The six CRC morphological patterns of interest (morphotypes). Left: example of an original annotation used for macrodissection and RNA extraction. 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 (23,24).

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 (core samples). (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. For equivalent plots for all samples, including non-core, see Supp.Fig. 6.

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.

Morphotype distribution per case (unique tumor) and intersections thereof: some cases had several morphotypes profiled.

A: Epithelial signatures from Pelka et al., 2021. Only statistically significant scores (NES) are shown.

B: immune signatures from Pelka et al., 2021. Only statistically significant scores (NES) are shown.

C: Stromal signatures from Pelka et al., 2021. Only statistically significant scores (NES) are shown.

Principal component analysis of hallmark pathways GSEA scores: loadings for the first two principal components, i.e., contribution of pathways to the first two axes.

Hallmark pathways differential activation between pairs of morphotypes. Here we compare the results from GSEA applied to differentially expressed genes between pairs of morphotypes originating from all cases to results of GSEA applied to differentially expressed genes between pairs of morphotypes originating from the same section (tumor) (i.e., matched pairs of morphotypes). All results are shown, including the statistically not significant one. First four columns correspond to pairs of morphotypes from all cases, while the last four to matched pairs of morphotypes.

A Case P1949

B Case P1811

(A) Normalized enrichment scores from GSEA for selected resistance signatures (from C2 section of MSigDB). Only significant scores are shown.

(B) Resistance scores (GSVA) per patient and morphotype for a number of cases where the whole-tumor prediction is contradicted by some regional score.

Molecular subtypes and morphotypes in all samples, including non-core samples.

Main clinical parameters of the study cohort.

Distribution of main clinical parameters per morphotype (and tumor-adjacent normal and supportive stroma).

List of prognostic signatures tested.