Anoikis

By integrating the data of the TCGA-UCEC in The Cancer Genome Atlas (TCGA, https://portal.gdc.cancer.gov/), we obtained gene expression profiles and clinical data of 520 EC patients. A total of 434 anoikis-related genes (ARGs) were extracted from GeneCards, and genes with a relevance score >0.4 were selected (Supplementary Table S1). In order to obtain differentially expressed gene (DEG) in EC, we conducted differentiation analysis for the expression of all genes in the normal and tumor samples by limma in R software (|fold change (FC)| = 1.0 and p value <0.05). Then, differentially expressed ARGs were collected by interacting with DEG sets.

PDOs were derived from ascites drainage samples from four patients with GCPM. Ascites drainage was filtered with a 70-um cell strainer then centrifuged at 300 g for 5 min. Pellets were resuspended into single-cell suspensions with PBS and incubated with red blood cell lysis buffer for 5 min on ice, then centrifuged again at 300 g for 5 min. Finally, the cells were embedded in Matrigel, seeded onto pre-warmed 24-well culture plates, and cultured in Advanced DMEM/F12 medium (GIBICO) with 50% L-WRN conditioned medium supplemented with 10 mM HEPES (GIBICO), 10 mM Nicotinamide (Sigma), 1X N2 (GIBICO), 1X B27 (GIBICO), 1X Glutamax (GIBICO), 1.25 mM N-Acetylcysteine (Sigma-Aldrich), 50 ng/mL EGF (Peprotech), 200 ng/mL FGF10 (Peprotech), 10 nM gastrin (R&D Systems), and 1X Primocin (Invivogen). 10 uM ROCK inhibitor (Y-27632, R&D Systems) was provided for the first generation of PDOs to prevent anoikis. PDOs were overlaid with culture medium and incubated at 37 °C in humidified air containing 5% CO₂. Culture medium was changed every 3 days. For passaging, PDOs were collected by mechanically dissociating the Matrigel, centrifuged at 150 g for 5 min, incubated with pre-warmed TrypLE express for 7 min, then pipetted about 100 times. All PDOs were trypsinized to single cells under a light microscope then transferred into new matrigel with culture medium and plated in pre-warmed 24 well-plates, incubated at 37 °C in humidified air containing 5% CO₂. PDOs were passaged every 7 days until at least the 15th generation. PDOs were treated with culture media and inhibitors including Hydroxychloroquine Sulfate (1uM), DC661 (4uM), TORIN1 (2uM), or Rapamycin (400 nM) for 7 days before analysis. Culture medium and drugs were changed every 3 days. To embed PDOs, culture media was removed, PDOs were washed in DPBS on ice for 15 mins, then PDOs were fixed in 10% formalin on ice for 2 h. Finally, PDOs were moved to 75% ethanol at 4 °C overnight, mounted in 3% agar, and embedded in paraffin.

The gene expression profiles for TCGA liver hepatocellular carcinoma (LIHC), including 50 normal and 371 tumor samples, were downloaded using the R package “TCGAbiolinks”. The phenotype and survival data of the LIHC cohorts were downloaded from UCSC Xena (https://xenabrowser.net/datapages/, (accessed on 21 September 2022)). The DEGs were analyzed using the R package “TCGAbiolinks”. The p-values were adjusted based on the false discovery rate (FDR) correction method, and the DEG cutoff was set as |log₂FC| > 1 and adjusted to a p-value < 0.05.
The gene expression profiles of the GSE84402 and GSE101685 datasets, including 22 normal and 38 tumor samples, were used as validation cohorts for the prognostic model. The gene expression profiles and clinical data for the ICGC Liver Cancer-RIKEN, JP (LIRI-JP) cohorts, including 232 tumor samples, were also used as validation cohorts for the prognostic model.
A total of 794 anoikis-related genes was acquired from GeneCards (https://www.genecards.org/ (accessed on 22 September 2022)), and 448 of these genes were selected based on a score > 0.4. The overlap of DEGs for LIHC and anoikis-related genes was visualized using the R package “VennDiagram”.