We next focused on stromal lineages, with no further subclassification performed for endothelial cells. Fibroblasts, CAFs and CSCs were subclassified into 10 classes, with only one class (S10) discovered in the KUL3 cohort (Additional file
13: Fig. S6A, Additional file
14: Fig. S7A). Subclasses were annotated according to the classification system provided by Lambrechts et al. [
19] S1-4 and S10 were all exclusive to adjacent normal tissue (Additional file
13: Fig. S6A, Additional file
14: Fig. S7A). The marker genes in each cluster are shown in Additional file
13: Fig. S6B and Additional file
14: Fig. S7B (Additional file
3: Table S3). Specifically, S1 and S2 featured adipocyte markers (
CFD and
APOD), resembling the phenotype of lipofibroblasts in lung tissue [
53]. S3 contained mesenchymal cells located in the colon lamina propria (
APOE and
ADAMDEC1 markers), and S10 was characterized by coexpression of
KCNN3 and
P2RY1, which have been reported to regulate multiple neuromuscular transmission processes in the colon [
19].S4 expressed Wnt signaling genes (
FRZB),
SOX6 and
PDGFRA, which function in maintaining the epithelial stem cell niche. Previous studies have described
PDGFRA+ fibroblasts as progenitors that give rise to lipofibroblasts and myofibroblasts [
54].The differentiation trajectory of fibroblast lineages in our study also indicated that S4 include highly plastic cells with the potential to give rise to other subtypes (Additional file
13: Fig. S6C, Additional file
14: Fig. S7C). For CAFs, a minor subclass (S6) characterized by
SERPINE1, IGF1,
WT1 and
KRT19 expression was isolated.
IGF1 has been reported to be associated with survival in bladder urothelial carcinoma, [
41] and higher expression of
SERPINE1 has also been reported to be an adverse factor in lung cancer[
50] .In addition, S6 expressed
KRT19 and
WT1, indicating that S6 resembles the mesothelial phenotype [
55].The remaining CAF subgroup (S5) was characterized by enrichment of collagens (
COL12A1,
COL1A1 and
COL3A1),
INHBA and MMPs (
MMP1 and
MMP11) (Additional file
13: Fig. S6B, Additional file
14: Fig. S7B). SCENIC analysis also identified unique TFs for S6, indicating that the underlying molecular network of S5 is completely different from that of S6 (Additional file
13: Fig. S6D, Additional file
14: Fig. S7D).
CAFs have been reported to generate a modified extracellular matrix (ECM) environment to promote cancer cell survival. Consistently, patients with high infiltration levels of S5 and S6 subtype cells showed worse survival (Additional file
13: Fig. S6E). The correlation of the C4 cell proportion with the frequency of CAFs indicated that the S5 subtype contributes to an aggressive phenotype of the epithelium (Additional file
13: Fig. S6F, Additional file
14: Fig. S7E). With regard to the contractile genes (
ACTA2 and
TAGLN) expressed by CSCs, we identified three classes (S7-9). S7 corresponded to pericytes because of its high expression of the characteristic genes
RGS5, collagens (
COL4A1,
COL4A2 and
COL18A1) and
NOTCH3 (which is related to vessel maturation). S8 was deemed myofibroblasts and was characterized by smooth muscle-related contractile genes (
MYH11 and
PLN), while S9, which represented smooth muscle cells, coexpressed contractile genes and cytoskeletal genes (
MYH11,
SYNPO2,
CNN1 and
DES) (Additional file
13: Fig. S6B. Fig. S7B). Overall, our analysis of colorectal stromal cells suggests that S5 subtype cells play a role in promoting cancer progression.