Cell culture and reagents

Four human GC cell lines (HGC-27, AGS, MKN45, and SNU-1) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO2. Cancer-associated fibroblasts (CAFs) were isolated from fresh GC tissues and immortalized after transformation. CAFs were maintained in DMEM (Gibco) with 10% FBS under identical culture conditions. The primary antibodies used in this study are listed in Table 1.

Table 1 The primary antibodies applied in this study were shown as followsTissue microarray sample collection

A total of 143 GC tissue samples were collected from patients who underwent surgical resection at the First Affiliated Hospital of Zhejiang University between January and December 2016. These samples were used to construct a tissue microarray (TMA) for immunohistochemical (IHC) analysis. Clinicopathological data and follow-up survival information were obtained, including assessments of peritoneal metastasis based on surgical records, pathology reports, and postoperative evaluations (e.g., abdominal CT, MRI, PET-CT, and biopsy results). Patients were informed about the use of their samples for scientific research and the requirement for informed consent was waived by the Clinical Research Ethics Committee of the First Affiliated Hospital of Zhejiang University (Approval No: 2024–0956).

Cell transfection

The FERMT2 and SOX2 lentiviral expression vectors were constructed by cloning the FERMT2 cDNA fragment (NM_006832.3) and SOX2 cDNA fragment (NM_003106.4), respectively, into shuttle vectors. For gene silencing, specific short hairpin RNAs (shRNAs) targeting FERMT2 and small interfering RNAs (siRNAs) targeting SOX2 were designed (sequences provided in Table 2). Recombinant lentiviral vectors were packaged and purified by Genomeditech (Shanghai, China). GC cells were subsequently infected with the lentiviral vectors and subjected to selection with puromycin or blasticidin for 2 weeks.

Table 2 Primer, siRNA and shRNA sequences in this study were shown as followsInduction of anoikis

GC cells were resuspended at a density of 1 × 10^5 cells/ml in RPMI-1640 medium with 10% fetal bovine serum and plated (2 ml/well) onto ultra-low attachment 6-well plates (Corning, #3471). At specified suspension intervals, cells were collected for protein extraction or viability analysis.

Cell migration and invasion assays

Cell migration and invasion were assessed using 24-well Transwell chambers with 8.0-μm pore polycarbonate filters (Corning, #3422). For migration assays, 5 × 10^4 cells were seeded into uncoated upper chambers, while 1 × 10^5 cells were used for invasion assays in Matrigel-coated chambers (Corning, #354,234). The upper chambers contained 200 µL of serum-free medium, and the lower chambers 500 µL of complete medium. After 24 h, cells on the lower filter surface were fixed with 4% paraformaldehyde, stained with 0.05% crystal violet, and washed with distilled water. Non-migrated cells were removed using a cotton swab. Migrated cells were counted in three random fields per sample at 20 × magnification under a light microscope.

Soft agar colony formation assay

Prepare 1.2% and 0.7% agar solutions and keep them in liquid form at 37 °C after autoclaving. For the bottom layer, mix 1.2% agar with 20% RPMI-1640 medium (1:1), add 2 mL per well of a 6-well plate, and allow it to solidify at room temperature. For the top layer, mix 0.7% agar with 20% RPMI-1640 medium (1:1), incorporate 10^3 cells, and overlay this mixture onto the solidified bottom layer. After the top layer solidifies, incubate the plate at 37 °C for 2–3 weeks, adding 200 µL of medium to each well every three days to maintain culture conditions.

Western blotting

Total proteins were extracted from cultured cells using RIPA buffer (Beyotime Inc., China) with phosphatase and protease inhibitors (1:100 dilution). The lysates were centrifuged at 12,000 × g for 20 min at 4 °C, and the supernatant was collected. Equal protein amounts (20 μg) were loaded per lane, separated by SDS-PAGE, and transferred to PVDF membranes (Millipore, USA). Membranes were incubated overnight at 4 °C with primary antibodies, followed by incubation with an HRP-conjugated secondary antibody for 1 h at room temperature. β-actin was used as a loading control. Protein bands were visualized using the ECL Chemiluminescence Detection Kit (Yeasen Biotechnology, China). Primary antibodies are listed in Table 1.

RNA extraction and RT-qPCR analysis

The RNA Purification Kit (EZBioscience, B0004DP) was used to extract RNA from cells. The RT Reagent Kit (AG) was applied for reverse transcription using 1 μg of RNA in a total reaction volume of 20 μL. Universal SYBR Green Fast qPCR Mix (ABclonal) was used for qPCR on the CFX96 Real-Time PCR Detection System (StepOnePlus). The relative expression levels of each gene were normalized to GAPDH expression, and each experiment was repeated at least three times.

