Integrating single-cell and spatial analysis reveals MUC1-mediated cellular crosstalk in mucinous colorectal adenocarcinoma

Abstract

Background: Mucinous colorectal adenocarcinoma (MCA) is a distinct subtype of colorectal cancer (CRC) with the most aggressive pattern, but effective treatment of MCA remains a challenge due to its vague pathological characteristics. An in-depth understanding of transcriptional dynamics at the cellular level is critical for developing specialised MCA treatment strategies.

Methods: We integrated single-cell RNA sequencing and spatial transcriptomics data to systematically profile the MCA tumor microenvironment (TME), particularly the interactome of stromal and immune cells. In addition, a three-dimensional bioprinting technique, canonical ex vivo co-culture system, and immunofluorescence staining were further applied to validate the cellular communication networks within the TME.

Results: This study identified the crucial intercellular interactions that engaged in MCA pathogenesis. We found the increased infiltration of FGF7⁺/THBS1⁺ myofibroblasts in MCA tissues with decreased expression of genes associated with leukocyte-mediated immunity and T cell activation, suggesting a crucial role of these cells in regulating the immunosuppressive TME. In addition, MS4A4A⁺ macrophages that exhibit M2-phenotype were enriched in the tumoral niche and high expression of MS4A4A⁺ was associated with poor prognosis in the cohort data. The ligand-receptor-based intercellular communication analysis revealed the tight interaction of MUC1⁺ malignant cells and ZEB1⁺ endothelial cells, providing mechanistic information for MCA angiogenesis and molecular targets for subsequent translational applications.

Conclusions: Our study provides novel insights into communications among tumour cells with stromal and immune cells that are significantly enriched in the TME during MCA progression, presenting potential prognostic biomarkers and therapeutic strategies for MCA.

Key points: Tumour microenvironment profiling of MCA is developed. MUC1⁺ tumour cells interplay with FGF7⁺/THBS1⁺ myofibroblasts to promote MCA development. MS4A4A⁺ macrophages exhibit M2 phenotype in MCA. ZEB1⁺ endotheliocytes engage in EndMT process in MCA.

Keywords: mucinous colorectal adenocarcinoma; scRNA‐seq; spatial transcriptomics; tumour microenvironment.

FIGURE 1

Cell landscape of paired human MCA tissues and normal colon tissues. (A) Graphic overview of the presented study design. (B) UMAP plots of 40 424 and 48 081 cells originating from tumour and normal tissues of six MCA patients. Fourteen cell clusters were identified. (C) Bar plot showing distributions (left) and proportions (right) of cells derived from every sample for each cell cluster. (D) Expressive distribution of signature genes in overall cells for each cell cluster. (E) Dot plot showing the expression profile of signature genes in each cell cluster. (F) Cell proportions of the normal and tumour groups in each cell cluster.

FIGURE 2

Dynamic changes in cell populations and communication in the tumour microenvironment. (A and B) Percentage comparison between normal and tumour groups in elevated (A) and decreased (B) cell clusters with a significant difference. (C) Communication comparison between normal and tumour groups among cell clusters. (D) Communication comparison between normal and tumour groups among immune, fibroblast, myeloid, endothelium, and tumour modules. (E) Strength of links (including incoming and outgoing links) among cell clusters. (F) Information flow comparison between normal and tumour groups in signalling pathways. (G) Comparison of ligand‐receptor pair interactions between the normal and tumour groups at the pathway level among cell clusters.

FIGURE 3

Subsets of myofibroblasts are associated with tumour progression and clinical outcome. (A) UMAP plot of myofibroblasts coloured by cell subsets. (B) Fraction of each cell subset in every sample. (C) Cell proportions of normal and tumour groups in each cell subset. (D) Expressive pattern of CAF canonical genes in each cell subset. (E) The expression level of collagen‐related genes, integrins, and fibronectin in myofibroblast subsets and Rho signalling in tumour cells. (F) Biological processes of up‐ and downregulated genes for myofib‐1. (G) Physiological processes of highly expressed genes for myofib‐1. (H). Biological processes of up‐and downregulated genes for myofib‐2. (I) Physiological processes of highly expressed genes for myofib‐2. (J) The Kaplan–Meier curves showed that MCA patients with higher expression of AEBP1 and POSTN were associated with worse prognosis. (K) The graphic pattern of fibroblast growth factors in myofib‐1 and myofib‐2. (L) Expression comparison between normal and tumour groups for CAF canonical genes in myofib‐1. Green, normal group; orange, tumour group.

