Clinical Neurosciences, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK.
Neuropathol Appl Neurobiol. 2013 Apr;39(3):243-55. doi: 10.1111/j.1365-2990.2012.01283.x.
Microglia form a high proportion of cells in glial tumours but their role in supporting or inhibiting tumour growth is unclear. Here we describe the establishment of an in vitro model to investigate their role in astrocytomas.
Rat hippocampal slices were prepared and, after 7 days to allow microglia to become quiescent, rat C6 astrocytic tumour cells were added. Over the following 7 days, infiltration and cell death were studied using fluorescent C6 tumour cells and confocal microscopy; immunophenotyping of microglia was performed using CD68 (phagocytosis), MHCII (antigen-presentation) and Iba1 (microglial marker regardless of functional state). Cell proliferation was assessed using Ki67 and qPCR to detect cytokine expression. Sham and control groups were included.
Microscopy showed proliferation of C6 tumour cells with both infiltration of tumour cells into the hippocampal tissue and of microglia among the tumour cells. Confocal experiments confirmed increasing tumour cell infiltration into the hippocampal slice with time (P<0.001), associated with cell death (σ=0.313, P=0.022). Ki67 showed increased proliferation (P<0.001), of both tumour cells and Iba1+ microglia and increased microglial phagocytosis (CD68: P<0.001). Expression of pro-inflammatory cytokines IL1, IL6 and TNFα were downregulated with expression of the anti-inflammatory cytokine TGFβ1 maintained.
This model allows study of the proliferation and infiltration of astrocytic tumour cells in central nervous system tissue and their interaction with microglia. Our data suggest that microglial function is altered in the presence of tumour cells, putatively facilitating tumour progression. Manipulation of the microglial functional state may have therapeutic value for astrocytic tumours.
小胶质细胞在神经胶质肿瘤中构成了很大比例的细胞，但它们在支持或抑制肿瘤生长方面的作用尚不清楚。本研究建立了一种体外模型来研究其在星形细胞瘤中的作用。
制备大鼠海马切片，在 7 天内让小胶质细胞静止，然后加入大鼠 C6 星形胶质细胞瘤细胞。在接下来的 7 天内，使用荧光 C6 肿瘤细胞和共聚焦显微镜研究浸润和细胞死亡；使用 CD68（吞噬作用）、MHCII（抗原呈递）和 Iba1（无论功能状态如何的小胶质细胞标志物）对小胶质细胞进行免疫表型鉴定。使用 Ki67 和 qPCR 评估细胞增殖以检测细胞因子表达。包括假手术组和对照组。
显微镜显示 C6 肿瘤细胞增殖，肿瘤细胞浸润海马组织，小胶质细胞浸润肿瘤细胞之间。共聚焦实验证实，随着时间的推移，肿瘤细胞不断向海马切片中渗透（P<0.001），并伴随着细胞死亡（σ=0.313，P=0.022）。Ki67 显示肿瘤细胞和 Iba1+小胶质细胞增殖增加（P<0.001），小胶质细胞吞噬作用增强（CD68：P<0.001）。促炎细胞因子 IL1、IL6 和 TNFα 的表达下调，抗炎细胞因子 TGFβ1 的表达维持。
该模型允许研究星形细胞瘤在中枢神经系统组织中的增殖和浸润及其与小胶质细胞的相互作用。我们的数据表明，肿瘤细胞存在时小胶质细胞的功能发生改变，可能促进肿瘤进展。对小胶质细胞功能状态的操纵可能对星形细胞瘤具有治疗价值。