Immunohistochemistry

Paraffin-embedded sections were baked at 65 °C for 2 h for tissue fixation. They were then deparaffinized with xylene, dehydrated through graded alcohols, and subjected to antigen retrieval in sodium citrate buffer. The primary antibody was applied based on tissue size and incubated at 37 °C for 60 min. Afterward, an enzyme-labeled goat anti-rabbit IgG polymer was added and incubated at 37 °C for 20 min. DAB chromogenic solution was applied for 2 min at room temperature. Sections were counterstained with hematoxylin for 20 s, rinsed with tap water for bluing, air-dried, and coverslipped with neutral resin.

Immunoprecipitations

Whole-cell protein from GC cells cultured in a 10 cm dish was extracted using 1 mL of RIPA buffer (Sigma). The extract was incubated overnight at 4 °C with BeyoMag™ Protein A + G Magnetic Beads (Beyotime) and the corresponding antibody on a vertical rotator. The precipitates were collected using a magnetic rack. After adding 1 × loading buffer, the samples were heated at 100 °C for 15 min. Co-precipitated proteins were analyzed by Western blotting.

Chromatin immunoprecipitation (ChIP)

The BeyoChIP™ Enzymatic ChIP Assay Kit (Protein A/G Beads, P2083S) was used for chromatin immunoprecipitation. Micrococcal Nuclease (MNase) digestion was employed for chromatin fragmentation, replacing the traditional sonication method. SOX2 antibody and normal Rabbit IgG (negative control) were used to precipitate chromatin fragments bound to the target protein. After column purification, the precipitated chromatin fragments were analyzed using methods including PCR followed by agarose gel electrophoresis and RT-qPCR. All experimental procedures were performed according to the manufacturer’s protocol.

Immunofluorescence

GC cells (1 × 10^4) were seeded in confocal dishes and fixed with 4% paraformaldehyde for 15 min. After permeabilization with 0.5% Triton X-100 in PBS for 20 min, cells were blocked with 5% BSA for 30 min. Without washing off the blocking solution, cells were incubated overnight at 4 °C with primary antibody (1:200 in 1% BSA). Afterward, cells were incubated with fluorescent secondary antibody (1:200 in 1% BSA) for 1 h at room temperature, protected from light. DAPI was used for nuclear staining for 5 min. Samples were mounted with an anti-fade medium and imaged using a Leica-S5 confocal microscope.

In vivo assays for tumor anoikis

Four-week-old female nude mice were purchased and maintained according to ethical guidelines approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Zhejiang University (Approval No. 2024–1299). In this experiment, mice were randomly assigned to experimental groups using the computer-generated randomization method from the “randomizeR” package (version 3.0.2) to ensure unbiased allocation. The researcher administering treatments and assessing outcomes was blinded to the group assignments to minimize bias in data analysis and interpretation. For the peritoneal metastasis model, 5 × 10^6 cells in 200 µL of PBS were injected into the peritoneal cavity of mice. The TGF-β receptor kinase inhibitor SB-431542 (10 μM, diluted in 1 µL of DMSO) was administered via intraperitoneal injection on the 1 st, 3 rd, 5th and 7 th days following tumor cell inoculation in 200 µL of PBS. After 4 weeks, mice were sacrificed, and the peritoneal cavities were examined. Tumor nodules were carefully dissected, pooled, photographed, and weighed.

Acquisition and processing of single-cell sequencing data

Single-cell sequencing data from primary GC and peritoneal metastasis lesions were retrieved from the GEO database (GSE210347 and GSE163558). Batch effects across samples were corrected using the "Harmony" R package. Cell and gene filtering, as well as normalization and scaling, followed protocols described in a previous study[13]. Cell subpopulations were identified using the "FindClusters" function in the "Seurat" R package, with a default resolution of 0.5, and renamed based on canonical markers. FERMT2 expression levels were extracted from the gene expression matrix and compared between primary and peritoneal metastasis lesions.

Statistical analysis

All experiments were performed independently at least three times, and all data are expressed as “mean ± SD.” For comparisons between two groups, we used Student’s t-test for normally distributed quantitative data and the nonparametric Mann–Whitney U test for quantitative data that did not follow a normal distribution. Statistical analyses were conducted using R (version 4.3.1), with P < 0.05 considered statistically significant.