Processing of Bulk RNA-seq data

CRC RNA-seq data and clinical information from the TCGA database were retrieved via the UCSC Xena browser (http://xena.ucsc.edu/). Gene expression data obtained from the GEO database (GSE29621, GSE39582, GSE17536, GSE17537, GSE33382, and GSE72970) were processed with log2(x + 1) transformation to acquire samples with normalized RNA-seq or microarray data.

Single-cell RNA-seq data collection and analysis

A single-cell RNA sequencing (scRNA-seq) dataset comprising 23 patients was obtained from the GEO database (GSE132465). Cells with > 1,000 unique molecular identifier (UMI) counts > 200 genes < 6,000 genes, and < 10% of mitochondrial gene expression in UMI counts were filtered. Following the standard dimensionality reduction and clustering procedures, clusters were annotated with classical cell markers.

For the sub-clustering analysis of myeloid cells and macrophages, we extracted cell populations initially annotated as myeloid cells and macrophages and repeated the aforementioned workflow. Myeloid cells were annotated based on classical myeloid markers, while macrophages were further annotated and classified according to their function.

To differentiate between malignant and non-malignant epithelial cells, the ‘InferCNV’ R package was used to deduce large-scale chromosomal copy-number variations (CNVs) from single-cell transcriptome profiles. The R package ‘CellChat’ with default parameters was employed to compute the communication intensity between cuproptosis malignant epithelial cells and other cell types, while the ‘Monocle2’ package, also with default settings, was used to delineate the phenotypic transition trajectory of macrophages.

Construction of the ICDS model gene set

Selection of Immune-Related Genes: single-sample gene set enrichment analysis (ssGSEA) via the R package GSVA was employed to quantify the relative infiltration of 28 immune cell types in the TCGA-CRC cohort (97 genes for 28 immune cells downloaded from the Molecular Signatures Database (MSigDB)). Subsequently, clustering was performed using the ‘Consensus Cluster Plus’R package based on the infiltration profiles of these immune cells. Thereafter, a comprehensive assessment utilizing the consensus score matrix and CDF curve was conducted to determine the optimal number of clusters. The ‘WGCNA’R package was then employed to perform unsigned weighted gene co-expression network analysis (WGCNA) to identify immune-related gene modules (n = 4295).

Selection of programmed cell death and stress genes: The R package ‘GSVA’ was used for ssGSEA to evaluate the enrichment of four pathways associated with programmed cell death and stress responses in the TCGA-CRC cohort. Clustering was then carried out with the ‘Consensus Cluster Plus’ R package based on the enrichment data of these pathways. The method for determining the optimal number of clusters is the same as described above. Finally, differential analysis was performed using the R package ‘limma’ (|logFC|> 0.2), resulting in the identification of 2,769 differentially expressed genes associated with programmed cell death and stress.

Construction of the ICDS Model Gene Set: The final ICDS model gene set (n = 579) was established as the intersection of programmed cell death and stress genes with immune-related genes, deemed a reliable representation.

Construction of the ICDS machine learning model

We employed a three-step strategy to construct the ICDS machine learning model, with each patient's final score considered the ICDS index. Step 1: Univariate Cox Regression: Prognostic genes were identified through univariate Cox regression analysis, defining genes with hazard ratios (HR) and 95% confidence intervals (CI) with p < 0.05. Step 2: LASSO Cox Regression: To prevent overfitting, LASSO Cox regression was applied using the ‘glmnet’ R package to narrow down the candidate genes. Genes with the minimal lambda value were retained in the model. Step 3: Stepwise Cox Regression: Stepwise Cox regression was ultimately used to select the genes most suitable for model construction. The model with the minimal Akaike Information Criterion (AIC) for survival genes was designated as the final:

where n denotes the number of model genes, Coefi denotes each model gene risk coefficient, and Expi denotes each model gene mRNA level.

Prognostic evaluation of ICDS index

Kaplan–Meier survival curves were generated using the ‘survival’ package in R, followed by univariate and multivariate Cox regression analyses. Forest plots were created using the ‘forestplot’ package. Clinical nomograms were constructed utilizing the ‘survival’ and ‘rms’ packages.

Analysis of immune cell and cytokine infiltration

Utilizing the R package ‘GSVA’, ssGSEA was employed to quantify the relative abundance of various TME-infiltrating cells and cytokines within ICDS subgroups [24].

Predictive analysis of immunotherapy

Utilizing the R package ‘easier’, we computed immunotherapy response scores for ICDS subgroups based on cellular components, pathway activity, transcription factor activity, ligand-receptor weights, and intercellular interactions.

