striking elevations of IL2RG both in the mRNA and protein levels in GC tissues and human gastric cancer SGC-7901 cell line compared, respectively, with the adjacent normal gastric tissues and normal GES-1 cells, and higher IL2RG expression was associated with lower survival. Analyses on the GSE29272 and GSE15459 datasets from Gene Expression Omnibus verified that IL2RG was highly expressed in GC patients and was associated with poor overall survival. In addition, molecular docking showed that a small molecule, resatorvid (TAK 242), might be an inhibitor of IL2RG. We conclude that IL2RG is overexpressed in advanced GC and is associated with low survival. IL2RG may serve as a biomarker of GC progression and poor prognosis.

BackgroundIntertumoral heterogeneity represents a significant hurdle to identifying optimized targeted therapies in gastric cancer (GC). To realize precision medicine for GC patients, an actionable gene alteration-based molecular classification that directly associates GCs with targeted therapies is needed.MethodsA total of 207 Japanese patients with GC were included in this study. Formalin-fixed, paraffin-embedded (FFPE) tumor tissues were obtained from surgical or biopsy specimens and were subjected to DNA extraction. We generated comprehensive genomic profiling data using a 435-gene panel including 69 actionable genes paired with US Food and Drug Administration-approved targeted therapies, and the evaluation of Epstein-Barr virus (EBV) infection and microsatellite instability (MSI) status.ResultsComprehensive genomic sequencing detected at least one alteration of 435 cancer-related genes in 194 GCs (93.7%) and of 69 actionable genes in 141 GCs (68.1%). We classified the 207 GCs into four The Cancer Genome Atlas (TCGA) subtypes using the genomic profiling data; EBV (N = 9), MSI (N = 17), chromosomal instability (N = 119), and genomically stable subtype (N = 62). Actionable gene alterations were not specific and were widely observed throughout all TCGA subtypes. To discover a novel classification which more precisely selects candidates for targeted therapies, 207 GCs were classified using hypermutated phenotype and the mutation profile of 69 actionable genes. We identified a hypermutated group (N = 32), while the others (N = 175) were sub-divided into six clusters including five with actionable gene alterations: ERBB2 (N = 25), CDKN2A, and CDKN2B (N = 10), KRAS (N = 10), BRCA2 (N = 9), and ATM cluster (N = 12). The clinical utility of this classification was demonstrated by a case of unresectable GC with a remarkable response to anti-HER2 therapy in the ERBB2 cluster.ConclusionsThis actionable gene-based classification creates a framework for further studies for realizing precision medicine in GC.

<div>Abstract<p>Gastric cancer is the world's third leading cause of cancer mortality. In spite of significant therapeutic improvements, the clinical outcome for patients with advanced gastric cancer is poor; thus, the identification and validation of novel targets is extremely important from a clinical point of view. We generated a wide, multilevel platform of gastric cancer models, comprising 100 patient-derived xenografts (PDX), primary cell lines, and organoids. Samples were classified according to their histology, microsatellite stability, Epstein–Barr virus status, and molecular profile. This PDX platform is the widest in an academic institution, and it includes all the gastric cancer histologic and molecular types identified by The Cancer Genome Atlas. PDX histopathologic features were consistent with those of patients' primary tumors and were maintained throughout passages in mice. Factors modulating grafting rate were histology, TNM stage, copy number gain of tyrosine kinases/<i>KRAS</i> genes, and microsatellite stability status. PDX and PDX-derived cells/organoids demonstrated potential usefulness to study targeted therapy response. Finally, PDX transcriptomic analysis identified a cancer cell–intrinsic microsatellite instability (MSI) signature, which was efficiently exported to gastric cancer, allowing the identification, among microsatellite stable (MSS) patients, of a subset of MSI-like tumors with common molecular aspects and significant better prognosis. In conclusion, we generated a wide gastric cancer PDX platform, whose exploitation will help identify and validate novel “druggable” targets and optimize therapeutic strategies. Moreover, transcriptomic analysis of gastric cancer PDXs allowed the identification of a cancer cell–intrinsic MSI signature, recognizing a subset of MSS patients with MSI transcriptional traits, endowed with better prognosis.</p>Significance:<p>This study reports a multilevel platform of gastric cancer PDXs and identifies a MSI gastric signature that could contribute to the advancement of precision medicine in gastric cancer.</p></div>

<div>Abstract<p>Gastric cancer is the world's third leading cause of cancer mortality. In spite of significant therapeutic improvements, the clinical outcome for patients with advanced gastric cancer is poor; thus, the identification and validation of novel targets is extremely important from a clinical point of view. We generated a wide, multilevel platform of gastric cancer models, comprising 100 patient-derived xenografts (PDX), primary cell lines, and organoids. Samples were classified according to their histology, microsatellite stability, Epstein–Barr virus status, and molecular profile. This PDX platform is the widest in an academic institution, and it includes all the gastric cancer histologic and molecular types identified by The Cancer Genome Atlas. PDX histopathologic features were consistent with those of patients' primary tumors and were maintained throughout passages in mice. Factors modulating grafting rate were histology, TNM stage, copy number gain of tyrosine kinases/<i>KRAS</i> genes, and microsatellite stability status. PDX and PDX-derived cells/organoids demonstrated potential usefulness to study targeted therapy response. Finally, PDX transcriptomic analysis identified a cancer cell–intrinsic microsatellite instability (MSI) signature, which was efficiently exported to gastric cancer, allowing the identification, among microsatellite stable (MSS) patients, of a subset of MSI-like tumors with common molecular aspects and significant better prognosis. In conclusion, we generated a wide gastric cancer PDX platform, whose exploitation will help identify and validate novel “druggable” targets and optimize therapeutic strategies. Moreover, transcriptomic analysis of gastric cancer PDXs allowed the identification of a cancer cell–intrinsic MSI signature, recognizing a subset of MSS patients with MSI transcriptional traits, endowed with better prognosis.</p>Significance:<p>This study reports a multilevel platform of gastric cancer PDXs and identifies a MSI gastric signature that could contribute to the advancement of precision medicine in gastric cancer.</p></div>