Stratification and pathway enrichment analysis of TCGA-BLCA based on ICD-related genes.

A-C. Gene expression heatmap (A), clinical heatmap (B), and survival analysis (C) of ICD-high and ICD-low groups. D-E. Differential gene heatmap (D) and volcano plot (E) of ICD-high and ICD-low groups. F-G. GO enrichment analysis. H. KEGG enrichment analysis. I-L. GSEA enrichment analysis.

The immune characteristics within the tumor microenvironment vary between ICD-high and ICD-low groups.

ESTIMATE scores (A), immune scores (B), stromal scores (C), and tumor cell purity (D) of ICD-high and ICD-low groups. E. Immune cell infiltration in the two subgroups. F-H. Expression profiles of the two subgroups in 13 immune-related pathways (F), 8 immune checkpoints (G), and HLA gene family (H). I. Efficacy of immunotherapy in the two subgroups.

Construction and validation of risk-scoring model.

A. Hazard ratio of four ICD-related genes identified by Cox regression analysis. B-C. LASSO regression analysis for constructing the prognostic model. D-E. Survival analysis of high-risk and low-risk groups in TCGA (D) and GEO (E) databases. Analyzed the correlation between risk-scores and patients, along with four key genes, in both TCGA (F-H) and GEO databases (I-K).

The association between risk-scores and clinical features and immune characteristics.

A-D. The association between the four genes composing the risk-scores and patient prognosis. E. Clinical heatmap of the two subgroups. F-G. Differences in stage (F) and grade (G) between the two subgroups. H-J. Constructed nomogram (H) and ROC curve (I-J). K-L. Cox regression analysis.M.Stromal scores, immune scores, and ESTIMATE scores of the high-risk and low-risk groups. N. Immune infiltration in the two subgroups.

The association between the risk-score and the sensitivity to chemotherapy (A-P) and immunotherapy (Q-T).

Validate the risk-scoring model through tissue arrays and single-cell sequencing.

A-B. Analyzing the expression of CALR, IFN1B, IFNG, and IL1R1 using immunofluorescence staining. C-D. Survival analysis of the four genes (C) and risk-scores (D) in patients. E-I. The relationship between risk-scores and CD39, CD8+, CD8+LAG3+, Tumor Size, and T Stage. J-K. Expression analysis of CALR, IFN1B, IFNG, and IL1R1 in single-cell sequencing data.

Somatic mutations in ICD-high and ICD-low groups.

A. Tumor mutation burden in the two subgroups. Most common mutated genes in the ICD-high (B) and ICD-low (C) groups.

Prognostic analysis of somatic mutations in high-risk and low-risk groups.

A-B. Most common mutated genes in high-risk (A) and low-risk (B) groups. C. Tumor mutation burden in the two subgroups. D. Prognostic analysis of different risk burdens with risk scores.

Relationship between risk scores and different clinical features.

A-G. Relationship between Age, Gender, Grade, M, Stage, T, N, and risk scores. H-S. Survival analysis of subgroups based on Age, Gender, Grade, M, Stage, T, N, and risk scores.

The relationship between risk scores and immune-related pathways and immune infiltration.

A. The relationship between risk scores and immune-related pathways. B. The relationship between risk scores and immune infiltration.

The association between risk-scores and immune cells.

A-H. Correlation analysis of risk scores with immune cells. I-P. Analysis of the correlation between risk scores and immune cells using various algorithms.

The correlation of CALR, IFNG, and IL1R1 with immune cell infiltration.

A-B. The correlation of CALR with immune cell infiltration. C-H. The correlation of IL1R1 with immune cell infiltration. I-P. The correlation of IFNG with immune cell infiltration.

Pathway enrichment analysis and correlation with immune checkpoints of CALR, IFN1B, IFNG, IL1R1, and risk score.

A-B. Pathway enrichment analysis of CALR, IFN1B, IFNG, IL1R1, and risk score. C. Correlation of CALR, IFN1B, IFNG, IL1R1, and risk score with immune checkpoints.

Expression of CALR, IFN1B, IFNG, IL1R1 in tissues.

A. Expression of the four genes in cancer and adjacent tissues in tissue microarrays. B-E. Expression of CALR, IFN1B, IFNG, IL1R1 in cancer and adjacent tissues in TCGA. F-G. Expression of CALR, IFNG, IL1R1 in public database single-cell sequencing data (GSE135337).