Giannattasio Ariane, Weil Sandra, Kloess Stephan, Ansari Nariman, Stelzer Ernst H K, Cerwenka Adelheid, Steinle Alexander, Koehl Ulrike, Koch Joachim
NK Cell Biology, Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt, Germany.
Institute for Cellular therapeutics, IFB-Tx, Hannover Medical School, Hannover, Germany.
BMC Cancer. 2015 May 3;15:351. doi: 10.1186/s12885-015-1321-y.
The complex cellular networks within tumors, the cytokine milieu, and tumor immune escape mechanisms affecting infiltration and anti-tumor activity of immune cells are of great interest to understand tumor formation and to decipher novel access points for cancer therapy. However, cellular in vitro assays, which rely on monolayer cultures of mammalian cell lines, neglect the three-dimensional architecture of a tumor, thus limiting their validity for the in vivo situation.
Three-dimensional in vivo-like tumor spheroid were established from human cervical carcinoma cell lines as proof of concept to investigate infiltration and cytotoxicity of NK cells in a 96-well plate format, which is applicable for high-throughput screening. Tumor spheroids were monitored for NK cell infiltration and cytotoxicity by flow cytometry. Infiltrated NK cells, could be recovered by magnetic cell separation.
The tumor spheroids were stable over several days with minor alterations in phenotypic appearance. The tumor spheroids expressed high levels of cellular ligands for the natural killer (NK) group 2D receptor (NKG2D), mediating spheroid destruction by primary human NK cells. Interestingly, destruction of a three-dimensional tumor spheroid took much longer when compared to the parental monolayer cultures. Moreover, destruction of tumor spheroids was accompanied by infiltration of a fraction of NK cells, which could be recovered at high purity.
Tumor spheroids represent a versatile in vivo-like model system to study cytotoxicity and infiltration of immune cells in high-throughput screening. This system might proof useful for the investigation of the modulatory potential of soluble factors and cells of the tumor microenvironment on immune cell activity as well as profiling of patient-/donor-derived immune cells to personalize cellular immunotherapy.
肿瘤内部复杂的细胞网络、细胞因子环境以及影响免疫细胞浸润和抗肿瘤活性的肿瘤免疫逃逸机制，对于理解肿瘤形成和破解癌症治疗的新切入点具有重要意义。然而，依赖哺乳动物细胞系单层培养的细胞体外试验忽略了肿瘤的三维结构，因此限制了其在体内情况的有效性。
从人宫颈癌细胞系建立三维体内样肿瘤球体作为概念验证，以研究96孔板形式下自然杀伤细胞（NK细胞）的浸润和细胞毒性，该形式适用于高通量筛选。通过流式细胞术监测肿瘤球体的NK细胞浸润和细胞毒性。浸润的NK细胞可通过磁性细胞分离回收。
肿瘤球体在数天内保持稳定，表型外观变化较小。肿瘤球体表达高水平的自然杀伤（NK）细胞2D受体（NKG2D）的细胞配体，介导原代人NK细胞对球体的破坏。有趣的是，与亲代单层培养相比，三维肿瘤球体的破坏所需时间长得多。此外，肿瘤球体的破坏伴随着一部分NK细胞的浸润，这些细胞可被高纯度回收。
肿瘤球体代表了一种通用的体内样模型系统，可用于在高通量筛选中研究免疫细胞的细胞毒性和浸润。该系统可能有助于研究肿瘤微环境中的可溶性因子和细胞对免疫细胞活性的调节潜力，以及对患者/供体来源的免疫细胞进行分析以实现细胞免疫治疗的个性化。
