An emerging problem in the treatment of breast cancer is the increasing incidence of metastases to the brain years after diagnosis. Treatment options for brain metastases are mainly limited to radiotherapy and neurosurgery, so better understanding of this niche, and the cellular mechanisms involved, is urgently required. Microglia are the resident macrophage population in the brain, undertaking a surveillance role, and becoming “activated” by neuronal injury or lesion, promoting an inflammatory response. Microglia have been reported to promote proliferation, angiogenesis and invasion in brain tumours. However, the mechanisms underlying microglial involvement appear complex and better models are needed to study microglial activation in the context of brain metastases. Here, we sought to address this need by developing a novel protocol for visualisation of tumour cell-microglial interactions in vivo using multiphoton intravital imaging.
We used an inverted Zeiss 780MP 2-photon microscope with a purpose adapted LSM Tech InverterScope objective inverter modified for wavelengths >900 nm with the addition of a reflective interior and MaxMirror dielectric mirrors. We used heterozygous Cx3cr1GFP/+ mice which express GFP in microglia under control of the endogenous Cx3cr1 promoter. We evaluated implantation of an optical window on the parietal bone as the best method for examining cellular behaviour in situ in the outer cortex. We detected GFP-expressing microglia in Cx3cr1GFP/+ mice up to 250 µm below the window without significant loss of resolution. In a subset of animals, we implanted dsRed-expressing MDA-MB-231 breast cancer cells suspended in Matrigel under the optical window. Using this approach, we visualised accumulation of activated microglia and tumour cells up to 6 days following implantation. We propose that this novel in vivo approach may have use for the interrogation of molecular mechanisms underlying microglial involvement in the progression of brain metastases.
This work was supported by Medical Research Council and the Wellcome Trust through the Centre for Chronic Diseases and Disorders (C2D2) at the University of York.
|Tumour Microenvironment, Basic Science To Novel Therapies
|15/06/17 → 16/06/17