Related Experiment Video
Updated: May 18, 2026
Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion
Published on: October 20, 2016
Importance of direct macrophage-tumor cell interaction on progression of human glioma
Yoshihiro Komohara1, Hasita Horlad, Koji Ohnishi
1Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.
Abstract:
We previously showed tumor-associated macrophages/microglia (TAMs) polarized to the M2 phenotype were significantly involved in tumor cell proliferation and poor clinical prognosis in patients with high grade gliomas. However, the detailed molecular mechanisms involved in the interaction between TAMs and tumor cells have been unclear. Current results reveal that, in coculture with human macrophages, BrdU incorporation was significantly elevated in glioma cells, and signal transducer and activator of transcription-3 (Stat3) activation was found in both cell types. Direct mixed coculture led to stronger Stat3 activation in tumor cells than did indirect separate coculture in Transwell chamber dishes. Screening with an array kit for phospho-receptor tyrosine kinases revealed that phosphorylation of macrophage-colony stimulating factor receptor (M-CSFR, CD115, or c-fms) is possibly involved in this cell-cell interaction; M-CSFR activation was detected in both cell types. Coculture-induced tumor cell activation was suppressed by siRNA-mediated downregulation of the M-CSFR in macrophages and by an inhibitor of M-CSFR (GW2580). Immunohistochemical analysis of phosphorylated (p)M-CSFR, pStat3, M-CSF, M2 ratio, and MIB-1(%) in high grade gliomas revealed that higher staining of pM-CSFR in tumor cells was significantly associated with higher M-CSF expression and higher MIB-1(%). Higher staining of pStat3 was associated with higher MIB-1(%). High M2 ratios were closely correlated with high MIB-1(%) and poor clinical prognosis. Targeting these molecules or deactivating M2 macrophages might be useful therapeutic strategies for high grade glioma patients.
Insights
Tumor-associated macrophages (TAMs) drive glioma growth via M-CSFR signaling, activating Stat3 in tumor cells. Targeting M-CSFR or M2 macrophages may offer new high-grade glioma therapies.
Area of Science:
- Immunology
- Oncology
- Neuroscience
Background:
- Tumor-associated macrophages (TAMs) polarized to the M2 phenotype are implicated in high-grade glioma progression and poor prognosis.
- The precise molecular mechanisms governing TAM-glioma cell interactions remain incompletely understood.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying the interaction between TAMs and glioma cells.
- To investigate the role of macrophage-colony stimulating factor receptor (M-CSFR) signaling in this crosstalk.
- To evaluate potential therapeutic targets for high-grade gliomas.
Main Methods:
- Coculture of human macrophages and glioma cells.
- Assessment of cell proliferation (BrdU incorporation) and signal transducer and activator of transcription-3 (Stat3) activation.
- Phospho-receptor tyrosine kinase array screening.
- siRNA-mediated M-CSFR downregulation and M-CSFR inhibitor (GW2580) treatment.
- Immunohistochemical analysis of pM-CSFR, pStat3, M-CSF, M2 ratio, and MIB-1 in patient tumor samples.
Main Results:
- Coculture enhanced glioma cell proliferation and Stat3 activation in both cell types, with direct contact yielding stronger Stat3 activation.
- M-CSFR activation was identified in both cell types and found to be crucial for coculture-induced tumor cell activation.
- siRNA-mediated M-CSFR knockdown and M-CSFR inhibition suppressed tumor cell activation.
- In high-grade gliomas, pM-CSFR in tumor cells correlated with M-CSF expression and proliferation marker MIB-1(%).
- pStat3 staining correlated with MIB-1(%), and high M2 ratios were associated with high MIB-1(%) and poor prognosis.
Conclusions:
- M-CSFR signaling plays a critical role in the crosstalk between TAMs and glioma cells, promoting tumor cell proliferation via Stat3 activation.
- Targeting M-CSFR or deactivating M2 macrophages represents a promising therapeutic strategy for high-grade glioma patients.
