Ethics statement

Protocols for fresh organoid culture of gastric cancer were approved by the Ethics Committee for Scientific Research of Ruijin Hospital, and informed consents were obtained from the patients. An additional number was used to register the samples in the database with no link to patient names or personal information.

Establishment of organoid lines

The primary cancer and lymphatic metastatic cancer of the station six were collected after radical gastrectomy within 30 min ex vivo and stored in advanced DMEM/F12 medium (12,634,010, Thermo Fisher Scientific). The tissues were washed with PBS containing 1×penicillin-streptomycin (C0222, Beyotime) and 1×puromycin (A1113802, Thermo Fisher Scientific) at least four times, and then minced to 1–2 mm3 for further digestive incubation in buffer including 1 mg/ml collagenase(40507ES60, Yeasen), 1 mg/ml collagenase IV (40510ES60, Yeasen), and 1 U/ml dispase II (40104ES60, Yeasen) for 1.5 h at 37℃ with shaking. The incompletely digested tissue pieces were discarded and then cells were centrifuged at 1000 rpm for 5 min. The cell precipitates were mixed with DMEM and matrigel (356,231, Corning) at a 1:1 ratio and seeded in a 24-well plate (50 µl/well). Once the matrigel was polymerized after 30 min incubation at 37 °C, complete medium (Table S1) was added (500 µl/well). The organoid culture was performed at 37 °C, 5% CO2, and the medium was replaced every 5 days. The well-grown organoids were obtained after 7 to 10 days of cultivation. The organoids were digested with TrypLE Express enzyme (12,605,010, Thermo Fisher Scientific) for 0.5–1 h. The cell suspensions were collected and centrifuged at 1000 rpm for 5 min. The cell precipitates were mixed with matrigel at a 1:1 ratio and seeded in a 24-well plate (50 µl/well, 5 × 104 cells) again for passaging or subsequent study.

Hematoxylin and eosin (H&E) staining

Well-grown organoids (diameter > 100 μm) were blown thoroughly at least 10 times and centrifuged at 1500 rpm for 5 min. The organoid spheres were collected and fixed in 4% paraformaldehyde for 30 min, put in 10% agarose, and embedded in paraffin. The paraffin block was cut into 8-µm sections. The sections were treated in xylene twice for 10 min, and immersed in pure ethanol for 5 min, 95% ethanol for 5 min, and 75% ethanol for 5 min. The sections were heated with 1×antigen repair reagent at 100℃ for 20 min, and washed with clean water. After washing, the sections were stained in hematoxylin for 10 min, immersed in hydrochloric acid and ammonium hydroxide for several seconds, and washed in running water for 1 h. The sections were stained in alcohol eosin for 2 min (G1005, Servicebio), and dehydrated in 70% and 90% alcohol for 10 min, and further immersed in 95% ethanol and pure ethanol for 5 min, respectively. The sections were transparent in xylene for 10 min twice, and sealed with neutral resin.

Immunohistochemistry (IHC) and western blotting

In immunohistochemical assays, the sections were prepared in the same way as in H&E stain. The staining methods were performed as previously described [28]. The primary antibodies included E-cadherin (GB11082, Servicebio, 1:200), N-cadherin, GB111273, Servicebio, 1:200), and Ki67 (GB111141, Servicebio, 1:200). The western blot assay was performed as previously described [29]. The primary antibodies included p-mTOR (5536, CST, 1:1000), mTOR (2972, CST, 1:1000), p-AKT (4060, CST, 1:200), AKT(4691, CST, 1:1000), HRP-conjugated goat anti-rabbit IgG (H + L) (SA00001-2, Proteintech, 1:3000), and HRP-conjugated anti-GAPDH (HRP-60,004, Proteintech, 1:3000).

