CELL INTELLIGENCE · 肿瘤细胞治疗研究
肿瘤细胞治疗研究
英文原题：A High-Throughput, Three-Dimensional Multiple Myeloma Model Recapitulating Tumor-Stroma Interactions for CAR-Immune Cell-Mediated Cytotoxicity Assay.
A High-Throughput, Three-Dimensional Multiple Myeloma Model Recapitulating Tumor-Stroma Interactions for CAR-Immune Cell-Mediated Cytotoxicity Assay.
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多发性骨髓瘤（MM）的特征是骨髓（BM）中克隆性浆细胞过度增殖。骨髓生态位成分会促成免疫抑制性肿瘤微环境（TME），但目前仍缺少能重现复杂 TME、并适用于嵌合抗原受体（CAR）工程化免疫细胞高通量细胞毒性检测的三维（3D）MM 模型。
研究者利用荧光素酶/红色荧光蛋白（Luc/RFP）双报告系统制备稳定表达荧光素酶（Luc）的靶向 MM 细胞，以追踪 MM 生长。在 96 孔板中构建 3D 球状体，可含或不含癌相关成纤维细胞（CAF）样基质细胞；后者由 MM 来源条件培养液激活。通过多功能微孔板读数仪进行荧光素酶检测，评估第三代抗 CD138 CAR-NK-92 细胞的细胞毒性；使用多种荧光染料并在荧光显微镜下观察免疫细胞浸润。
研究首先表明，荧光素酶检测可相对简便且稳健地特异性监测共培养体系中 Luc 标记肿瘤细胞的生长，从而实现 CAR 免疫细胞细胞毒性的高通量评估。通过该检测发现，CAF 样基质细胞可在二维培养和 3D 球状体中损害 NK 细胞效应功能，可能通过旁分泌信号和物理屏障作用实现。重要的是，MM 细胞与 CAF 样基质细胞组成的 3D 球状体，提供了更全面且具生理相关性的肿瘤免疫学模型。该模型也可用于研究免疫细胞向球状体核心的迁移和浸润。研究还显示，加入 CAR 确实提高了 NK 细胞浸润 3D 球状体的能力。
所建立的 3D 球状体模型部分重现了具有免疫抑制环境的复杂 TME，适用于 CAR 免疫细胞细胞毒性的高通量筛选。鉴于 MM 领域迫切需要创新 CAR 免疫细胞，该模型可能对加快肿瘤免疫药物发现具有重要意义。
Multiple myeloma (MM) is characterized by an excessive proliferation of clonal plasma cells in the bone marrow (BM). Components in BM niche contribute to the immunosuppressive tumor microenvironment (TME), but three-dimensional (3D) MM models that recreate the complex TME and enable high-throughput cytotoxicity assay of chimeric antigen receptor (CAR)-engineered immune cells are still lacking.
Stable, luciferase (Luc)-labeled target MM cells were generated using Luc/RFP dual reporter system to track MM growth. 3D spheroids were formed in a 96-well plate in the presence or absence of cancer-associated fibroblast (CAF)-like stromal cells activated by MM-derived conditioned medium and the cytotoxicity of CAR-immune cells, which were represented by third-generation anti-CD138 CAR-NK-92 cells, was evaluated by luciferase assay using a multimode microplate reader. Immune cell infiltration was visualized under a fluorescence microscope by using multiple fluorescent dyes.
We first showed that luciferase assay provides a relatively simple and robust means to specifically monitor Luc-labeled tumor cell growth in a coculture system, allowing the high-throughput assessment of CAR-immune cytotoxicity. Through this assay, we demonstrated that CAF-like stromal cells impaired NK cell effector function in 2D culture and 3D spheroids, likely via paracrine signaling and physical barrier function. Importantly, we showed that 3D spheroids consisting of MM cells and CAF-like stromal cells provide a more comprehensive, physiologically relevant immuno-oncology model. Our established model could also be used to investigate the trafficking and infiltration of immune cells into the core of spheroids. Herein, we showed that CAR incorporation did improve the ability of NK cells to infiltrate 3D spheroids.
Our established 3D spheroid model, which partially recapitulates the complex TME with immunosuppressive environment, is suitable for high-throughput screening of CAR-immune cytotoxicity and could be important in accelerating immuno-oncology drug discovery for MM since there is a pressing need to establish innovative CAR-immune cells.
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