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Updated: Feb 17, 2026
In Vitro Assay to Study Tumor-macrophage Interaction
Published on: August 1, 2019
M2 macrophage-based classification identifies DOK3 as a driver of pro-tumoral polarization and migration in
Chang-Yuan Ren1, Ji Shi2,3, Chang-Lin Yang1
1Beijing Neurosurgical Institute, Capital Medical University, Beijing, China.
Background:
Glioblastoma (GBM) is the most aggressive primary brain tumor, characterized by limited therapeutic options and dismal prognosis. Among the components of the immunosuppressive tumor microenvironment (TME), M2-polarized macrophages are pivotal mediators of tumor progression, yet the molecular mechanisms underlying their polarization and pro-tumoral functions remain inadequately understood.
Methods:
We integrated bulk and single-cell RNA sequencing datasets from CGGA, TCGA, and GSE131928. M2 macrophage infiltration was quantified using the xCell algorithm, and unsupervised clustering of M2-associated genes was first performed to define macrophage-centered immune subtypes, further refined by XGBoost and LASSO modeling. Based on the molecular features of the most immunosuppressive subtype, a macrophage-associated risk score based on CTSB, LITAF, and DOK3 was constructed and validated across independent cohorts for prognostic and immune relevance. Functional validation was performed by silencing DOK3 in THP-1-derived macrophages, followed by co-culture with glioma cells to assess macrophage polarization and tumor cell behavior.
Results:
Elevated M2 macrophage infiltration correlated with reduced tumor purity, spatial heterogeneity, and worse survival. Three immune subtypes (C1-C3) were identified; notably, the C1 subtype exhibited the highest M2 infiltration, strongest immunosuppressive features, and poorest prognosis. The derived macrophage-based risk score robustly stratified patient survival and correlated with CD163 expression and immune checkpoint activation. Single-cell analysis revealed predominant DOK3 expression in macrophages and microglia. Functional assays demonstrated that DOK3 knockdown reduced CD163 expression and attenuated glioma cell invasiveness, supporting its role in promoting M2 polarization and tumor aggressiveness.
Conclusion:
This integrative analysis identifies DOK3 as a pivotal regulator of M2 macrophage polarization and a driver of glioblastoma progression. Together, immune subtyping and the simplified macrophage-based risk model represent complementary strategies, with the latter providing a practical tool for prognostic stratification. Targeting DOK3 offers a promising therapeutic strategy to reprogram the TME and improve clinical outcomes in patients with GBM.
Insights
This study identifies DOK3 as a key factor in M2 macrophage polarization, driving glioblastoma (GBM) progression. Targeting DOK3 may offer a new therapeutic strategy for GBM patients by reprogramming the tumor microenvironment.
Area of Science:
- Oncology
- Immunology
- Genomics
Background:
- Glioblastoma (GBM) is an aggressive brain tumor with poor prognosis.
- M2 macrophages in the tumor microenvironment (TME) promote GBM progression.
- Molecular mechanisms of M2 macrophage polarization in GBM are not fully understood.
Purpose of the Study:
- To investigate the role of M2 macrophages in GBM progression.
- To identify molecular drivers of M2 polarization in the GBM TME.
- To develop a prognostic model based on macrophage-associated genes.
Main Methods:
- Integrated bulk and single-cell RNA sequencing data from multiple cohorts.
- Quantified M2 macrophage infiltration using the xCell algorithm.
- Defined immune subtypes and developed a risk score using machine learning (XGBoost, LASSO).
- Validated findings through functional assays involving DOK3 knockdown.
Main Results:
- Elevated M2 macrophage infiltration correlated with reduced tumor purity, increased heterogeneity, and worse survival.
- Identified three immune subtypes, with C1 showing highest M2 infiltration and poorest prognosis.
- Developed a macrophage-based risk score that stratified patient survival and linked to immune checkpoint activation.
- DOK3 knockdown reduced M2 polarization markers (CD163) and glioma cell invasiveness.
Conclusions:
- DOK3 is a key regulator of M2 macrophage polarization and GBM progression.
- A macrophage-based risk score provides a practical tool for prognostic stratification.
- Targeting DOK3 presents a potential therapeutic strategy to improve GBM outcomes.
