Stem Cell and Neurotherapies Laboratory, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.
Department of Neurosurgery, Salford Royal Hospital, Salford, UK.
Neuropathol Appl Neurobiol. 2019 Feb;45(2):119-140. doi: 10.1111/nan.12489. Epub 2018 May 17.
Resident and peripherally derived glioma associated microglia/macrophages (GAMM) play a key role in driving tumour progression, angiogenesis, invasion and attenuating host immune responses. Differentiating these cells' origins is challenging and current preclinical models such as irradiation-based adoptive transfer, parabiosis and transgenic mice have limitations. We aimed to develop a novel nonmyeloablative transplantation (NMT) mouse model that permits high levels of peripheral chimerism without blood-brain barrier (BBB) damage or brain infiltration prior to tumour implantation.
NMT dosing was determined in C57BL/6J or Pep3/CD45.1 mice conditioned with concentrations of busulfan ranging from 25 mg/kg to 125 mg/kg. Donor haematopoietic cells labelled with eGFP or CD45.2 were injected via tail vein. Donor chimerism was measured in peripheral blood, bone marrow and spleen using flow cytometry. BBB integrity was assessed with anti-IgG and anti-fibrinogen antibodies. Immunocompetent chimerised animals were orthotopically implanted with murine glioma GL-261 cells. Central and peripheral cell contributions were assessed using immunohistochemistry and flow cytometry. GAMM subpopulation analysis of peripheral cells was performed using Ly6C/MHCII/MerTK/CD64.
NMT achieves >80% haematopoietic chimerism by 12 weeks without BBB damage and normal life span. Bone marrow derived cells (BMDC) and peripheral macrophages accounted for approximately 45% of the GAMM population in GL-261 implanted tumours. Existing markers such as CD45 high/low proved inaccurate to determine central and peripheral populations while Ly6C/MHCII/MerTK/CD64 reliably differentiated GAMM subpopulations in chimerised and unchimerised mice.
NMT is a powerful method for dissecting tumour microglia and macrophage subpopulations and can guide further investigation of BMDC subsets in glioma and neuro-inflammatory diseases.
驻留和外周衍生的神经胶质瘤相关的小胶质细胞/巨噬细胞（GAMM）在驱动肿瘤进展、血管生成、侵袭和削弱宿主免疫反应方面发挥着关键作用。区分这些细胞的来源具有挑战性，目前的临床前模型，如基于辐射的过继转移、联体共生和转基因小鼠存在局限性。我们旨在开发一种新的非清髓性移植（NMT）小鼠模型，该模型允许在肿瘤植入前，在外周嵌合率不损害血脑屏障（BBB）或脑浸润的情况下，实现高水平的外周嵌合。
在接受从 25mg/kg 到 125mg/kg 浓度的白消安预处理的 C57BL/6J 或 Pep3/CD45.1 小鼠中确定 NMT 剂量。通过尾静脉注射标记有 eGFP 或 CD45.2 的供体造血细胞。使用流式细胞术在外周血、骨髓和脾脏中测量供体嵌合率。用抗 IgG 和抗纤维蛋白原抗体评估 BBB 完整性。免疫功能正常的嵌合动物被原位植入小鼠神经胶质瘤 GL-261 细胞。使用免疫组织化学和流式细胞术评估中枢和外周细胞的贡献。使用 Ly6C/MHCII/MerTK/CD64 对外周细胞的 GAMM 亚群进行分析。
NMT 在 12 周内实现了>80%的造血嵌合率，而没有 BBB 损伤和正常的寿命。骨髓来源的细胞（BMDC）和外周巨噬细胞约占 GL-261 植入肿瘤中 GAMM 群体的 45%。现有的标记物，如 CD45 高/低，证明不准确，以确定中枢和外周群体，而 Ly6C/MHCII/MerTK/CD64 可靠地区分嵌合和非嵌合小鼠中的 GAMM 亚群。
NMT 是一种用于剖析肿瘤小胶质细胞和巨噬细胞亚群的强大方法，并可以指导对神经胶质瘤和神经炎症性疾病中 BMDC 亚群的进一步研究。
