We collected fresh GC tissues from patients of Jiangsu Province Hospital of Chinese Medicine that had surgery to remove tumors. Organoids were kept in the recovery solution (Bio Genous Technology, E238006). Patient-specific baseline attributes such as histological classification, cancer stage, and therapeutic interventions were documented. Histopathological analysis revealed adenocarcinoma, comprising 13 cases of PCC and 14 cases of non-poorly cohesive carcinoma (NPCC).

After sample collection, the tissue was washed 10 times in PBS solution containing penicillin-streptomycin-gentamicin (1%, 100×, Solarbio P1010), dissociated using sterile surgical scissors to cut into tissue particles 2–3 mm³ and digested with Tumor Tissue Dissociation Kit (Bio Genous Technology, K601003-A100/K601008-B100) at 37°C for 30 min while gently shaking (Ouyang et al., 2022).

Filtered in series on 70 μm nylon gauze (Falcon, 352,350), spun down (250 × g for 3min at 4°C). If there are red pellet after spinning, then lysis needs to be done using Red Blood Cell Lysis Buffer (Bio Genous Technology, E238010) as previously described (Tong et al., 2023).

Cell pellets were subsequently resuspended in Growth Factor-Reduced Matrigel (Corning, 356,231) at a density of 1 × 10⁴ cells/50 μL and plated in 48-well culture plates (Corning, 3,548). Following matrix polymerization (10 min at 37°C, 5% CO₂), GC organoid culture medium (Bio Genous Technology, K2179-GC-A500) was carefully overlaid. Culture maintenance included medium replacement every 72–96 h and routine passaging every 7–14 days using Organoid Dissociation Reagent (Bio Genous Technology, E238001) (Yu et al., 2023).

Harvested tumor tissues were placed in a 6 cm culture dish, mechanically chopped into pieces of 1 mm, and gently shaken and digested at 37°C for 1 h. We made the digesting solution with phosphate-buffered saline containing calcium and magnesium ions, 1 mg/mL collagenase Ⅳ, and 1 mg/mL DNase Ⅰ. Tumor tissue homogenate was centrifuged for 7–10 min at 4°C and 350× g before being rinsed three times with DMEM medium containing 10% FBS. After digestion, tumor homogenates were passed through a 70 μm strainer and rinsed with new PBS containing 2% FBS. The cell suspension was centrifuged for 7–10 min at 4°C and 350× g, followed by erythrocyte lysis. In summary, pellets were resuspended with 1 mL of lysis buffer and agitated for 1 min. Then, PBS containing serum was added and the suspension was centrifuged for 7–10 min (4°C, 350× g). We resuspended the cell pellets in 5 mL of fresh PBS containing FBS and counted them using 0.4% trypan blue solution (w/v).

IHC staining was performed on 4 μm FFPE sections following antigen retrieval in citrate buffer (10 mM, pH 6.0, 95°C, 20 min). After peroxidase blocking and serum incubation, sections were treated with primary antibodies (dilutions in Supplementary Table S1) at 4°C overnight, followed by HRP-conjugated secondary antibodies (1:500) and DAB visualization. Two blinded pathologists independently scored staining intensity (0–3) and positive area (0–4), with composite H-scores (0–12) calculated as intensity × percentage. Digital imaging was conducted using a Nikon Eclipse Ni-E microscope (200×, DS-Fi3 camera) (Steele et al., 2019).

Tissues fixed in 4% paraformaldehyde underwent graded ethanol dehydration, paraffin embedding, and sectioning (4 μm). Sections were dewaxed in xylene, rehydrated through ethanol gradients, acid-conditioned (5% acetic acid, 1 min), and sequentially stained with Mayer’s hematoxylin (5 min, 25°C) and eosin (1 min). After ethanol-xylene dehydration, slides were mounted with neutral resin for histomorphological analysis using an Olympus IX81 microscope (DP74 camera).

Following experimental interventions, organoids in 48-well microplates were collected and rinsed thrice with ice-cold PBS, prior to fixation with 4% paraformaldehyde and then permeabilized with 0.3% Triton X-100 at room temperature for 30 min. Following the application of Immunol staining blocking buffer, the cells were incubated overnight at 4°C with primary anti-CDX-2 and anti-CEA antibodies, then incubated with coupled CoraLite®488-conjugated or CoraLite®594-conjugated secondary antibodies (Koh et al., 2021). The nuclei were stained with DAPI. Organoid were imaged in z-axis steps of 2 mm with a laser confocal microscope (FV4000, Olympus, Tokyo, Japan).

A direct contact co-culture model was established to investigate cellular interactions between GC organoids and cancer-associated fibroblasts (CAFs), labeling by different fluorescence (GC cells: mCherry, MSCs: GFP). Human CAFs were isolated from GC tissues following established protocols (Zhao et al., 2022). CAFs used for experimental studies at passages 4–6. CAFs and organoids at a ratio of 1:2 embedded in Matrigel (Zhao et al., 2024). After 5 days of co-culture, the co-culture state was photographed in z-axis steps of 2 mm with a laser confocal microscope (FV4000, Olympus, Tokyo, Japan).

