Gastric organoids: A promising model for studying “inflammation-cancer” transition in atrophic gastritis

Abstract

Current experimental models struggle to simulate the complex process of the transformation from atrophic gastritis to gastric cancer, while gastric organoid technology, especially region-specific modeling, provides a more precise in vitro platform for studying this carcinogenic mechanism. Helicobacter pylori activates carcinogenic signaling pathways through virulence factors, inducing DNA damage, epigenetic dysregulation, and immune microenvironment imbalance, driving inflammation-cancer conversion. Intestinal metaplasia and spasmolytic polypeptide-expressing metaplasia serve as critical precursor lesions, gradually developing into dysplasia and adenocarcinoma under the influence of chronic inflammation and genetic instability through intestinal cell transformation and high trefoil factor 2-expressing cell expansion. The immune suppression, metabolic reprogramming, and matrix remodeling within the tumor microenvironment collaboratively create a pro-cancer ecosystem that accelerates inflammation-carcinogenesis transformation. The gastric organoid model successfully simulates the spatiotemporal dynamics of the carcinogenesis process in atrophic gastritis, and its future integration with single-cell omics, real-time imaging technologies, and artificial intelligence technologies could provide a more precise platform for elucidating molecular mechanisms and screening intervention strategies. These advances position gastric organoids as pivotal tools for clinical translation, enabling personalized risk stratification, early intervention targeting precancerous transitions, and ex vivo prediction of patient-specific therapeutic responses to guide precision management of gastric cancer.

Key Words: Gastric organoids; Atrophic gastritis; Inflammation-cancer transition; Gastric cancer; Helicobacter pylori; Tumor microenvironment; Spasmolytic polypeptide-expressing metaplasia

Core Tip: Existing models are difficult to simulate the complex process of atrophic gastritis to gastric cancer, and gastric organoid technology provides a more accurate platform for studying this mechanism. Helicobacter pylori infection triggers activation of key oncogenic signaling pathways, DNA damage, and epigenetic alterations through its virulence factors and induced persistent inflammation. The gradual development of intestinal metaplasia and spasmodic polypeptide-expressing metaplasia accelerates the completion of inflammation-cancer transition under the combined action of tumor microenvironment of immunosuppression, metabolic abnormalities, and matrix remodeling. The gastric organoid model successfully simulates the spatiotemporal dynamics of inflammation-cancer transition, and provides a powerful in vitro research platform.

INTRODUCTION

Atrophic gastritis, characterized by the loss of gastric glandular structures and mucosal thinning, represents a critical precancerous lesion in the Correa cascade of gastric carcinogenesis, which outlines the multi-step progression from normal mucosa to adenocarcinoma, encompassing chronic gastritis, atrophy, intestinal metaplasia, and dysplasia[1]. Epidemiological studies highlight its higher prevalence in regions with elevated gastric cancer burden[2,3], and reveal that the global prevalence estimates of atrophic gastritis, intestinal metaplasia, and dysplasia were 25.4%, 16.2%, and 2.0%, respectively. Notably, the prevalence of all these precursor lesions exhibited an escalating trend in countries with high and medium gastric cancer incidence compared to those with low incidence[4]. Population-based studies reveal that the annual incidence-rate person year is 0.5%, 0.6%, 2.8%, and 3.9% for gastric cancer/high-grade intraepithelial neoplasia, low-grade intraepithelial neoplasia, type 1 neuroendocrine tumors, and all gastric neoplastic lesions, respectively[5].

A significant challenge in modeling the inflammation-cancer transition in atrophic gastritis lies in the inability of traditional two-dimensional cell cultures to capture the spatial heterogeneity and multicellular crosstalk of the gastric mucosa, a limitation that is especially pertinent in the context of Helicobacter pylori (H. pylori) infection and the ensuing metaplasia-dysplasia sequence[6]. Animal models, while useful for in vivo studies, often exhibit interspecies differences in gastric physiology, immune responses, and microbial interactions, limiting their translational relevance to human disease[7]. Patient-derived xenograft models, though retaining tumor heterogeneity, suffer from high costs, prolonged development timelines (> 6 months), and inability to model early premalignant stages of atrophic gastritis[8].

The dynamic complexity of the chronic inflammatory microenvironment remains elusive to current experimental systems. For example, prevailing models are incapable of sustaining long-term H. pylori colonization or of accurately replicating the critical bidirectional interactions between infected epithelium and stromal cells, which are pivotal drivers of carcinogenesis[9,10]. Additionally, conventional methods fail to accurately account for the clinically relevant atrophy regression following H. pylori eradication, which is a crucial step in the evaluation of interventions targeting premalignant lesions[11]. Interstudy variability in experimental results is further increased by the lack of unified protocols to model intestinal metaplasia, a hallmark precancerous lesion in atrophic gastritis[12]. Collectively, these deficiencies prevent the development of therapeutic techniques and the explication of molecular mechanisms underlying inflammation-cancer transition.

