Yanysheva E P, Melnikov P A, Chudakova D A, Shirmanova M V, Baklaushev V P, Yusubalieva G M
Junior Researcher, Laboratory of Solid Tumor Immunotherapy; Federal Center of Brain Research and Neurotechnologies of the Federal Medical Biological Agency of Russia, 1, Bldg. 10, Ostrovityanova St., Moscow, 117513, Russia; Junior Researcher, Laboratory of Cell Technologies; Federal Scientific and Clinical Center for Specialized Types of Medical Care and Medical Technologies of the Federal Medical Biological Agency of Russia, 28 Orekhovy Blvd., Moscow, 115682, Russia.
PhD, Researcher, Laboratory of Neurobiology; Serbsky Federal Medical Research Centre of Psychiatry and Narcology, 23 Kropotkinsky Lane, Moscow, 119034, Russia.
Sovrem Tekhnologii Med. 2025;17(1):70-78. doi: 10.17691/stm2025.17.1.07. Epub 2025 Feb 28.
Glioblastoma is the most aggressive primary brain tumor with poor prognosis characterized by resistance to standard treatments and immune evasion. Regulatory T lymphocytes (Tregs) play a key role in immune suppression in the tumor microenvironment and can be used as targets for malignant gliomas therapy. is to study migration of Tregs to the tumor site in the process of dynamic glioblastoma growth on the transgenic C57Bl/6-FoxP3-eGFP mouse line.
The study was performed using the C57Bl/6-FoxP3-eGFP mouse strain, which allows for the detection of FoxP3-positive Tregs by fluorescent signal. Orthotopic glioblastomas were implanted by stereotactic injection of fluorescently labeled GL-261-BFP and GL-261-mScarlet tumor cell lines. Intravital confocal microscopy was used to monitor infiltration of the tumor site by immune cells, visualized by intravenous injection of fluorescently labeled antibodies against CD45. The results of intravital microscopy were confirmed by histological and immunohistochemical examination on days 3, 6, 9, 14, and 16 after the implantation. To assess the immunological status, tumor-infiltrating lymphocytes (TILs) were isolated from the brain and Tregs were counted using a flow cytometer (immediately after isolation and after cultivation for 2 weeks).
Intravital microscopy and brain slice studies have demonstrated infiltration of the glioblastoma site by Tregs, with the proportion of Tregs increasing with tumor progression (the increase in the absolute number of Treg was proportional to the increase in the number of glioma cells). Subsequent co-cultivation of isolated TILs with glioma cells revealed increase of Treg population within 2 weeks from 2.8% to >40%, confirming the activating effect of glioblastoma with respect to Tregs.
The dynamics of GL-261 glioma microenvironment infiltration by Tregs has been investigated. The glioblastoma cells were shown to activate Tregs in the peritumor space and to promote their selective expansion when co-cultured with TILs . These data can be used for further studies on C57Bl/6-FoxP3-eGFP mice to find approaches to inactivate Tregs in glioblastoma.
胶质母细胞瘤是最具侵袭性的原发性脑肿瘤，预后较差，其特征是对标准治疗有抗性且能逃避免疫。调节性T淋巴细胞（Tregs）在肿瘤微环境的免疫抑制中起关键作用，可作为恶性胶质瘤治疗的靶点。本研究旨在利用转基因C57Bl/6-FoxP3-eGFP小鼠模型，研究胶质母细胞瘤动态生长过程中Tregs向肿瘤部位的迁移情况。
本研究使用C57Bl/6-FoxP3-eGFP小鼠品系，该品系可通过荧光信号检测FoxP3阳性Tregs。通过立体定向注射荧光标记的GL-261-BFP和GL-261-mScarlet肿瘤细胞系，植入原位胶质母细胞瘤。采用活体共聚焦显微镜监测免疫细胞对肿瘤部位的浸润情况，通过静脉注射荧光标记的抗CD45抗体使其可视化。在植入后第3、6、9、14和16天，通过组织学和免疫组化检查对活体显微镜检查结果进行确认。为评估免疫状态，从脑中分离肿瘤浸润淋巴细胞（TILs），并使用流式细胞仪对Tregs进行计数（分离后立即及培养2周后）。
活体显微镜检查和脑切片研究表明，Tregs浸润胶质母细胞瘤部位，且Tregs比例随肿瘤进展而增加（Treg绝对数量的增加与胶质瘤细胞数量的增加成比例）。随后将分离的TILs与胶质瘤细胞共培养，结果显示Treg群体在2周内从2.8%增加到>40%，证实了胶质母细胞瘤对Tregs的激活作用。
研究了Tregs对GL-261胶质瘤微环境的浸润动态。结果表明，胶质母细胞瘤细胞可激活肿瘤周围空间的Tregs，并在与TILs共培养时促进其选择性扩增。这些数据可用于对C57Bl/6-FoxP3-eGFP小鼠的进一步研究，以寻找使胶质母细胞瘤中Tregs失活的方法。