Nude mice (BALB/c-nu/nu) aged 4–6 weeks were procured from Beijing Vital River Laboratory Animal Technology Co., Ltd. All experimental animals were free of specific pathogens. Patient 1- and patient 8-derived organoids were retrieved using organoid recovery solution (Bio Genous Technology, E238006). A specific quantity of organoids was resuspended in 50% Matrigel based on the number of mice available. Subsequently, 100 μL of the mixture was subcutaneously injected into the right forelimb of nude mice (Qu et al., 2021). Regular weighing of the mice was conducted, and measurements of tumor length and width were taken to determine tumor volume (tumor volume = length × width × width/2). The experiment was terminated if the tumor diameter exceeded 20 mm or if significant weight loss was observed, at which point the mice were euthanized (Yoshida, 2020).

Three days after seeding, the culture medium was replaced with media containing varying concentrations of 5-fluorouracil (5-FU), oxaliplatin, irinotecan, and docetaxel. Following a 72-h incubation period, the half-maximal inhibitory concentration (IC₅₀) was determined to assess drug sensitivity (Li et al., 2022).

All experimental procedures were independently replicated three times in triplicate. Quantitative data were presented as mean ± standard error of the mean (SEM) and demonstrated normal distribution. Intergroup comparisons were conducted using T-tests or one-way ANOVA, with the choice of post hoc testing determined by variance homogeneity. Post hoc analyses were performed using either Tukey’s multiple comparison test or Dunnett’s T3 test. Statistical analyses were carried out using GraphPad Prism® software (GraphPad Inc., USA). Statistical significance was defined as P < 0.05 for all tests. The statistical staff performed analyses in a blinded manner.

With the aid of 3D organoid culture technique, we successfully built 23 GC organoid lines out of 27 tissue specimens (Construction efficiency: 85.19%) (Table 1). The Inclusion/exclusion criteria for participants and sample collection rules are listed in Supplementary Material S1. The cohort comprised 11 NPCC and 12 PCC cases, with success rates of 78.57% (11/14) and 92.31% (12/13), respectively. Clinical and pathological characteristics of the samples are detailed in Supplementary Table S2. Notably, organoid culture success rates showed no correlation with clinical characteristics. After 3D culturing within several days, GC organoids developed circular cavity-like structures. To evaluate organoid growth dynamics, we conducted longitudinal morphological analyses (Figures 1A,B). After 7 days of culture, individual organoids attained diameters of up to 500 μm, with PCC-derived organoids exhibiting significantly faster growth rates and larger sizes compared to NPCC organoids (Figures 1C,D). Following successful establishment, both PCC and NPCC organoids demonstrated long-term culture viability while maintaining stable morphological characteristics (Figure 1E). Furthermore, cryopreservation and consequent revival of organoids did not affect significant difference among morphological attributes nor changed related to overall phenotypic features or appearances. Transmission electron microscopy revealed that the organoids formed spherical hollow structures consisting of tightly adherent cells (Liu et al., 2024b), where intercellular contacts were dominated by tight junction morphology (Figure 1G).

We conducted hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) analyses on the matched organoids and tissues (Figure 2A). Immunofluorescence (IF) was utilized to examine the expression of the typical GC markers CEA and CDX-2 (Figure 2B). The results show that the level of pan-CK and CEA was high expression in GC tissue and organoids, but opposite, CDX-2 displayed in NPCC tissue and organoids with low level in PCC tissue and organoids. The HE and IHC staining of rest PCC organoids were in Supplementary Figure S1, and NPCC organoids in Supplementary Figure S2.

To assess the tumorigenicity of GC organoids, we transplanted the typical PCC and NPCC organoids into nude mice and monitored their growth. After about 40 days, the organoids formed subcutaneous tumors in the mice. The subcutaneous xenografts of PCC organoids were larger than those of NPCC (Figures 3A–C). HE staining results of organoid-based xenotransplantation were also presented (Figure 3D).

GC organoids of distinct histological subtypes were assessed for in vitro sensitivity to four chemotherapeutic agents: 5-FU, oxaliplatin, docetaxel, and irinotecan. Two organoid cohorts, including 8 NPCC and 6 PCC cases, were treated with fixed drug concentrations (0.2, 1, 5, 10, 50 μmol/L). Dose-response curves from biological replicates of the same organoid line showed high correlation (Pearson’s R ² > 0.99), confirming culture system stability and consistency across passages (Figure 4A). Organoids from different GC patients demonstrated varied responses to the drugs. No significant differences in half-maximal inhibitory concentration (IC₅₀) values for 5-FU, oxaliplatin, and irinotecan were found between NPCC and PCC organoids. However, PCC organoids had lower IC₅₀ values for docetaxel, indicating greater sensitivity to this treatment (Figures 4B,C). The response of the two groups of organoids to different drugs was presented by heatmap (Figure 4D). In a word, PCC organoids showed heightened sensitivity to docetaxel in vitro with lower IC₅₀.

To investigate the potential for organoid-fibroblast co-culture, we established co-cultures of GC organoids (PCC-G008 and NPCC-G001) with CAFs. After 5 days of co-culture, CAFs formed an extensive network surrounding the organoids (Figure 5A). Notably, co-culture with CAFs significantly enhanced organoid growth, as evidenced by increased organoid size in both PCC and NPCC lines (Figure 5B).We further examined the influence of CAFs on organoid response to four chemotherapeutic agents (5-FU, oxaliplatin, docetaxel, and irinotecan). Organoids co-cultured with CAFs exhibited significantly greater chemoresistance compared to organoids cultured alone (Figure 5C). These findings demonstrate that CAFs modulate organoid sensitivity to chemotherapy.