The existing models have fundamental limitations in the spatiotemporal dynamics of cancer transformation in atrophic gastritis. Gastric organoid technology has shown transformative value in this context, and its integration with single-cell omics, real-time imaging technology, and artificial intelligence technology in the future is expected to establish a more accurate platform to elucidate molecular mechanisms and screen intervention strategies, ultimately reducing the global disease burden of inflammation-driven gastric cancer.

CONSTRCUCTION METHODS OF GASTRIC ORGANOIDS

Foundation for model establishment

The foundation of gastric organoid modeling relies on three pillars: Stem cell biology, three-dimensional microenvironment simulation, and functional validation systems. Current protocols predominantly utilize adult gastric stem cells [leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5)+ populations] or human induced pluripotent stem cells as starting materials, with successful derivation reported from both surgical specimens and endoscopic biopsies[10,13,14]. The self-organization capacity of these progenitor cells enables recapitulation of gastric epithelial polarity and glandular architecture when cultured in Matrigel-based matrices supplemented with niche factors [epidermal growth factor (EGF), Noggin, and R-spondin][10,15,16].

Advanced three-dimensional culture systems now permit maintenance of cellular vitality through 10-15 serial passages while preserving original tissue transcriptomic signatures, as evidenced by conserved expression of mucin 5AC, oligomeric mucus/gel-forming (mucus secretion) and H(+)/K(+) ATPase subunit A (a proton pump subunit) in antral-derived organoids[17,18]. Crucially, the integration of decellularized extracellular matrix (ECM) components has enhanced mechanical support for muscularis mucosae development in fundic models[19,20]. Functional validation through H. pylori infection models demonstrates the platform’s capacity to replicate inflammatory cascades preceding carcinogenesis[21].

Region-specific modeling in gastric organoids

The anatomical precision of gastric organoid modeling is crucial to faithfully recapitulate the distinct biological and pathological features of different stomach compartments, such as the acid-secreting corpus and the mucus-producing antrum. Leveraging inherent differences in stem cell populations and niche factors across these regions, advances in three-dimensional culture systems now enable the generation of compartment-specific organoids[22,23]. Comparative marker analyses demonstrate that corpus-derived organoids have distinctive transcriptional signatures that correlate with elevated pepsinogen C expression, whereas antral counterparts exhibit prominent mucin 6 mucin production[17]. Importantly, studies using patient-matched corpus-antral organoid pairs have demonstrated that their responses to Wnt/catenin pathway activation[22] mirror fundamental signaling distinctions inherent to these anatomical regions.

The exact isolation and growth of geographically defined gastric stem cells are the driving force behind region-specific modelling. Antral organoids, for example, require Wnt signaling activation combined with R-spondin and Noggin treatment to maintain their proliferative capacity, whereas corpus organoids depend on EGF and fibroblast growth factor 10 signals for long-word culture[20]. Studies demonstrate that antral biopsy-derived organoids exhibit higher proliferation rates and organoid-forming efficiency than their corpus-derived counterparts[17]. Additionally, advancements in human induced pluripotent stem cell-derived gastric organoids have enabled the generation of region-specific epithelia, such as fundic lineages marked by parietal cell differentiation and antral lineages expressing GATA binding protein 4/6 transcription factors[16]. In addition to mimicking the native tissue architecture, these models now incorporate region-specific stromal components like muscular mucosa in antral organoids[20].

Critical technical components include: (1) Regional stem cell isolation: Identification of Lgr5+ stem cells in the antrum and Troy+ chief cells in the corpus through region-specific biomarkers such as SRY-box transcription factor 2 in antral while trefoil factor 2 (TFF2) in corpus epithelium[23]; (2) Microenvironment modulation: Stepwise withdrawal of EGF/Noggin/R-spondin for corpus modeling vs sustained Wnt activation for antral organoid maintenance[22,24]; and (3) Pathological transformation: Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) engineering of E-cadherin/TP53 mutations in region-specific organoids to simulate the inflammation-cancer transition cascade (Figure 1)[24,25].

Figure 1 Region-specific modeling using gastric organoids. TFF2: Trefoil factor 2; Lgr5: Leucine-rich-repeat-containing G-protein-coupled receptor 5; SoX2: SRY-box transcription factor 2; EGF: Epidermal growth factor.

The standardization of protocols for generating region-specific gastric organoids constitutes a major challenge in the field. Significant heterogeneity, stemming from discrepancies in initial biopsy site selection, culture medium composition, and ECM scaffolds, directly contributes to the phenotypic and functional variability observed across different organoid systems[17]. In order to replicate region-specific pathological features like corpus-dominant atrophic gastritis and antral intestinal metaplasia during incendiary to cancerous development, it is crucial to address these issues.

MECHANISM OF “INFLAMMATION-CANCER” TRANSITION IN ATROPHIC GASTRITIS

A varied interaction between ongoing immunoinflammatory activation, epithelium reprogramming, and pathological signaling pathway activation underlies the inflammation-driven harmful change in atrophic gastritis. Driven by dysregulated oncogenic pathway stimulation and impaired tumor suppressor functions, chronic H. pylori colonization, host-directed immune aggression, and redox imbalance collectively cause extensive mucosal degeneration, culminating in consecutive histological transformations from atrophy to metaplasia and malignant precursors.

