The six CRC morphological patterns of interest (morphotypes). Left: example of an original annotation used for macrodissection and RNA extraction. Note that the original annotations in the image are not identical to the ones used in the main text. Here, A-SE stands for serrated (SE) in the text, B-DE for desmoplastic (DE) in the text, C-MUC for mucinous (MU) in the text, and D-ST for solid/trabecular (TB) in the text, respectively. Also, N indicates a tumor-adjacent normal epithelial region and S a supportive stroma region, respectively. Right: examples of morphotypes – complex tubular (CT), desmoplastic (DE), mucinous (MU), papillary (PP), serrated (SE), and solid/trabecular (TB).

CRC morphotypes: in silico decomposition of the cellular admixture. (A) Boxplots of the tumor purity (epithelial content – ESTIMATE method) in each tumor morphotype and the two non-tumor regions, ordered by increasing median values. (B) Signatures specific to colon crypt compartments and major cell types estimated from gene expression data in terms of normalized enrichment scores (NES): only statistically significant scores are shown. (C) Immune cell fractions (and unassigned fractions) inferred from gene expression data using quanTIseq method. (D) Types of cancer-associated fibroblasts (CAFs) as estimated from gene expression using the signatures from (24,25).

Top differentially expressed genes and hallmark pathways. (A) GSEA scores for hallmark pathways in the six morphotypes and two non-tumoral regions. Only pathways with statistically significant scores are shown. (B) Principal component analysis of hallmark pathways: the median profiles of the six morphotypes (CT: complex tubular, DE: desmoplastic, MU: mucinous, PP: papillary, SE: serrated, and TB: solid/trabecular) and the two non-tumoral regions (NR: tumor-adjacent normal and ST: supportive stroma) are projected onto the space defined by first two principal components (74% of the total variance). The top pathways contributing to the principal axes are shown as well. See also Supplemental Figure SF3. (C) Heatmap of top 5 up- and down-regulated genes for each of the six morphotypes.

Results of comparison of each morphotype (and the two non-tumoral regions) with the average profile. The table shows top 20 up- and down- regulated genes and significantly activated hallmark pathways and processes (as result of GSEA). The genes not significant after p-value adjustment (at FDR=0.15) have their symbols greyed. See also Supplemental Tables ST5 and ST6.

Intra-tumoral heterogeneity and the morphotypes (for all core samples, including those unassigned by the classifiers). Only cases with at least two distinct morphotypes present are shown. (A) left: CMS assignment for tumors represented by multiple regions. right: CMS assignment per morphotype (and two non-tumoral patterns). (B) left: iCMS assignment for tumors represented by multiple regions. right: iCMS assignment per morphotype (and two non-tumoral patterns). (C) Differences between paired signatures: morphotypes vs whole tumor (each signature was normalized to [0,1] prior to computing the differences). Only four (morphotype, whole tumor) pairs were represented enough in the data. (D) Boxplots for the ten (normalized) signatures across morphotypes. The “Eschrich” and “Jorissen” signatures vary significantly (Kruskal-Wallis’s test) across morphotypes. For equivalent plots for all samples, including non-core, see Supp.Fig. 7.

Intra-tumoral heterogeneity case study. For the same case, different CMS labels are assigned to regions and whole tumor profile. The hallmark pathways show various levels of activation (as computed by GSVA) within same section. The relative change in prognostic scores indicate potential underestimation of risk for some signatures, while others appear to be stable across tumor. See also Supplemental Figure SF5A- B. Note that in the pathology section image, the original annotations were preserved, and they are not identical to the ones used in the main text. Here, MUC stands for mucinous (MU) in the text. Also, N indicates a tumor-adjacent normal epithelial region and S a supportive stroma region, respectively