Tumor-associated myeloid cells assist glioblastoma invasion via plexin-B2

Introduction: Glioblastoma (GBM) is highly infiltrative, and reprograms microglia and macrophages into tumor-associated myeloid cells (TAMs) to support dissemination. However, its interaction with stromal cells, and the precise role of TAMs in mediating invasion, are unclear. Here, we retrace the early stages of GBM-TAM interaction using KR158, a highly-infiltrative murine GBM model with phenotypic resemblance to human GBM. We also elucidate a key role of the collision guidance receptor, plexin-B2, in this pathological process.

Methods: KR158 cells were intracranially transplanted into immunocompetent adult C57Bl/6 mice; tumors formed by 8 weeks. Myeloid-specific plexin-B2 conditional knockout C57Bl/6 mice were generated via tamoxifen-inducible CreER. Single-cell RNA sequencing (scRNA-seq) was performed using the 10X Genomics platform, and clustering analysis using the Seurat R package. The Ivy Glioblastoma Atlas Project database was used for gene expression profile analysis, and the NIH Glioma BioDiscovery Portal to match expression signatures to patient survival.

Results: TAMs intertwined with invading GBM cells exhibited altered orientation, morphology, and ECM deposition patterns, while microglia even far upfield showed activation. Plexin-B2 was upregulated in TAMs, and its deletion resulted in GBM-TAM cell misalignment, decreased invasion, and altered infiltration pattern. This phenotypic difference was paralleled by transcriptional changes demonstrated through scRNA-seq analysis, with GBM in PB2cKO associated with a three-fold decrease of the tumor cell infiltrating edge cluster, and a two-fold increase in the tumor bulk cluster. Gene analysis revealed expression profiles corresponding to distinct, region-specific tumor and TAM subpopulations. Additionally, these expression signatures were associated with poor patient survival.

Conclusion : TAM transformation occurs in distinct stages, beginning with widespread myeloid cell mobilization ahead of invasion (onco-field). When in proximity to migrating GBM cells, myeloid cells align to entrap nascent microinfiltrates (onco-nets). Finally, through physical contact, they transform and restructure ECM into invasion tracks (onco-streams). Plexin-B2 is central in this pathological remodeling; its deletion in TAMs was associated with reduced invasion and drastic alteration of tumor architecture. Understanding these mechanisms — and how they can be perturbed — may open up novel therapeutic avenues.