Role of H. pylori infection in inflammation-cancer transition

H. pylori virulence factors in genomic instability and neoplastic niche formation: Complex chemical relationships mediated by important virulence factors play a vital role in the pathogenesis of H. pylori-associated gastric carcinogenesis. Biologically, cytotoxin-associated protein A (CagA) orchestrates malignant signaling through phosphotyrosine-controlled interactions with Src homology-2 domain-containing phosphatase 2, resulting in fundamental activation of Wnt/β-catenin signaling cascades that drive dysregulated epithelial proliferation[26]. Vacuolating cytotoxin A simultaneously causes mitochondrial toxicity by targeted disruption of membrane potential gradients, which results in an excessive generation of reactive oxygen species and impaired apoptotic regulation (Figure 2)[26,27]. Through dual systems of strong DNA harm and chromatin remodeling errors, these simultaneous chemical perturbations result in combined genomic lesions, creating a permissive neoplastic niche. Patients with concurrent atrophic gastritis and intestinal metaplasia, where sustained oxidative microenvironments interact with epigenetic instability to promote malicious growth, are particularly vulnerable, according to recent studies[28,29].

Figure 2 Role of Helicobacter pylori in inflammation-cancer transition. P13K: Phosphoinositide 3-kinase; AKT: Protein kinase B; CagA: Cytotoxin-associated protein A; MAPK: Mitogen-activated protein kinase; VacA: Vacuolating cytotoxin A; ROS: Reactive oxygen species; Nrf2: Nuclear factor erythroid-2-related factor 2; FOXO: Forkhead box O; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; STAT3: Signal transducer and activator of transcription 3; IL: Interleukin; EMT: Epithelial-mesenchymal transition.

Pro-inflammatory signaling, transcriptional reprogramming, and dysbiosis: Persistent H. pylori infection triggers sustained activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription 3 pathways, upregulating pro-inflammatory cytokines that promote epithelial-mesenchymal transition (EMT)[28,30]. Single-cell RNA sequencing analyses demonstrate that H. pylori-positive atrophic gastritis exhibits unique transcriptional signatures, including overexpression of cancer-related genes and downregulation of tumor suppressors[28]. Meanwhile, multi-omics studies have demonstrated that H. pylori-induced dysbiosis is a crucial precursor of the inflammation-cancer transition[31]. The bacteria’s urease activity elevates gastric pH, facilitating colonization by nitrate-reducing species that produce carcinogenic N-nitroso compounds[31]. In atrophic mucosa, H. pylori downregulate microRNA 148a to activate DNA methyltransferase 1, leading to the hypermethylation of the tumor suppressor gene promoters[27]. These epigenetic alterations persist even after H. pylori eradication, explaining the residual cancer risk in post-eradication patients with established atrophic gastritis[32,33].

Intestinal metaplasia and spasmolytic polypeptide-expressing metaplasia

Intestinal metaplasia: Intestinal metaplasia, characterized by the replacement of gastric mucosa with intestinal-type epithelium, represents a critical precancerous lesion in the stepwise progression from atrophic gastritis to gastric cancer. Histologically, intestinal metaplasia is classified into “complete” and “incomplete” subtypes, with the latter exhibiting higher malignant potential due to its closer association with genetic instability and dysplasia[34]. Molecular studies reveal that intestinal metaplasia arises from chronic inflammation driven by H. pylori infection, which induces oxidative DNA damage and aberrant activation of signaling pathways such as Wnt/β-catenin and bone morphogenetic protein (BMP). These pathways promote the transdifferentiation of gastric stem cells into intestinal-like epithelial cells, a process mediated by transcription factors like caudal-related homeobox transcription factor 2 and SRY-box transcription factor 2[34,35]. Notably, the persistence of H. pylori-induced inflammation accelerates the accumulation of somatic mutations in tumor suppressor genes and oncogenes, creating a mutagenic microenvironment that facilitates malignant transformation[36,37].

The transition from intestinal metaplasia to dysplasia and invasive carcinoma is further driven by epigenetic reprogramming. Hypermethylation of promoter regions in genes regulating cell cycle control and DNA repair is frequently observed in intestinal metaplasia tissues, leading to loss of tumor suppressor function[35,38]. Additionally, alterations in the gastric microbiota—particularly the depletion of H. pylori and enrichment of pro-carcinogenic species such as Streptococcus and Lactobacillus—synergize with mucosal inflammation to sustain epithelial proliferation and immune evasion[37,39]. Recent single-cell RNA sequencing studies have identified a subset of metaplastic cells expressing both gastric and intestinal markers, suggesting a “hybrid” phenotype that may serve as a transitional state during cancer initiation[24,40].

Despite its established role in gastric carcinogenesis, intestinal metaplasia regression remains controversial. While H. pylori eradication reduces inflammation and partially reverses atrophy, metaplastic epithelia often persist as “field defects” with residual molecular abnormalities[32,41]. Emerging evidence highlights the potential of targeting metaplasia-associated pathways or modulating the microbiome to interrupt the inflammation-cancer cascade[24,37]. However, the lack of reliable in vitro models for intestinal metaplasia has hindered therapeutic development, underscoring the need for advanced platforms such as gastric organoids to recapitulate metaplastic progression and test intervention strategies[40,42].