Pathway enrichment analysis

Through the R package ‘clusterProfiler’, pathways from the Gene Ontology (GO) Biological Process Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) were subjected to over-representation analysis (ORA) and gene set enrichment analysis (GSEA). Differential genes in the single-cell dataset were identified using the FindAllMarkers function (p < 0.01, avg_Log2FC ≥ 0.25), while bulk transcriptomic differential genes were analyzed with the R package ‘limma’ (p < 0.05, |logFC|< 0.5).

Single-cell ICDS differential gene scoring

Quantitative analysis of differential gene expression between ICDS subgroups in the TCGA-CRC cohort was conducted using the R package ‘AUCell’. Visualization of cell type-specific scores was achieved through the use of violin plots and ridge plots, produced by the R packages ‘ggplot2’ and ‘ggridge’, respectively.

Cell culture

The human colon cancer cell lines DLD1 and HCT116 were obtained from the American Type Culture Collection (ATCC). The HCT116 cell line was cultured in DMEM medium (Gibco, Carlsbad, USA) supplemented with 10% fetal bovine serum, while the DLD1 cell line was maintained in RPMI-1640 medium (Gibco, Carlsbad, USA) with 10% fetal bovine serum. THP-1 cells were purchased from Procell (Wuhan, China). THP-1 cells were maintained in RPMI 1640 medium (Gibco, Carlsbad, USA) augmented with 0.05 mM 2-mercaptoethanol (Sigma-Aldrich) and 10% fetal bovine serum. All cell lines were kept in a cell incubator at 37 °C with a 5% CO2 atmosphere.

Macrophage induction and polarization

Initially, THP-1 cells were exposed to 100 nM PMA (Sigma-Aldrich) fo 24 h to facilitate their differentiation into M0 phase macrophages. Subsequently, the M0 macrophage culture medium was supplemented with 20 ng/ml IL-4 (PeproTech) and 20 ng/ml IL-13 (PeproTech), followed by a 48-h incubation to induce polarization into M2 macrophages.

Collection of cuproptosis cell fragments

The HCT116 and DLD1 colon cancer cell lines were uniformly plated in six-well plates and cultured until they reached 80% confluence. Then, 0.5 µM Elesclomol (Selleck, S1052) was added, and the cells were incubated for 48 h. After 48 h, extensive cell death was observed. The cell culture medium was then subjected to centrifugation at 1000 g for 10 min, and the supernatant was collected for co-culturing with M2 macrophages.

Real-time quantitative PCR (qRT-PCR) assay

The reverse transcription of total RNA was performed utilizing Hifair® III Reverse Transcriptase (Yeasen Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer's specifications. Subsequently, the resultant product was immediately stored at −80 °C for future use. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted using a QuantStudio 3 Real-Time PCR System (Thermo Fisher) in conjunction with Hieff® qPCR SYBR Green Master Mix (Yeasen Biotechnology Co., Ltd., Shanghai, China). The qRT-PCR protocol entailed an initial denaturation step at 95 °C for 5 min, followed by 40 amplification cycles, each comprising denaturation at 95 °C for 10 s, primer-specific annealing at 60 °C for 20 s, and extension at 72 °C for 20 s. Primer sequences are provided in Table S1. The relative expression levels of the target genes were determined using the 2 − ΔΔCt method, with GAPDH employed as the internal reference gene.

Western blot

Proteins were extracted from cell lines using RIPA buffer, and equivalent protein samples (30 μg, quantified by the BCA Protein Assay Kit) were separated on SDS-PAGE gels. The proteins were subsequently transferred onto PVDF membranes. Post-transfer, the membranes were blocked with TBST containing 5% BSA, followed by overnight incubation at 4 °C with primary antibodies targeting GAL3ST4 (1:3000). The membranes were then hybridized with secondary antibodies at 37 °C for 1 h, after which band intensities were detected using the ECL-plus kit.

Immunofluorescence of cells

Inoculate the prepared cells into a 24-well plate with coverslips, cultivating until a suitable cell density is attained; aspirate the culture medium and wash with pre-cooled PBS for 5 min, repeated three times; fix the cells with 4% paraformaldehyde, followed by washing with PBS for 5 min, repeated three times; treat with a permeabilization solution containing 0.1% Triton X-100 for 15 min, followed by washing with PBS for 5 min, repeated three times; treat with a permeabilization solution containing 0.1% Triton X-100 for 15 min, followed by washing with PBS for 5 min, repeated three times; aspirate the blocking solution and add the primary antibody (1:200), incubating overnight at 4 °C; the next day, aspirate the primary antibody, wash with PBS for 5 min, repeated three times, then add the fluorescent secondary antibody, incubating in the dark at room temperature for 1 h, followed by PBS washing for 5 min, repeated three times; use a pipette tip to apply an antifade mounting medium containing DAPI onto a glass slide, and gently place the coverslip on top; observe the cells under a fluorescence microscope.

