Fig. 3: Regions of interest definition and GeoMx spatial transcriptomic analysis results in responders versus non-responders.

A Representation of immunofluorescent staining of one region of interest. Pan cytokeratin (green) was used to identify tumor epithelium for selection of the tumor epithelial compartment. Syto-13 (blue) was used to label nuclei and CD45 (red) was used to label leukocytes. Negative selection approach was used to identify the stroma compartment reflective of the tumor microenvironment that includes CD45 positive and pan-cytokeratin negative cells. B Heatmap showing the scaled Q3-normalized expression of the differentially expressed genes (DEGs) between responders (R) and non-responders (NR) in stroma compartment. Patients are depicted by key (ID). C Volcano plot for the DEGs in R versus NR in stroma compartment. Significant DEGs are labeled and shown in red, with log₂FC > 1 and adjusted p-value < 0.05. NS non-significant. D Heatmap showing the scaled Q3-normalized expression of the DEGs between R and NR in tumor epithelial compartment. Patients are depicted by key (ID). E Volcano plot for the DEGs in R versus NR in tumor epithelial compartment. Significant DEGs are labeled and shown in red, with log₂FC > 1 and adjusted p-value < 0.05. NS non-significant. The scaled bar (color key) in the heatmaps represents the scaled Q3-normalized expression of the identified DEGs. Volcano plots used generalized linear models to identify the differentially expressed genes between AOIs and p-values were corrected for multiple comparison using Benjamini and Hochberg method. All experiments have been repeated twice to ensure reproducibility.