Lin Yuan-Na, Nasir Apsra, Camacho Sharon, Berry Deborah L, Schmidt Marcel O, Pearson Gray W, Riegel Anna T, Wellstein Anton
Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University.
Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University;
J Vis Exp. 2020 Jun 23(160). doi: 10.3791/61392.
Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells. Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.
免疫疗法在癌症治疗方面取得了重大进展，尽管仍有大量癌症对治疗具有抗性。仅有有限的几种检测方法能够直接监测肿瘤细胞与免疫细胞之间的相互作用并深入了解其机制，其中，T细胞在执行适应性免疫系统对癌细胞的细胞毒性反应中发挥着重要作用。由于使用相对简便，大多数检测方法基于细胞的二维（2D）共培养，但癌细胞的侵袭性生长表型（癌细胞的特征之一）的呈现有限。当前的三维（3D）共培养系统要么需要特殊设备，要么需要对共培养的癌细胞和相互作用的T细胞的侵袭进行单独监测。在此，我们描述了一种同时监测癌细胞球体在3D环境中的侵袭行为以及共培养中T细胞细胞毒性的方法。球体形成是由具有U形底部的无支架琼脂糖微孔铸型中增强的细胞间相互作用驱动的。T细胞共培养和癌细胞向I型胶原基质中的侵袭均在琼脂糖铸型的微孔内进行，无需转移细胞，从而在整个检测过程中维持完整的3D共培养系统。胶原基质可与琼脂糖铸型分离，便于进行免疫荧光（IF）染色和细胞的共聚焦成像。此外，细胞可被分离用于进一步培养，或进行诸如基因表达分析或荧光激活细胞分选（FACS）等分析。最后，3D共培养物在包埋和切片后可通过免疫组织化学（IHC）进行分析。该检测方法的可能改进包括改变细胞外基质（ECM）的组成，以及在癌细胞中加入不同的基质细胞或免疫细胞。