FIGURE 4

Co‐culture assay of MCA cancer cells and tumor‐associated fibroblasts. (A) Schematic illustration of the co‐culture platform. (B and C) Representative images and analysis of mCherry‐labeled COLO205 (red) cultured alone or in co‐culture with GFP‐labeled CAFs (green) and imaged by confocal microscopy after 48 h (n = 3). (D) Kinetic curves of COLO205 or CAFs cultured alone or in co‐culture, as assessed by xCELLigence RTCA‐DP. (E and G) Representative images and analysis of COLO205 cultured alone or in co‐culture with CAFs treated with doxorubicin for 24 h. (F and H) Transwell images and analysis of COLO205 cells cultured alone or treated with THBS1 (400 ng/mL), FGF7 (50 ng/mL), and THBS1 (400 ng/mL) in combination with FGF7 (50 ng/mL). (I) The quantitative migration rate of COLO205 when co‐cultured with CAFs or siFGF7 CAFs. (J) The quantitative migration rate of CAFs when co‐cultured with COLO205 or siMUC1 COLO205.

FIGURE 5

Macrophages are associated with tumour progression and immune evasion. (A) UMAP plot of myeloid cells coloured by cell subtypes. (B) Dot plots showing the expression profile of signature genes in each cell subtype. (C) Cell percentage change of each cell subtype in normal and tumour samples. (D) Heatmap showing expression profiles of feature genes in overall cells for each cell subtype. (E) Differentiation trajectory of cell subtypes. Distribution (left) and proportion (right) of each cell subtype over state of differentiation. (F) Overall gene (upper panel) and feature gene (lower panel) expression profiles of each cell subtype along with pseudotime of differentiation. (G) Expression information of tumor‐associated macrophage (TAM) canonical genes in macrophage subset 1 and 2. (H) Expression comparison of TAM canonical genes between normal and tumour samples in macrophage subset 1. (I) The Kaplan–Meier curve showed that MCA patients with higher expression of MS4A4A were associated with poorer prognosis. (J). qPCR analysis of M1 and M2 macrophage markers in monoculture THP1 cells, THP1 cells co‐cultured with COLO205, or THP1 cells co‐cultured with siMUC1 COLO205. (K) qPCR analysis of MUC1, ICAM, and IL6 mRNA levels in COLO205 cells after MUC1 knockdown.

FIGURE 6

Endothelial tip cells are involved in endothelial‐to‐mesenchymal transition and are associated with tumorigenesis. (A) UMAP plot of endothelial cells coloured by cell subtypes. (B) Dot plot of signature genes in each cell subtype. (C) Fraction of each cell subtype in every sample. (D) Proportion of each cell subtype in normal and tumour groups. (E) Copy number variations (CNVs) profile in normal and tumour endothelial cells. (F) Violin plot showing the expression patterns of signature genes of each cell subtype. (G). Kaplan‐Meier curve showing differences in hazard ratios for various groups classified by the expression of key genes derived from endothelial tip cells. (H) Dot plot showing the expression profiles of EndMT‐related genes in each cell subtype. (I) Expression comparison of EndMT‐related genes between normal and tumour groups in endothelial tip cells. (J) Highly expressed genes of endothelial tip cells enriched in the process of epithelial‐to‐mesenchymal‐like transition. (K and L) Representative images and analysis of ZEB1 knockdown on HUVEC tube formation co‐cultured with COLO205.

FIGURE 7

Spatial transcriptomics reveals cell crosstalk between CAFs, macrophages, and tumour cells. (A) UMAP plot of ST spots coloured by the cell type of tumour tissues from one MCA patient. (B) Clustering of ST spots and assigning a major cell type for each spot in MCA tumour tissues. (C) Dot plots of signature genes in each cell type for ST spots in tumour tissues. (D) UMAP plot of ST spots coloured by the cell type of matched normal tissues. (E) Clustering of ST spots and assigning a major cell type for each spot in normal tissues. (F) Dot plots of signature genes in each cell type for ST spots in normal tissues. (G, H) Expression and spatial distribution of signature genes in overall ST spots of tumour tissues (G) and normal tissues (H). (I) Correlation between tumour and CAF scores evaluated by the expression of the MUC1 and FGF7 genes. (J) Correlation between tumour and CAF scores evaluated by the expression of the MUC1 and THBS1 genes. (K) Correlation between tumour and TAM scores evaluated by the expression of the MUC1 and MS4A4A genes.

FIGURE 8

Highly infiltrated FGF7 ⁺/THBS1 ⁺ myofibroblasts, MS4A4A ⁺ macrophages, and ZEB1 ⁺ endothelial cells correlated with an advanced stage and worse outcome of MCA. (A–F) Expressive information of miR‐26a‐5p (A), miR‐214‐3p (B), miR‐7‐5p (C), miR‐18a‐5p (D), miR‐19b‐3p (E), and miR‐744‐5p (F) in the high and low gene expression groups. (G, H). Methylation scores of the genes MGMT (G) and WNT5A (H) in the high and low gene expression groups. (I). Representative immunofluorescence staining of human MCA tissues and normal tissues. (J). Kaplan–Meier curves present the overall survival analysis for THBS1 and MS4A4A in Chinese MCA patients. (K) Biological model of cell communications among tumour cells, CAFs, TAMs, and endothelial cells. The plot was created using BioRender (https://biorender.com/).