Spasmolytic polypeptide-expressing metaplasia: Spasmolytic polypeptide-expressing metaplasia (SPEM), as a distinct metaplastic pattern different from intestinal metaplasia, has been identified as a novel origin in the inflammation-cancer transformation process[43]. SPEM typically occurs in the gastric corpus glands, with cellular morphology resembling pyloric gland cells or duodenal Brunner’s gland cells. Historically termed pseudo-pyloric metaplasia, mucin-secreting cell metaplasia, or corpus-antral transition, SPEM cells exhibit high expression of TFF2. Under chronic inflammatory conditions, the identification of SPEM relies on positive TFF2 transcriptional expression coupled with negative mature chief cell gene markers. Serum TFF2 levels in chronic atrophic gastritis patients show a positive correlation with both occurrence and severity of SPEM[44,45]. Gastrokine 3, a novel transcription factor, serves as a biomarker distinguishing chief cells, neck mucous cells, and SPEM. This factor remains undetectable in healthy gastric corpus but demonstrates high expression in SPEM cells at gland bases of autoimmune gastritis or H. pylori-associated chronic atrophic gastritis patients, while showing low/no expression in other corpus cells[44]. SPEM is detectable in most precancerous lesions and remnant gastric cancer biopsies, showing a close association with early gastric cancer[46,47]. Experimental studies confirm SPEM cell lineage’s potential to progress to dysplasia and adenocarcinoma[48,49]. During acute/chronic gastric mucosal injury, SPEM mobilizes reparative cells to damaged areas, reprograms gastric epithelial cells, and reinforces epithelial defense through mucus/cytokine secretion. However, persistent inflammation promotes sustained metaplastic reprogramming, ultimately leading to dysplasia and carcinogenesis[50,51] (Figure 3), with cluster of differentiation 44-expressing SPEM cells demonstrating higher malignant potential[52].

Figure 3 Spasmolytic polypeptide-expressing metaplasia in inflammation-cancer transition. SPEM: Spasmolytic polypeptide-expressing metaplasia.

Remarkably, SPEM may progress to intestinal metaplasia. In glands containing both SPEM and intestinal metaplasia cells, SPEM typically localizes to gland bases while metaplastic cells occupy luminal regions. At SPEM-intestinal metaplasia transition zones, intestinal metaplasia cells exhibit strong Ki-67 positivity and hyperproliferation, suggesting possible SPEM-derived origin[53]. Aggregated proteins and selectively activated macrophages may facilitate this progression[54,55], positioning SPEM as the initial step in inflammation-carcinoma transformation compared to intestinal metaplasia.

During the diffuse injury stage induced by H. pylori infection, the gastric mucosa undergoes significant pathological alterations, including atrophy of oxyntic glands, reduction in parietal cell numbers, depletion of zymogenic chief cells, and emergence of SPEM cell lineage[56,57]. However, H. pylori infection not only serves as an initiating factor for SPEM but also exacerbates its progression through mechanisms involving chronic inflammatory responses and immune activation[57]. Additionally, SPEM facilitates the aggregation and dissemination of H. pylori[58].

Tumor microenvironment in inflammation-cancer transition

Tumor microenvironment restructuring and pro-tumorigenic signaling in inflammation-cancer transition: Tumor microenvironment (TME) is a crucial factor in the pathological transition from atrophic gastritis to gastric cancer. Sustained inflammatory signaling within atrophic gastritis induces progressive TME restructuring, as evidenced by alterations in the stromal compartment, leukocyte recruitment dynamics, and ECM reorganization. Phenotypic conversion of native fibroblasts into pro-tumorigenic variants generates paracrine signaling mediators including mitogenic factors and interleukins (IL), which collectively drive the epithelial-epithelial change and neovascularization processes essential for malignant growth[59]. The activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) and NF-κB pathways by hypoxia-mediated stabilization of hypoxia inducible factor 1 within inflamed mucosal niches contribute to the formation of a self-sustaining circuit that supports malignant phenotypic acquisition[60,61].

Immune dysregulation and evasion in the atrophic gastritis-associated TME: The TME associated with atrophic gastritis displays symptomatic immune regulatory dysfunction, wherein H. pylori-mediated chronic inflammation signaling drives the invasion of immunosuppressive cellular components. Notably, regulatory T lymphocyte populations and tumor-educated macrophages establish dominant immunosuppressive niches through functionally impaired cytotoxic lymphocyte-mediated surveillance mechanisms, a pathological process supported by clinical studies[26,62]. Immune evasion of neoantigens during the early malignant transition is promoted by the constitutive upregulation of immune checkpoint regulators, particularly programmed death-ligand 1 expression on gastric epithelial interfaces, as characterized by molecular analyses[38,63]. Biologically, persistent H. pylori colonization initiates pathogen-driven oxidative assault on mucosal epithelia, resulting in accumulated DNA integrity compromise that synergizes with TME-promoted chromosomal instability through chemical crosstalk between DNA damage response pathways and inflammatory signaling cascades, thus establishing a mutagenic continuum that potentiates carcinogenic progression[64,65].