Cell growth analysis

The well-grown organoids were digested with TrypLE Express enzyme for 0.5–1 h. The single-cell suspension was centrifuged at 1000 rpm for 5 min. The cell precipitate was mixed with matrigel at a 1:1 ratio and seeded in a 96-well plate (6 µl /well, 6000 cells, n = 3). After polymerization of the matrigel for 30 min at 37 °C, the complete medium (60 µl/well) was added. Cellular vitality of the organoids was measured by a CCK8 assay (CK04, DOJINDO) every 48 h for 8 days. The growth curves were plotted using time (hour) as the x-axis and cell vitality from the 450-nm OD value as the y-axis.

Short tandem repeats (STRs) analysis

The well-grown organoids were digested with TrypLE Express enzyme for 15 min and centrifuged at 1500 rpm for 5 min to collect the organoid spheres. DNA was extracted with a genome extraction kit (Ap-mn-p-500, Axygen), amplified by the 21-STR amplification protocol, and detected on an ABI 3730XL. We focused on eight core STR loci (D5S818, vWA, D7S820, D16S539, TH01, D13S317, TPOX and CSF1PO) and the sex chromosome Amelogenin. The STR results were matched with the STR data library in DSMZ tools, which covers STR data of 2455 cell lines from the ATCC, DSMZ, JCRB and RIKEN databases. When the STR matching rate was above the cut-off value (≥ 75%), the two matched lines were judged to be homologous, and otherwise, unique [30].

Karyotypes analysis

Colchicine solution (0.2 µg/ml) was added to well-grown organoids and incubated for 1.5 h at 37℃. Organoids were then digested with TrypLE Express enzyme for 0.5–1 h. The single-cell suspensions were centrifuged at 1500 rpm for 5 min. The cell precipitates were preheated with 6 ml of KCL hypotonic solution (0.075 mol/L) at 37℃ in a water bath for 30 min with gentle agitation and were then thoroughly dried. The fixative solution (1 ml of methanol and glacial acetic acid at a ratio of 3:1) was slowly added for 30 min, and then the cells were centrifuged at 1500 rpm for 5 min. The cell precipitates were further fixed by the above fixative solution (5 ml) for 30 min and then centrifuged at 1500 rpm for 5 min. The cells were resuspended in 0.5 ml fixative solution. One or two drops of the cell suspension were added to a cooled slide and fixed by an alcohol flame. After drying at room temperature, the cells were stained with Giemsa for 10 min, the dye was washed and the slide was dried at room temperature. The cells in the division phase were observed and captured using the ZEISS karyotyping system (Ikaros, ZEISS).

Whole-exome sequencing (WES)

The paired organoid lines (at passage 18) and cryopreserved primary cancer tissues were used for DNA extraction (DP304, Tiangen). The DNA was fragmented by enzyme digestion and adenine deoxynucleotide and a sequencing adaptor were added. By PCR amplification, the enriched library was hybridized by Agilent SureSelect Human All Exon V6 to capture exonic regions. Genomic sequencing was performed using an Illumina PE150.The FASTQ format of sequencing data was mapped to the human reference genome (hg38) by the BWA Tool. Single nucleotide polymorphisms (SNPs; Sentieon DNAseq 201808.05), copy number variation (CNV; CNVkit, 0.9.6), somatic single nucleotide variants (SNVs; Sentieon DNAseq 201808.05), and insertions and deletions (InDel; Sentieon DNAseq 201808.05) were analyzed. The driver gene mutations were screened based on the CGC513, Bert Vogelstein125, SMG127 and Comprehensive435 databases. The raw data (PRJNA1015608) were uploaded to SRA database (PRJNA1015608).

Single-cell transcriptome sequencing (scRNA-seq)Cell dissociation and single-cell capture

The well-grown organoids were digested with TrypLE Express enzyme for 0.5–1 h. The single-cell suspensions with cell vitality greater than 70% were collected into nuclease-free water.

Library preparation and sequencing

The Chromium Single Cell 3ʹ Gel Beads-in-emulsion (GEM) Library &Gel Bead Kit v3 was used for library construction, and sequencing was performed on a Novaseq 60,000 (Illumina). The raw data (GSE242893) were updated to GEO database.

