Work

IMMU-50. A NOVEL CHIMERIC MODEL TO ACCURATELY IDENTIFY TAMMs IN GLIOBLASTOMA

Year: 2017

Type: article

Abstract: Glioblastoma is the most aggressive primary brain cancer with poor survival, but treatment strategies to improve prognosis have failed to materialise in over 30-years. Immune cells, in particular Tumour associated macrophages and microglia (TAMM) populate up to 40% of tumour bulk and are potential immunotherapy targets. However, functional roles of TAMM subpopulations still remain elusive and contradictory, due to limitations of existing mouse models and non-specific cell-surface markers used in human and rodent studies. To address these issues, we developed non-myeloablative chemotherapy conditioning regimen to transplant host mice expressing pan-leukocyte epitope CD45.1 with haematopoietic donor cells expressing congenic CD45.2, whilst retaining an intact recipient blood-brain barrier. Once 90% bone marrow chimerism was achieved at 12 weeks, we intracranially implanted GL261 murine glioblastoma cells into chimeric mice, recapitulating normal tumour development. Chimerism was used to overcome the bias of cell surface markers and reliably determine the origin of TAMM subpopulations. Immunofluorescence and multicolour hemisphere flow cytometry was used to accurately distinguish between peripheral macrophages and microglia. We show not only TAMMs, but multiple immune populations migrating to tumours and highlight marker sets such as CD11b/CD45 and Siglec-H/CX3CR1 ordinarily used to distinguish between microglia and macrophages to have significant co-staining from other leukocyte populations. Furthermore, TAMM microglia and macrophages are morphologically indistinguishable. To solve this problem, we identify a novel 4-marker set that separates TAMM into four subpopulations with ≥ 90% donor/recipient chimerism purity and succinctly illustrate the peripheral monocyte-macrophage differentiation axis. Overall, we have developed a novel chimeric model that preserves homeostatic brain microenvironment and achieves superior chimerism levels. Our results also emphasise a new 4-marker approach to subcategorising TAMM in glioblastoma. Applying this marker set for RNA sequencing of TAMM subpopulations and translating markers to human specimens will validate findings from an experimental glioblastoma model.

Source: Neuro-Oncology

DOI: 10.1093/neuonc/nox168.508