Intravital 2-photon imaging reveals distinct morphology and infiltrative properties of glioblastoma-associated macrophages

Abstract

Characterized by a dismal survival rate and limited response to therapy, glioblastoma (GBM) remains one of the most aggressive human malignancies. Recent studies of the role of tumor-associated macrophages (TAMs) in the progression of GBMs have demonstrated that TAMs are significant contributors to tumor growth, invasion, and therapeutic resistance. TAMs, which include brain-resident microglia and circulating bone marrow derived-monocytes (BMDMs), are typically grouped together in histopathological and molecular analyses due to the lack of reliable markers of distinction. To develop more effective therapies aimed at specific TAM populations, we must first understand how these cells differ both morphologically and behaviorally. Furthermore, we must develop a deeper understanding of the mechanisms encouraging their infiltration and how these mechanisms can be therapeutically exploited. In this study, we combined immunocompetent lineage tracing mouse models of GBM with high-resolution open-skull 2-photon microscopy to investigate the phenotypical and functional characteristics of TAMs. We demonstrate that TAMs are composed of 2 morphologically distinct cell types that have differential migratory propensities. We show that BMDMs are smaller, minimally branched cells that are highly migratory compared with microglia, which are larger, highly branched stationary cells. In addition, 2 populations of monocytic macrophages were observed that differed in terms of CX3CR1 expression and migratory capacity. Finally, we demonstrate the efficacy of anti-vascular endothelial growth factor A blockade for prohibiting TAM infiltration, especially against BMDMs. Taken together, our data clearly define characteristics of individual TAM populations and suggest that combination therapy with antivascular and antichemotaxis therapy may be an attractive option for treating these tumors.

Ontogeny of Tumor-Associated Macrophages

“…Functional differences might be linked with the differential cues from the TME that polarize the macrophages in a niche-specific manner in addition to their ontogeny-specific features. Live imaging studies represent a complementary approach to compare functional difference between TAM subsets as reported in the lungs (37) and recently in the brain (62). Further studies using fluorescent strains and lineage-tracing models (63) will be necessary to better address the functional features of TAM subsets to better understand their role in tumor development as well as resistance to anti-cancer therapies and unveil key target for immunotherapy.…”

Human Mesenchymal glioblastomas are characterized by an increased immune cell presence compared to Proneural and Classical tumors

“…For more comprehensive analyses and depictions of microglial processes, we refer to previously published work. [42][43][44][45] We measured the number of primary processes that extend from TAM, since lower numbers are associated with activation. 46 The highest number of processes were noted in adjacent tumor-free brain tissue, with an average of 2.2 primary processes per cell.…”

Comparison of the effect of culture medium on unfixed rat pancreatic tissue in two-photon excitation microscopy

“…In RPMI 1640 and HEPES, the blue and green fluorescence intensities were stronger at short wavelengths than at long wavelengths, and the red fluorescence intensity was stronger at long wavelengths than at short wavelengths. An advantage of 2PM over traditional confocal microscopy is increased imaging depth [10]. We analyzed images of pancreatic tissues at different depths.…”

“…The advantages of 2PM over traditional confocal microscopy include reduced AF and photobleaching effects, increased imaging depth, and minimal photodamage to the surrounding tissues [10,11]. Confocal microscopy provides depth discrimination and improves spatial resolution within the plane of focus by forming the image through the same scanning optics used for illumination and through a pinhole placed in front of the detector, which acts as a spatial filter to select emission from the plane of focus [12,13].…”