Quality control and data preprocessing

ScRNA-seq data analysis was performed using the 10×Genomics software package (Cell Ranger version 2.1.1) for data quality control and alignment. SCfastp was used for sequence filtering and sequence quality assessment. CellrangerCounts was used to count the number of cells. A total of 7662 cells and 6257 cells were collected from SPDO1LM and SPDO1P organoid lines, respectively. Gene expression matrixes were generated using unique molecular identifiers (UMIs). The alignment of transcriptome data to the GRCh38 reference genome (GRCh38_Ensembl_Ensembl104) was performed by CellrangerAggr. The UMIs were mapped to genes. Low-quality cells that matched the following were removed: (1)expressed gene number of cells are < 200 or > 7500.2 expressed mitochondrial genes or ribosomal genes of cells were > 25%. A total of 10,578 cells from SPDO1P and SPDO1LM were used for analysis.

Data normalization

The Seurat package (4.1.1) was used for data normalization, including “NormalizeData” and “LogNormalize”. The log-transformed UMI counts were computed to obtain the normalized gene expression value.

Dimension reduction

A subset of 2500 highly variable genes (HVGs) was selected for analysis by “FindVariableFeatures” and “ScaleData”. The “RunPCA” of the Seurat package was used for principal components analysis (PCA). The “JackStraw” was used to determine 20 PCA. The “ScoreJackStraw” was used to obtain principal components scores.

Cell clustering and gene set enrichment analysis (GSEA)

The “FindClusters” was used for cell clustering analysis. The linear dimensionality reduction and plotting of tSNE was performed by “RunTSNE” with a resolution of 0.2. The “FindAllMarkers” (min.pct = 0.25, logfc.threshold = 0.25) was used to obtain differential genes of the subclusters. The GSEA was performed to obtain enriched pathways.

Transcriptome sequencing (RNA-seq)

Tissues (5 × 5 mm) and organoid cells (1.5 × 106) were collected for RNA-seq assays. Total RNA was extracted with TRIzol Reagent. The RNA-seq libraries were prepared using the Fast RNA-seq Lib Prep Kit V2(RK20306, ABclonal, China) according to the manufacturerˊs instructions. The sequencing was performed by the NovaSeq xplus. Gene counts were normalized to transcripts per million (TPM) for data analysis.

3D invasion assay

The well-grown organoids were digested with TrypLE Express enzyme for 15 min and then centrifuged at 1000 rpm for 5 min. The precipitates were mixed with matrigel (354,231, Corning) at a ratio of 1:1 and seeded in a 24-well plate (50 µl/well, 1000 organoid spheres). After matrigel polymerization for 30 min at 37℃, complete medium (500 µl/well) was added. Images were captured at 0 and 12 h using an inverted microscope.

2D migration assay

The well-grown organoids were digested with TrypLE Express enzyme for 15 min and centrifuged at 1000 rpm for 5 min. Cells (300 µl) were resuspended in serum-free DMEM (2 × 105 cells) and added to the upper chamber of the transwell chamber. Complete medium (600 µl) was added to the lower chamber. The cells were incubated at 37℃ and 5% CO2 for 72 h, then stained with 1% crystal violet for 30 min. The cells in the upper chamber were wiped off and dried at room temperature. The cells were counted, and images were captured under a microscope.

2D invasion assay

The well-grown organoids were digested with TrypLE Express enzyme for 15 min, and centrifuged at 1000 rpm for 5 min. Matrigel (10% 100 µl) was added to the upper chamber of the transwell chamber and polymerized for 30 min at 37℃. Cells (2 × 105) were resuspended in 200 µl of serum-free DMEM (cells) and added to the upper chamber of the transwell chamber. Complete medium (600 µl) was added to the lower chamber. The cells were incubated at 37℃ and 5% CO2 for 96 h, then stained with 1% crystal violet for 30 min. The cells in the upper chamber were wiped off and dried at room temperature. The cells were counted and images were captured under a microscope.

Tubule formation assay

Human umbilical vein endothelial cells (EA.HY926) were evenly seeded in a 96-well plate coated with matrigel. The supernatants of SPDO1LM and SPDO1P organoid lines were added for incubation for 48 h. The tubular length and branch nodes were counted under a microscope.

