Effect of areas of illumination (AOI) size and sequencing depth on biological conclusions from segmented tumor and tumor microenvironment. (A) Left: Representative images of the colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) samples. Tumor, invasive-margin, and hyperproliferative regions are highlighted. Tissues were stained with antibodies against PanCK, CD3E, and PTPRC. Right: Enlarged region of the CRC image to highlight the size titration and segmentation strategy. Circular regions of interest were automatically segmented into tumor (orange) and immune (blue) compartments. (B) Scatterplot of AOI area versus number of cells with points colored by area bin: Very small, <2300 μm2; small, 2300–7850 μm2; mid, 7850–49,000 μm2; large, >49,000 μm2. (C) Number of genes detected per AOI for tumor and immune compartments in each AOI size bin, colored as in B. (D) Principal component analysis of variation between samples using genes detected above background in >20% of AOIs. PC1 versus PC2 is plotted with points colored by tumor type and shaped by segment type. (E) Spearman's correlation of WTA counts from each AOI with all RNA-seq data sets in TCGA. AOIs are ordered by area on the x-axis, and each point is a pairwise comparison with a data set in TCGA. Points are colored by TCGA tumor type: colon adenocarcinoma (blue), rectal adenocarcinoma (green), lung adenocarcinoma (red), lung squamous cell carcinoma (orange), and other (gray). AOIs are labeled by area bin, colored as in B. (F) Correlation of counts, single-sample Gene Set Enrichment Analysis (ssGSEA) enrichment, and cell type deconvolution between AOIs. For each metric, Spearman's correlations were calculated between each AOI compared with the largest AOI sizes, and averaged within different AOI size bins. (G) Left: Spearman's correlation of counts for each subsampled read depth and AOI size relative to counts at 300 reads/μm2. Right: Number of genes detected above background for each read depth and AOI size.