Metabolic reprogramming, ECM remodeling, and therapeutic implications: The stromal-immune crosstalk in the TME also reshapes the metabolic landscape. For example, cancer-associated fibroblasts reprogram glucose metabolism to produce lactate, fostering an acidic microenvironment that promotes tumor cell survival and invasion[59,63]. Furthermore, ECM stiffening, driven by aberrant collagen deposition, activates mechanosensitive signaling pathways, thereby promoting cancer cell proliferation and conferring resistance to apoptosis[61]. These findings underscore the TME as a multifaceted driver of inflammation-cancer transition, offering potential therapeutic targets such as stromal normalization, immune checkpoint inhibitors, and anti-fibrotic agents[60,66]. The multifaceted mechanisms underlying the inflammation-cancer transition in atrophic gastritis, encompassing H. pylori-driven signaling cascades, tumor microenvironment remodeling, and cellular reprogramming events, are summarized in Figure 4.

Figure 4 Molecular and cellular mechanisms driving “inflammation-cancer” transition in atrophic gastritis. H. pylori: Helicobacter pylori; P13K: Phosphoinositide 3-kinase; AKT: Protein kinase B; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; IL: Interleukin; MAPK: Mitogen-activated protein kinase; ERK: Extracellular signal-regulated kinase; JNK: C-Jun N-terminal kinase; SHP-2: Src homology-2 domain-containing phosphatase 2; ROS: Reactive oxygen species; Nrf2: Nuclear factor erythroid-2-related factor 2; HIF: Hypoxia inducible factor; PD-L1: Programmed death-ligand 1; CTLA-4: Cytotoxic T lymphocyte-associated protein 4; TAMs: Tumor-associated macrophages; CAF: Cancer-associated fibroblasts; ECM: Extracellular matrix; EMT: Epithelial-mesenchymal transition; TME: Tumor microenvironment.

Signaling pathways in inflammation-cancer transition

Mitogen-activated protein kinase signaling pathway: The mitogen-activated protein kinase (MAPK) cascade, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 subfamilies, is a key mediator of inflammation-driven carcinogenesis. By inhibiting apoptosis and promoting epithelial cell proliferation and survival, chronic H. pylori infection activates MAPK through virulence factors like CagA[67,68]. In atrophic gastritis models, MAPK hyperactivation correlates with elevated tumor necrosis factor-alpha and IL-6 Levels, exacerbating mucosal damage and metaplastic changes. Rhein, a natural anthraquinone, mitigates atrophic gastritis progression by inhibiting MAPK phosphorylation and downstream pro-inflammatory cytokine release[64].

Wnt/β-catenin signaling pathway: Aberrant Wnt pathway activation is a hallmark of atrophic gastritis-associated intestinal metaplasia and gastric cancer. Chronic inflammation, which results in Wnt ligand overexpression and β-catenin nuclear translocation, causes stem cell growth and glandular atrophy[69]. Claudin-18 deficiency in atrophic gastritis models accelerates Wnt activation, creating a permissive microenvironment for tumor initiation[69]. An atrophic gastritis-upregulated oncogene, growth arrest and DNA damage-inducible beta, interacts with Wnt effectors to promote cell cycle progression and EMT[64].

PI3K/AKT/mammalian target of rapamycin axis: The PI3K/AKT pathway promotes cell survival and proliferation under chronic inflammatory stress. PI3K/AKT is activated by H. pylori infection through CagA-dependent mechanisms, leading to the immortalization and oxidative damage of epithelial cells[70,71]. PI3K/AKT hyperactivation speeds up dysplasia in atrophic gastritis patients[72,73].

NF-κB and IL-17 pathways: NF-κB serves as a master regulator of pro-inflammatory cytokine networks that sustain atrophic gastritis progression. IL-17 signaling further amplifies NF-κB activity, creating a feedforward loop that perpetuates mucosal injury and immune evasion[74,75]. EMT and early tumor invasion are promoted by high IL-17A levels in tissues, which are in line with signal transducer and activator of transcription 3 activation and E-cadherin suppression[74,76].

Oxidative stress-related pathways: Reactive oxygen species overproduction in atrophic gastritis activates nuclear factor erythroid-2-related factor 2 and forkhead box O pathways, initially as compensatory antioxidant responses. However, sustained oxidative stress damages DNA repair mechanisms and induces epigenetic silencing of tumor suppressors like phosphatase and tensin homolog deleted on chromosome ten[75,76]. Rhein counteracts this by activating nuclear factor erythroid-2-related factor 2-mediated antioxidant defenses while suppressing MAPK-driven inflammation, illustrating dual-target therapeutic strategies[64].

