5-ALA Fluorescence-guided Biopsies Reveal Different Myeloid Phenotypes at the Core and Margin of Glioblastoma, and Their Responses to Topotecan

Introduction: Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Intraoperative fluorescence imaging with 5-aminolevulinic acid (5-ALA) aids in surgical resection by labeling the highly cellular tumor core. However, the infiltrative margin of GBM is non-fluorescent, or weakly-fluorescent, and therefore evades detection with 5-ALA. Tissue at the margin hosts a mixture of neoplastic and nonneoplastic cells, including microglia and macrophages, which form the biological context for adjuvant therapy and tumor recurrence. This study examined myeloid cell phenotypes in both the tumor core and the infiltrative margin and assessed their responses to glioma-targeted therapies.

Methods: MRI-guided biopsies were obtained from primary GBM patients, sampling both the contrast enhancing tumor core and non-contrast enhancing infiltrative margin. Tissue slices were cultured ex vivo and treated with PBS (control) or Topotecan, an anti-proliferative chemotherapy. Immunofluorescence staining quantified Iba1, a pan-myeloid marker, and P2RY12, a quiescent microglial marker. Whole-slide 20X images were analyzed with QuPath and statistical significance was assessed using one-way ANOVA and post-hoc t-tests.

Results: The abundance of Iba1-positive myeloid cells was consistent between the tumor core and infiltrative margin. However, the 5-ALA-positive tumor core and 5-ALA-negative margin showed a significant reduction in resting microglial phenotype (P2RY12-positive cells) compared to the 5-ALA-negative margin (p=0.006). Topotecan treatment further reduced P2RY12-positive cells in the tumor core (p=0.03).

Conclusion : This study identified significant differences in the myeloid populations in the 5-ALA-positive core and 5-ALA-negative margin of GBM. Notably, P2RY12-positive microglia are more abundant at the tumor margin, but are depleted by Topotecan, revealing that cytotoxic therapies modulate the tumor microenvironment beyond their direct effects on glioma cells. Ongoing studies are investigating mechanisms of microglial/myeloid response to chemotherapy and the potential effects on tumor progression.