Extended Data Fig. 1: Extended analysis of TME spatial polarization.

a,b, Scatter plots showing cross-platform consistency of top differentially expressed genes between tumour and adjacent stroma in representative TME cell types, comparing genes identified from bulk ST data (10x Visium) reconstructed with scRNA-seq data using CytoSPACE²⁵ (x-axis) and validated using single-cell ST data (MERSCOPE) (a), and vice versa (b) (Supplementary Table 4). Concordance was assessed by Spearman correlation, linear regression with 95% confidence intervals, and fraction of genes with consistent directionality between platforms (upper left confusion matrices). P-values were determined by two-sided t tests. Point colours reflect the mean of both axes, with orange and purple denoting tumour-associated and adjacent stroma-associated genes, respectively. Top genes (up to a maximum of 10% of genes included in the MERSCOPE gene panel; 50 genes in total, and 25 genes per compartment) were selected from Visium data (panel a) or from MERSCOPE data (panel b) to satisfy differential expression requirements of Q < 0.05 and median log₂ fold change (FC) across samples >0.05. Four genes per compartment with the highest average rank across both axes are highlighted. c, Same as a and b but showing for all evaluable TME cell types (n = 9) the fraction of top genes with consistent directionality between platforms in relation to the discovery cohort. Significance was assessed by two-sided Wilcoxon signed-rank test. ns, not significant. For details, see Methods. d, Same as Fig. 1d, using CytoSPACE to integrate scRNA-seq and ST data, but shown for plasma cells. The mean log₂ FC of differentially expressed genes in plasma cells between discovery (Visium) and each validation cohort (MERSCOPE, legacy ST) was assessed by Spearman correlation, with significance determined by a two-sided t test. Significant concordance was observed for MERSCOPE but not legacy ST, which has 100 µm spots and the lowest spatial resolution among evaluated ST assays. e, Heat map showing spatial expression programmes that stratify tumour and adjacent stroma independent of TME cell type (n = 9; coloured as in c), cancer type (n = 10), or ST platform (n = 3) (Supplementary Table 6). Genes with asterisks (PKM and FOS) denote the top markers in the Visium discovery cohort that are contained within the MERSCOPE gene panel. f, Spatial polarization of PKM and FOS expression in tumour and adjacent stroma in representative liver cancer (Liver 2, top) and colon cancer (Colon 2, bottom) specimens profiled by MERSCOPE (Supplementary Table 8). Left: Annotated cell types, tumour/stroma regions, and specimen-wide expression of PKM and FOS. Right: Representative microregions (50 µm²) showing PKM and FOS mRNA transcripts in diverse cell types. g, Heat map depicting normalized enrichment scores (fgsea¹¹⁴) of hallmark pathways in tumour versus adjacent stroma for pan-cell-type markers related to panel e (Supplementary Table 7). Gene sets were applied to an ordered expression vector of log₂ fold changes between tumour and adjacent stroma, balanced by TME cell type and cancer type. Data vectors in Visium discovery and MERSCOPE validation cohorts were compared by Spearman correlation, with significance determined by a two-sided t test. In panel c, the box centre lines, bounds of the box, and whiskers denote medians, 1^st and 3^rd quartiles, and minimum and maximum values within 1.5 × IQR (interquartile range) of the box limits, respectively.