Gene knockdown by siRNA

The siRNAs for FLNA were used for gene knockdown (Sangon Biotech, China). The knockdown was detected using western blotting. The antibodies included anti-FLNA (67,133, Proteintech, 1:1000) and HRP-conjugated goat anti-mouse IgG (H + L) (SA00001-1, Proteintech, 1:3000).

Drug sensitivity assays using CCK8 method

The well-grown organoids were digested with TrypLE Express enzyme for 15 min and centrifuged at 1000 rpm for 5 min. After cell counting, the cell suspension was mixed with matrigel at a ratio of 1:1, and seeded in a 96-well plate (6 µl/well, 6000 cells). After matrigel polymerization for 30 min at 37℃, complete medium (60 µl/well) containing various concentrations of drugs was added (Table S2). Traditional gastric cancer cell lines (AGS and NCI-N87) were used as controls. The cells were digested by TrypLE Express Enzyme into single cell suspension and 60 µl/well cells were seeded in 96-well plates for CCK8 analysis. Concentration gradients of drugs (5-Fu, 0, 0.5, 0.75, 1, 2, 5, and 10 μm), oxaliplatin (OXA, 0, 2, 5, 10, 20, 30, and 40 μm), and paclitaxel (PTX, 0, 0.1, 0.5, 1, 2, 5, and 10 nm) were added. After 5 days incubation at 37℃, a 10% CCK8 solution (CK04, DOJINDO) was added, and the matrigel was blown off with tips to make the organoids fully exposed to the reagent. After incubation at 37℃ for 2 h, the OD450 values were measured using a microplate reader to calculate cell vitality and IC50 of drugs.

Hypoxia and normoxia culture

The well-grown organoids were digested with TrypLE Express enzyme for 0.5–1 h. The cell precipitate was mixed with matrigel at a 1:1 ratio and then seeded in a 96-well plate (6 µl /well, 6000 cells, n = 3). After the matrigel polymerization, complete medium (60 µl/well) was added and cells were cultured for 72 h in hypoxic (37℃, 94% N2, 5% CO2, and 1% O2) and normoxic (37℃, 20% O2, and 5% CO2) conditions. The cellular vitality of organoids was measured using a CCK8 assay.

Lactate assay upon inhibition of glycolysis

The well-growth organoids were digested with TrypLE Express enzyme for 0.5–1 h. Organoid cells were seeded in a 96-well plate (6 µl /well, 6000 cells, n = 3). The chemical 2-deoxy-D-glucose (1 mM, M5140, AbMole, China) was added for incubation at 37℃, 20% O2 and 5% CO2 for 72 h. The culture medium was collected and tested by the lactate assay using the Cedex Bio Analyzer (06343759001, Roche, Switzerland) following the manufacturer’s instructions.

Tumorigenesis of organoid lines in nude mice

Organoid cells of SPDO1P or SPDO1LM (100 µl, 5 × 105 cells) were injected into the armpit of male BALB/c-nu mice (4 weeks of age, 4 mice/group). After 4 weeks, the mice were sacrificed and the tumors were removed. The tumor volume was calculated by: V = π/6 × L (long diameter) × W (short diameter). The xenograft tumors (PDO-X) were fixed in 4% paraformaldehyde overnight, embedded in paraffin, and stained with H&E. This study was approved by the Institutional Animal Care & Use Committee of Shanghai Jiao Tong University School of Medicine (B-2022-010).

Statistics

GraphPad Prism 6.0 (GraphPad Prism, RRID: SCR_002798) was used for statistical analysis. The Shapro-Wilk test was used for normal test. The student’s t-test (homogeneity of variance) or unpaired t-test with Welch’s correction (heterogeneity of variance) was used for analysis of two group data, respectively. ANOVA or Brown-Forsythe ANOVA was used for data analysis for the above two groups. Each experiment was repeated at least three times independently. P < 0.05 was considered statistically significant.