GASTRIC ORGANOIDS IN MODELING INFLAMMATION-CANCER TRANSITION IN ATROPHIC GASTRITIS

Investigating the role of H. pylori in inflammation-cancer transition

Gastric organoids as tools for dissecting H. pylori pathogenesis and early carcinogenic events: By mirroring the complex interactions between H. pylori, the gastric epithelium, and the inflammatory niche, gastric organoids offer unprecedented insights into the mechanisms driving the inflammation-cancer transition in atrophic gastritis, illuminating bacterial virulence, host-pathogen dynamics, and epithelial reprogramming[77-79]. For instance, studies using human gastric organoids derived from atrophic gastritis patients demonstrated that H. pylori infection induces persistent DNA damage in gastric stem cells, even after bacterial eradication, suggesting a “hit-and-run” mechanism in early carcinogenesis[24,77]. This aligns with clinical observations that H. pylori eradication may not fully reverse premalignant lesions in advanced atrophic gastritis[32,64]. Furthermore, organoid models have revealed strain-specific effects of H. pylori virulence factors (e.g., CagA and vacuolating cytotoxin A) on epithelial cell proliferation, apoptosis resistance, and metaplastic transformation[10,15,79]. For example, co-culture of H. pylori with gastric organoids triggered NF-κB-mediated inflammatory responses and activated oncogenic pathways such as Wnt/β-catenin, mirroring molecular events observed in human atrophic gastritis progression[78-80].

Modeling spatiotemporal evolution: The utility of gastric organoids further encompasses modeling the spatiotemporal progression of H. pylori-induced mucosal damage. Longitudinal studies employing serially passaged organoids derived from atrophic gastritis patients have demonstrated that chronic infection promotes the accumulation of a senescence-associated secretory phenotype in gastric epithelial cells, thereby fostering a pro-tumorigenic niche[28,77]. Organoids derived from older sponsors exacerbated this phenomenon, demonstrating the link between microbiological persistence and age-related genomic instability[80,81]. Additionally, organoid-based transcriptomic analyses demonstrated H. pylori-mediated upregulation of intestinal stem cell markers in gastric epithelial cells, providing mechanistic support for the initiation of intestinal metaplasia[10,24]. Notably, CRISPR/Cas9-engineered organoids lacking tumor suppressor genes displayed accelerated malignant transformation upon H. pylori challenge, underscoring the link between bacterial inflammation and genetic susceptibility[24].

Patient-derived organoids for personalized research and drug discovery: Patient-derived organoids have further enabled personalized investigation of H. pylori-host interactions. Comparative studies using patient-derived organoids from atrophic gastritis patients with varying degrees of atrophy revealed that H. pylori infection severity correlates with dysregulation of gastric acid secretion pathways and expansion of inflammation-associated microbial communities[31,82,83]. These findings were validated in xenograft models, where H. pylori infected organoids transplanted into mice developed histopathological features resembling human inflammation-cancer progression[24,84]. Moreover, organoid-based drug screening identified novel therapeutic targets, such as necroptosis inhibitors and senescence modulators, which attenuated H. pylori induced epithelial damage in preclinical models[28,64,85].

Modeling the stepwise progression from inflammation to cancer

Organoid modeling of sequential pathological transitions from atrophic gastritis to gastric cancer: Gastric organoids enable systematic dissection of sequential pathological transitions from atrophic gastritis to gastric cancer. Studies utilizing patient-derived atrophic gastritis organoids demonstrated their capacity to mimic histological progression, including intestinal metaplasia and dysplasia, through niche factor withdrawal and CRISPR/Cas9-mediated genetic engineering[24]. By serially passaging organoids over 20 generations, researchers observed spontaneous malignant transformation markers resembling clinical progression timelines[86]. These models faithfully preserve the genomic instability and epigenetic alterations characteristic of human inflammation transition[87], providing a time-resolved platform to map molecular events during carcinogenesis[88].

Replication of histopathological milestones and biomarker signature: Advanced organoid models successfully replicate histopathological milestones in gastric cancer development. Immunohistochemical analyses revealed progressive loss of gastric differentiation markers such as claudin-11/23 and gain of intestinal markers such as caudal-related homeobox transcription factor 2 matching clinical specimens from superficial inflammation-cancer transitions[89,90]. Proteomic profiling of organoid series (chronic gastritis - low-grade neoplasia - gastric cancer) identified stage-specific protein signatures, including elevated pepsinogen II/gastrin-17 ratios and decreased pepsinogen I/II ratios mirroring human serum biomarker patterns[88,91]. Such phenotypic concordance validates organoids as living biobanks preserving disease continuum trajectories[21,92].

Genome-edited gastric organoids: Genome-edited gastric organoids reveal crucial genetic events influencing malignant transformation of atrophic gastritis. Combinatorial mutagenesis investigations in murine organoids identified transforming growth factor (TGF)-β signaling dysregulation and TP53 inactivation as essential drives for transition from dysplasia to poorly differentiated gastric cancer[87]. Investigations of patient-derived organoid libraries have uncovered subtype-specific drug susceptibilities associated with rare genomic variants, such as mutations in chromatin-remodeling genes[93]. Moreover, organoids from serious atrophic gastritis patients with homogeneous gastric cancer exhibited distinctive transcriptomic profiles distinct from differentiated gastric cancer types[94], allowing precision modeling of personal development challenges.

