gms | German Medical Science

Objective: CCR2-CCL2 signaling is involved in myeloid cell recruitment to tumors and is considered as being a crucial axis for new therapeutic strategies. In glioma, we observed that CCR2ko mice showed 30% less infiltration of myeloid cells within the glioma tissue. Myeloid cells in glioma are composed of microglia and macrophages that both express CD11b and CD45. Glioma-bearing wildtype mice show an upregulation of CD45 expression (CD45high) as a sign of activation. In CCR2ko mice, we found significantly reduced amounts of CD11b+CD45high cells. In our study, we aimed to characterize the contribution of microglia and macrophages to the CD45high population, and their specific behavior in reaction to the CCR2-CCL2 signal and the impact on tumor progression. Therefore, we generated bone marrow chimeras to distinguish between microglia and macrophages by GFP-labeling.

Methods: Chimeras were generated using lethal irradiation with head protection. CX3CR1-GFP mice were used as recipient or bone marrow donor allowing the identification of microglia and macrophages. We established chimeras with CCR2ko of either microglia (ko/wt) or macrophages (wt/ko) and with ubiquitous functional CCR2 (wt/wt) as control. After reconstitution, tumor cells were implanted. On day 21 of tumor growth, glioma volumes were measured by MRI. Flow cytometric analyses and immunofluorescence staining were performed.

Results: Flow cytometry revealed that influx of peripheral macrophages into glioma tissues was similar in wt/wt and ko/wt chimeras (26-31%), and the percentage of CD45^high cells was comparable (about 40%). In contrast, the group of wt/ko chimeras showed minor infiltration of macrophages within the CD11b⁺CD45⁺ population (approx. 2%) accompanied by a reduced proportion of CD45^high cells. Interestingly, the contribution of macrophages to CD45^high cells differed extremely. In controls, macrophages accounted for 55% of the CD45^high population. The CD45^high fraction in wt/ko chimeras consisted of below 7% of macrophages. Indeed, we detected mainly microglia on tumor sections. The ko/wt group showed up to 83% of macrophages within the CD45^{high population}. Nevertheless, brain sections indicated accumulation of macrophages and microglia in glioma. Wt/ko as well as ko/wt chimeras tended to have larger tumor volumes than the control group.

Conclusion: For the first time, we report a recruitment mechanism for macrophages in gliomas. We demonstrate a strictly CCR2-dependent infiltration of macrophages. In contrast, microglia accumulate independently of CCR2. Chimeras with CCR2ko of either microglia or macrophages show larger tumor volumes than control group, confirming the importance of the CCR2-CCL2 signaling in glioma growth. Finally, the predominance of microglia in wt/ko chimeric mice offer a great tool for analyzing the specific behavior of microglia during tumor progression.