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Updated: Jul 2, 2026
A Macrophage-Tumor Spheroid Co-Invasion Assay
Published on: January 24, 2025
Comprehensive Analysis of Macrophage Dynamics, CCBE1, and Their Implications in Colorectal Cancer Microenvironment:
XiaoFei Fan1, ChunYan Zhang1, RongRong Gu1
1Department of Pharmacy, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu, China, ahnmc.com.
Background:
The significance of M2 macrophages in cancer is well established, yet their specific role and the regulatory molecules involved in colorectal cancer remain unclear.
Methods:
Using publicly available single-cell RNA-seq data from four colorectal cancer datasets (EMTAB8107, GSE139555, GSE146771, and GSE166555), we evaluated macrophage proportions and their functional enrichment. Immune cell infiltration was quantified by CIBERSORT, quanTIseq, and xCell on TCGA-COAD bulk transcriptomes, with immune correlations adjusted for tumor purity. GSEA was performed with MSigDB v7.4 gene sets (Hallmark, GO, KEGG). Prognostic models were built using Kaplan-Meier and multivariate Cox regression. In vitro, CCBE1 was silenced in SW480 and HCT116 cells; proliferation was measured by CCK-8, colony formation, and EdU assays, and migration/invasion were measured by transwell assays. M2 polarization was assessed by flow cytometry.
Results:
Macrophages represented a substantial fraction (20%-35% across datasets) of the tumor microenvironment and were associated with upregulated coagulation and KRAS signaling. Consensus M2 macrophage infiltration was linked to 91 upregulated genes, among which CCBE1 emerged as an independent prognostic risk factor (multivariate Cox: HR = 1.305 [95% CI: 1.039-1.639], p < 0.05). CCBE1 was overexpressed in colorectal cancer cell lines compared to NCM460. Silencing CCBE1 significantly suppressed cell proliferation, migration, and invasion. Moreover, the conditioned medium from CCBE1-knockdown cancer cells decreased the proportion of CD206+ M2 macrophages and upregulated M1 markers while downregulating M2 markers, indicating that CCBE1 promotes M2 polarization.
Conclusions:
CCBE1 is an oncogenic driver in colorectal cancer that independently predicts poor survival and functionally enhances tumor cell proliferation, invasion, and M2 macrophage polarization. Targeting CCBE1 may represent a potential therapeutic strategy for colorectal cancer.