Organoids as testbeds for intercepting progression and precision chemoprevention: Long-term cultured atrophic gastritis organoids serve as testbeds for intercepting progression. Chemoprevention studies using N-methyl-N’-nitro-N-nitrosoguanidine-induced metaplasia models identified retinoid derivatives that reverse intestinal marker expression[95]. Serial drug exposure experiments in premalignant organoids demonstrated that early intervention with epidermal growth factor receptor inhibitors delays malignant transformation by 3-4 passages compared to untreated controls[96]. Phase II clinical trial validation showed that organoid-predicted drug sensitivities correlated with a 78% accuracy in patient responses[97], establishing their utility for developing stage-specific therapeutic regimens[10].

Modeling the dynamic microenvironment

Organoid modeling of chronic inflammation mechanisms in atrophic gastritis: Gastric organoids have become a ground-breaking platform for understanding atrophic gastritis’s powerful and varied environment, particularly in terms of investigating how chronic inflammation, epithelial damage, and stromal remodeling interact with one another. By integrating patient-derived cells and cutting-edge three-dimensional culture techniques, scientists can analyze the historical evolution of molecular and cellular events during the inflammation-cancer transition. For instance, research applying gastric organoids made from H. pylori-infected cells have demonstrated that microenvironment-dependent chronic inflammatory signals like persistent IL-1 and tumor necrosis factor signaling lead to squamous cell hyperproliferation and DNA damage[9,77]. This replicates the pathological progression observed in atrophic gastritis, where frequent disease affects gastric glandular architecture and promotes metaplastic changes.

Recapitulating dynamic gastric microenvironmental factors: Advanced gastric organoid systems now faithfully mimic the dynamic gastric microenvironment, including its pH gradients, mucus composition, and immune cell infiltration. For instance, three-dimensional co-culture models incorporating stromal fibroblasts and macrophages successfully replicate the bidirectional epithelial-stromal crosstalk critical to atrophy progression[13,98]. By demonstrating that TGF-β exacerbates epithelial senescence and macrophage-derived cytokines accelerate metaplasia, these co-culture findings underscore the power of organoid models for identifying microenvironmental drivers of premalignant lesions like parietal cell loss and metaplastic expansion[77,95].

Investigating microbial dysbiosis beyond H. pylori: Gastric organoids have also been used to investigate the role of microbial dysbiosis on the inflammatory environment. Beyond H. pylori, organoid models exposed to non-H. pylori microbiota (e.g., Streptococcus and Lactobacillus species) reveal distinct patterns of epithelial barrier disruption and immune activation, suggesting that microbial community shifts during atrophic gastritis may independently contribute to carcinogenesis[98,99]. Gastric organoids have also been used to investigate the role of microbial dysbiosis on the inflammatory environment. These discoveries serve as a molecular framework for preventing disease progression by targeting stromal, immune, and epithelial interactions.

TECHNICAL LIMITATIONS AND FUTURE DIRECTIONS

Gastric organoid research has established protocols through enzymatic digestion protocols and Wnt3a/R-spondin1/Noggin-supplemented medium, with antrum-derived systems being more reproducible than corpus-derived models due to intrinsic regional stem cell heterogeneity[10,17,100,101]. Three interrelated factors underlie the persisting issues. Biological heterogeneity between anatomical regions (antrum vs corpus) and donor tissues is further exacerbated by the widespread use of Matrigel^TM in 93% of the protocols, where batch-to-batch variation of TGF-β/EGF adversely affects experimental reproducibility[19,21,102]. Technical implementation barriers were also quantified in a 2023 multicenter study reporting mere 58% successful interlaboratory protocol replication, where inadequate documentation of the dissociation procedures and regulation of the microenvironment were reported as critical failure points[102-104]. No models currently integrate essential TME components—immune networks, stromal cross-talk, and interactions with microbiota—mechanistically implicated in the regulation of the inflammation-cancer transition along atrophic gastritis progression[9,21]. Progressive cellular dysfunction and phenotypic disruption ensue with chronic culture sustenance beyond limited passages, particularly in precancerous atrophic gastritis-derived models[10,86].

Developing technologies address protocol standardization through computational modeling of culture parameters and microfluidic automation platforms, with new proposed certification platforms in Chinese regulatory standards[105-107]. Dual-reporter systems based on Lgr5-enhanced green fluorescent protein and phospho-histone H2B-monomeric Cherry now enable longitudinal imaging of epithelial remodeling during the process of carcinogenic development[92]. TME complexity reconstitution is advancing through strategic convergence of immune effectors, microbial consortia, and three-dimensional bioprinter stromal matrices, more accurately recapitulating inflammatory microenvironments that support inflammation-cancer evolution[9,21,100]. Translationally, patient-derived atrophic gastritis organoid biobanks coupled with multi-omics profiling are being established as platforms for biomarker discovery and personalized therapy development[83,92]. CRISPR/Cas9 engineering approaches facilitates systematic investigation of mutation-specific contributions to malignant transformation[21,24].

“Stomach-on-a-chip” microfluidic devices model the gastric mucosa by integrating its multi-layered architecture and incorporating dynamic forces like peristalsis, thereby enabling studies of host-microbe dynamics and drug transport. The resultant fluid flow enhances epithelial-mesenchymal interactions, driving epithelial cell maturation and the formation of an in vivo-like mucus layer[108,109].

