Fig 2: BCAT2 negatively correlates with anticancer immunity in TME of BLCA. A) Expression pattern of chemokines, chemokine receptors, MHC molecules, and immunostimulators in high and low BCAT2 groups. B) Activity of cancer immunity cycle in high and low BCAT2 groups. Seven colors represent seven steps in the cycle. *p < 0.05; **p < 0.01; ***p < 0.001. C) Correlations between BCAT2 and multiple types of immune cell (CD8+T cell, CD4+T cell, DC cell, and NK cell) in six independent algorithms. D) Expression patterns of multiple subtypes of immune cell (CD8+T cell, NK cell, Macrophage, Th1 cell, and DC cell) related effector genes in high and low BCAT2 groups. E) Correlations between BCAT2 and ICB related effector genes. Number in the circle means correlation coefficient.

Fig 3: Exploring the mechanism of BCAT2 in shaping TME by bulk RNA-seq and scRNA-seq. A,B) tSNE plots of single-cell expression pattern of BCAT2 in Xiangya scRNA cohort and GSE135337 cohort. C) Violin plot of single-cell expression pattern of BCAT2 in GSE145137 cohort. D,E) GSEA analysis of GO term indicate activities of cytokine/chemokine related pathways and immune cells chemotaxis related pathways between different BCAT2 groups in GSE135337 cohort. F) GSEA analysis of KEGG term indicate activities of antigen presentation and interaction of cytokine and cytokine receptor pathway between different BCAT2 groups in GSE145137 cohort. G–I) GSEA analysis of GO term indicate activities of cytokine/chemokine related pathways, immune cells chemotaxis related pathways and immune cells migration related pathways between different BCAT2 groups in GSE145137 cohort. J,K) GO and KEGG analysis of DEGs between BCAT2 OE and BCAT2 KD cell lines.

Fig 4: Validation the function of BCAT2 by Xiangya BLCA cohort. A) Correlations between BCAT2/cancer immunity cycle (left) and BCAT2/TIICs (right). Solid and dotted lines mean positive and negative relationships, thickness of the lines mean coefficient of correlations, lines with different colors represent p-value of correlations. B) Correlations between BCAT2 and ICB related effector genes. C) Correlations between BCAT2 and TIS score related effector genes. Number in the circle represents correlation coefficient. D) Intersection of BCAT2, immune score and stromal score positive-related genes. E,F) Top 20 enrichment signaling pathways of GO and KEGG analyses in the BCAT2-related genes.

Fig 5: Validation of exclusive spatial relationships of BCAT2+ tumor cells and CD8+T cells by TMA of Xiangya BLCA cohort. A) IHC image of BCAT2 and CD8 in inflamed and noninflamed types of TME. Scale bar: 50 µm. B) Correlation between BCAT2 and CD8 on the basis of IHC scores of them in overall TMA. C) Multicolor IF image of BCAT2, CK19, CD8, and combination index in inflamed and noninflamed types of TME. BCAT2+ cell (pink), CK19+ cell (cyan), CD8+T cell (orange), and cell nucleus (blue). Scale bar: 50 µm. D,E) Detailed coexpression rates of BCAT2+CK19+ and BCAT2+CD8+ cells in the typical sample. F) Distance gradient analysis (0–25, 25–50, 50–100, and 100–150 µm) of CD8+T cells around BCAT2+CK19+ cells in the typical sample.