Immunofluorescence Assay for Tissue Sections

Subject the paraffin sections to a deparaffinization agent (Wuhan, Seville) until they achieve complete hydration; apply an antigen retrieval buffer (Wuhan, Seville) to the prepared tissue sections to restore antigenicity, subsequently rinsing with PBS for three repetitions of 5 min; administer a 3% BSA solution onto the sections for a blocking period of 30 min; prepare a mixed solution containing two primary antibodies targeting distinct protein markers, dispense the solution onto the sections, and incubate overnight at 4 °C in a humidified chamber, followed by washing with PBS for three cycles of 5 min each; introduce the appropriate secondary antibodies, incubating for 1 h at room temperature while shielded from light, then perform PBS washes for three repetitions of 5 min each; apply DAPI staining solution to the sections and incubate at room temperature for 10 min, subsequently washing with PBS for three repetitions of 5 min; apply DAPI staining solution to the sections and incubate at room temperature for 10 min, subsequently washing with PBS for three repetitions of 5 min; utilize an antifade mounting agent for the sections, and subsequently acquire images with a fluorescence scanner (Pannoramic MIDI, 3DHISTECH).

Immunohistochemistry

Immerse paraffin sections in a deparaffinization solution (Wuhan, Seville) until fully rehydrated; apply an antigen retrieval solution (Wuhan, Seville) to the processed tissue sections for antigen restoration, followed by three washes with PBS for 5 min each; incubate the sections in a 3% hydrogen peroxide solution at room temperature, shielded from light, for 25 min to inhibit endogenous peroxidase activity, followed by three washes with PBS for 5 min each; incubate the sections in a 3% hydrogen peroxide solution at room temperature, shielded from light, for 25 min to inhibit endogenous peroxidase activity, followed by three washes with PBS for 5 min each; incubate the sections in a 3% hydrogen peroxide solution at room temperature, shielded from light, for 25 min to inhibit endogenous peroxidase activity, followed by three washes with PBS for 5 min each; introduce the corresponding secondary antibody and incubate at room temperature for 1 h, followed by three washes with PBS for 5 min each; dispense freshly prepared DAB chromogen, controlling the color development time under a microscope, with positive staining appearing brown, and subsequently rinse with tap water to halt the reaction; subject the sections to hematoxylin counterstaining for approximately 3 min, followed by rinsing under running water; then apply a differentiation solution for a few seconds, rinse again, and use a bluing solution followed by another rinse; sequentially immerse the sections in 75% ethanol for 5 min, 85% ethanol for 5 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, n-butanol for 5 min, and xylene I for 5 min for dehydration and clearing; then remove the sections from xylene, allow them to air dry briefly, and mount with adhesive; observe the sections under a bright-field microscope.

Cell transfection

Transfection is conducted when the cell density reaches 70%. siRNA is directly mixed with the transfection reagent (Advanced DNA RNA Transfection Reagent, AD600150) in a 1:1 ratio. The mixture is gently pipetted 10–15 times to ensure thorough homogenization and left to stand at room temperature for 10–15 min. The nucleic acid complexes are then added to the cell culture medium for transfection. After 48 h, the mRNA expression levels are assessed using PCR. The si-RNA (si-GAL3ST4) sequences are detailed in Table S2.

Cell coculture and cell metastasis assays

Thereafter, DLD1 and HCT116 cells were introduced into Transwell chambers (8-μm pore membrane, Corning, NY, USA) at a density of 2 × 10^5 cells per chamber, with varying treatments applied according to experimental requirements. Following a 48-h incubation, the chambers were fixed using 4% paraformaldehyde, and cells adhering to the upper side of the membrane were carefully removed by gentle wiping. The membranes were subsequently stained with crystal violet and examined under an optical microscope.

The CCK8 assay

The cells were initially seeded into 96-well culture plates at a density of 2000 cells per well. After a 24-h incubation, the cells underwent various treatments. Cell viability was evaluated at 0, 24, 48, 72, and 96 h post-treatment using the CCK8 assay, following the manufacturer's protocol.

Human samples

For the internal CRC sample cohort, three patients with a CRC diagnosis were recruited, and their clinical data were presented in Table S3. Tissue samples were obtained from the First Affiliated Hospital of Zhengzhou University, and all procedures were performed in accordance with the approval granted by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Approval No: 2023-KY-0353-003). All participating patients provided informed consent.

Statistical Analysis

All data analyses were conducted using R software version 4.2.2. Pearson's correlation test was used for correlation analysis. Comparisons between the two groups were calculated using unpaired Student’s t-test or unpaired nonparametric Mann–Whitney test. Each assay was independently replicated more than three times, with results displayed as means ± standard error of the mean (SEM) in the figures. Multiple time point comparisons between two groups, as well as comparisons among more than two groups, were computed using analysis of variance (ANOVA).