Operator-dependent variation in gastric cancer organoid culture success rates remains heavily reliant on tissue source quality and technical competence[102]. Harmonization of protocols by multicenter validation studies and creation of quantitative quality measures for stem cell isolation, matrix composition, and preparation of niche factors constitute essential prerequisites for clinical translation (Table 1)[10,100].

Table 1 Key advances and limitations of gastric organoids in studying inflammation-cancer transition in atrophic gastritis.

Research dimension	Key advances	Limitations
Region-specific modeling	(1) Achieved region-specific organoid modeling preserving regional transcriptional signatures[17,22]; and (2) Generated fundic/antral lineages from hiPSCs[16]	(1) Lack of standardized protocols: Phenotypic heterogeneity due to biopsy site variations, medium formulations, and matrix compositions[17]; and (2) Lower efficiency/stability in corpus-derived vs antrum-derived organoids[17,20]
H. pylori infection mechanisms	(1) Revealed CagA/VacA-driven carcinogenesis via Wnt/β-catenin activation, DNA damage, and epigenetic dysregulation[26-28]; and (2) Confirmed persistent DNA damage and cancer risk post-H. pylori eradication[24,32,33]	(1) Failure to recapitulate long-term H. pylori colonization and epithelial-stromal crosstalk[9,10]; and (2) Inadequate integration of immune-stromal microenvironment[79,81]
Premalignant lesion modeling	(1) Successfully modeled IM and SPEM: Revealing CDX2/SOX2-mediated transdifferentiation in IM[34,35] and supporting the role of SPEM as a potential cancer origin; and (2) Demonstrated SPEM–IM-dysplasia sequence[53]	(1) Absence of unified IM modeling protocols, limiting interstudy comparability[12,40]; and (2) Molecular mechanisms of SPEM-to-IM transition require validation[54,55]
TME	(1) Identified CAF-mediated immunosuppression via metabolic reprogramming (lactate) and ECM remodeling[59,61]; and (2) Validated PD-L1 upregulation enabling immune evasion[38,63]	(1) Inability to integrate core TME components (immune cells, stromal crosstalk, microbiota)[9,21]; and (2) Poor simulation of hypoxic niches and mechanical stress[60,66]
Signaling pathway studies	(1) Elucidated synergistic oncogenic roles of MAPK, Wnt/β-catenin, and PI3K/AKT pathways in chronic inflammation[64,67,72]; and (2) Verified targeted interventions (e.g., Rhein inhibiting MAPK/Nrf2)[64]	(1) Difficulty in recreating dynamic cross-talk between multiple pathways[64,69]; and (2) Clinical relevance of pathway activation thresholds needs validation[72,73]
Technical optimization	(1) CRISPR/Cas9 editing simulated TP53/CDH1 mutations accelerating carcinogenesis[24,25]; and (2) PDOs enabled personalized drug screening[97]	(1) MatrigelTM batch variations compromise reproducibility[19,102]; and (2) Phenotypic drift in long-term cultures[86]

H. pylori: Helicobacter pylori; hiPSCs: Human induced pluripotent stem cells; CagA: Cytotoxin-associated protein A; VacA: Vacuolating cytotoxin A; IM: Intestinal metaplasia; SPEM: Spasmolytic polypeptide-expressing metaplasia; CDX2: Caudal-related homeobox transcription factor 2; SOX2: SRY-box transcription factor 2; CAF: Cancer-associated fibroblasts; ECM: Extracellular matrix; PD-L1: Programmed death-ligand 1; MAPK: Mitogen-activated protein kinase; P13K: Phosphoinositide 3-kinase; AKT: Protein kinase B; Nrf2: Nuclear factor erythroid-2-related factor 2; CRISPR/Cas9: Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats -associated protein 9; CDH1: E-cadherin; PDOs: Patient-derived organoids; TME: Tumor microenvironment.

CONCLUSION

Gastric organoids have been considered as essential tools for creating patient-specific models that examine mutation profiles and drug sensitivities and microenvironmental interactions during inflammation-cancer transitions. The retention of phenotypic and genotypic heterogeneity from primary tissues by gastric organoids allows for exact disease progression modeling which makes them suitable for studying molecular mechanisms and predicting therapeutic responses in individualized settings.

The future advancement of gastric organoid models hinges on the convergence of single-cell omics, real-time live imaging, and artificial intelligence. Single-cell omics provides a comprehensive molecular atlas of the cellular heterogeneity within atrophic gastritis, thereby enabling the identification of rare cell subpopulations and transcriptional programs that drive malignant transformation. Real-time imaging technology can help researchers track cell behavior and analyze the dynamic changes of inflammation-cancer transition. Multi-omics analysis through artificial intelligence will promote atrophic gastritis research towards intelligent intervention. On this basis, gastric organoids will play a significant role in the study of atrophic gastritis inflammation-cancer transition and provide help for improving patient outcomes.

ACKNOWLEDGEMENTS

Yong-Jing Li offered guidance and valuable suggestions on figure design.