BACKGROUND Glioblastoma (GBM), the most malignant primary brain tumor with a median survival less than two years, is characterized by aggressive invasive growth, presence of hypoxic and necrotic regions, and T-cell dysfunction. Mesenchymal cells with characteristics of cancer associated fibroblasts are present in GBMs, but it remains to be established whether they contribute to T cell sequestration in the tumor stroma and functional inactivation similarly to extracranial tumors. Murine models do not reflect several aspects of human immune system and 2D cell culture systems often fail to recapitulate complex interactions in the tumor microenvironment. 3D spheroids prepared from patient-derived cell cultures offer higher degree of complexity and biological relevance. The aim of this study was to establish a 3D GBM spheroid model to determine the effect of patient derived mesenchymal cells on human T cell infiltration and phenotype. MATERIAL AND METHODS Glioma stem-like cell and mesenchymal cell cultures were derived from human GBMs. Naïve T lymphocytes were isolated from buffy coats of healthy donors by Ficoll paque gradient separation, followed by negative magnetic isolation techniques using Naïve Pan T Cell Isolation Kit (Miltenyi Biotec). 3D spheroids were prepared by embedding the cells in 20 µl of Geltrex (Thermo Fisher Scientific). T cells activated by magnetic beads with anti-CD2, anti-CD3 and anti-CD28 antibodies (Miltenyi Biotec) and IL-2 were subsequently added to the spheroids. After 5 days of co-culture, spheroids were dissociated, and viable cells were counted using automated cell counter (Biorad). Expression of a T-cell activation marker CD69 and immune checkpoint molecules LAG3, and PD-1 in CD3+ cells was analyzed by flow cytometry. Mesenchymal, stem cell and lymphocyte markers TE-7, Nestin and CD45 respectively, were analyzed by immunohistochemistry (IHC) on frozen spheroid sections. RESULTS We prepared spheroids comprising glioma stem-like cells with and without mesenchymal cells. Glioblastoma-derived cells were evenly dispersed throughout the spheroids as demonstrated by IHC. T cells infiltrated into both types of spheroids, but their number in spheroids containing mesenchymal cells was about 40% (p=0.002) lower compared to the ones without mesenchymal cells. In both types of spheroids, the presence of activated allogeneic T cells led to more than a 50% reduction of the glioblastoma-derived cells. Ongoing studies evaluate the effect of mesenchymal cells on the phenotype of spheroid infiltrating T cells. CONCLUSION We have successfully established a 3D spheroid model to study the role of mesenchymal cells in GBM microenvironment. Our results indicate that mesenchymal cells may influence the infiltration of T cells and modulate their phenotype. SUPPORT Project was financed by the Center for Tumor Ecology (CZ.02.1.01/0.0/0.0/16_019/0000785).

中文翻译：

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P10.01 间充质细胞对 3D 胶质母细胞瘤球体模型中 T 细胞浸润和表型的影响

背景技术胶质母细胞瘤 (GBM) 是最恶性的原发性脑肿瘤，中位生存期少于两年，其特征是侵袭性生长、缺氧和坏死区域的存在以及 T 细胞功能障碍。具有癌症相关成纤维细胞特征的间充质细胞存在于 GBM 中，但它们是否有助于肿瘤基质中的 T 细胞隔离和类似于颅外肿瘤的功能失活仍有待确定。鼠模型不能反映人类免疫系统的几个方面，2D 细胞培养系统通常无法概括肿瘤微环境中的复杂相互作用。从患者来源的细胞培养物中制备的 3D 球体具有更高的复杂性和生物学相关性。本研究的目的是建立一个 3D GBM 球体模型，以确定患者来源的间充质细胞对人类 T 细胞浸润和表型的影响。材料和方法 胶质瘤干细胞和间充质细胞培养物来源于人 GBM。通过 Ficoll paque 梯度分离从健康供体的血沉棕黄层中分离幼稚 T 淋巴细胞，然后使用幼稚泛 T 细胞分离试剂盒 (Miltenyi Biotec) 进行负磁分离技术。通过将细胞嵌入 20 µl Geltrex (Thermo Fisher Scientific) 中制备 3D 球体。随后将由具有抗 CD2、抗 CD3 和抗 CD28 抗体 (Miltenyi Biotec) 和 IL-2 的磁珠激活的 T 细胞添加到球体中。共培养 5 天后，球体被解离，使用自动细胞计数器（Biorad）对活细胞进行计数。通过流式细胞术分析 T 细胞活化标志物 CD69 和免疫检查点分子 LAG3 和 PD-1 在 CD3+ 细胞中的表达。间充质、干细胞和淋巴细胞标志物 TE-7、Nestin 和 CD45 分别通过免疫组织化学 (IHC) 对冷冻球体切片进行分析。结果 我们制备了包含有和没有间充质细胞的胶质瘤干细胞样细胞的球体。如 IHC 所示，胶质母细胞瘤衍生细胞均匀分散在整个球体中。T 细胞浸润到两种类型的球体中，但与不含间充质细胞的球体相比，它们在含有间充质细胞的球体中的数量减少了约 40% (p=0.002)。在这两种类型的球体中，活化的同种异体 T 细胞的存在导致胶质母细胞瘤衍生细胞减少 50% 以上。正在进行的研究评估间充质细胞对球状浸润 T 细胞表型的影响。结论 我们成功建立了 3D 球体模型来研究间充质细胞在 GBM 微环境中的作用。我们的结果表明间充质细胞可能影响 T 细胞的浸润并调节它们的表型。SUPPORT 项目由肿瘤生态学中心 (CZ.02.1.01/0.0/0.0/16_019/0000785) 资助。我们的结果表明间充质细胞可能影响 T 细胞的浸润并调节它们的表型。SUPPORT 项目由肿瘤生态学中心 (CZ.02.1.01/0.0/0.0/16_019/0000785) 资助。我们的结果表明间充质细胞可能影响 T 细胞的浸润并调节它们的表型。SUPPORT 项目由肿瘤生态学中心 (CZ.02.1.01/0.0/0.0/16_019/0000785) 资助。