Phenotypic dynamics of microglial and monocyte-derived cells in glioblastoma-bearing mice

Abstract

Inflammatory cells, an integral component of tumor evolution, are present in Glioblastomas multiforme (GBM). To address the cellular basis and dynamics of the inflammatory microenvironment in GBM, we established an orthotopic syngenic model by grafting GL261-DsRed cells in immunocompetent transgenic LysM-EGFP//CD11c-EYFP reporter mice. We combined dynamic spectral two-photon imaging with multiparametric cytometry and multicolor immunostaining to characterize spatio-temporal distribution, morphology and activity of microglia and blood-derived infiltrating myeloid cells in live mice. Early stages of tumor development were dominated by microglial EYFP+ cells invading the tumor, followed by massive recruitment of circulating LysM-EGFP+ cells. Fluorescent invading cells were conventional XCR1+ and monocyte-derived dendritic cells distributed in subpopulations of different maturation stages, located in different areas relative to the tumor core. The lethal stage of the disease was characterized by the progressive accumulation of EGFP+/EYFP+ monocyte-derived dendritic cells. This local phenotypic regulation of monocyte subtypes marked a transition in the immune response.

Revealing and Harnessing Tumour-Associated Microglia/Macrophage Heterogeneity in Glioblastoma

“…However, under certain pathological conditions, such as in GBM, the local inflammatory environment can compromise the integrity of the blood brain barrier leading to the infiltration of inflammatory monocytes from the circulation, which subsequently differentiate into monocyte-derived macrophages once they enter the brain tissue [24]. Therefore, in GBM, tumour-associated macrophages encompass resident parenchymal microglia, perivascular macrophages and peripheral monocyte-derived cells [25]. As a general observation, although tumour-associated macrophage proportions may vary in an organ-dependent manner, they have emerged as one of the most critical cell types contributing to worse prognosis across the vast majority of cancers [26].…”

Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells

“…, EGFP or DsRed). Blue-emitting dyes such as Cascade Blue have been successfully used in intravital studies and can efficiently be excited by TPM (Ricard and Debarbieux 2014; Ricard et al 2016b). It must be considered, however, that in living tissues the maximum imaging depth is a function of the wavelengths of both the excitation and fluorescence beam.…”

“…Intravenous injection of fluorescent probes can also be used to determine the acidity of tissues using pH indicators ( cf. Table 4), or to measure physiological parameters such as cerebral blood flow (Chaigneau et al 2003) and blood-brain-barrier permeability (Ricard et al 2009) in different pathologies, including vascular occlusion (Schaffer et al 2006) and brain tumors (Ricard et al 2013b, 2016b). Investigating the properties of the blood vessel tree enables the assessment of side effects of treatments in preclinical trials (Ricard et al 2013a).…”

“…For example, using Thy1- CFP /LysM- GFP /CD11c- EYFP triple transgenic mice intravenously injected with Rhodamine B dextran or Quantum-dots 655, Fenrich et al have revealed a differential spatiotemporal recruitment of myelomonocytic cells after a spinal cord injury (Fenrich et al 2013). Recently, Ricard et al have described the combination of a LysM- EGFP /CD11c- EYFP mouse implanted with DsRed -expressing glioblastoma cells and intravenously injected with Cascade Blue dextran in order to study the recruitment of immune cells during glioblastoma progression (Ricard et al 2016b). …”

“…The laser power should be kept as low as possible and the experiment duration as short as possible, to limit photobleaching and phototoxicity. This is essential for multicolor timelapse experiments (Fenrich et al 2013; Ricard et al 2016b), in which the same region is scanned over and over, at the risk of exposing the tissue to an energy that would threaten its health and/or integrity. When different excitation wavelengths are required (for example, when both Cascade Blue with λ ex = 800 nm and eGFP with λ ex = 940 nm are present in the same biological sample), a sequential acquisition or the simultaneous use of two Ti:Sapphire femtosecond lasers is required.…”