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Background: 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.
Methods: 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.
Results: 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.
Conclusion: 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.
Natural killer (NK) cells are a promising tool for the use in adoptive immunotherapy, since they efficiently recognize and kill tumor cells. In this context, ex vivo cultivation is an attractive option to increase NK cells in numbers and to improve their antitumor potential prior to clinical applications. Consequently, various strategies to generate NK cells for adoptive immunotherapy have been developed. Here, we give an overview of different NK cell cultivation approaches and their impact on shaping the NK cell antitumor activity. So far, the cytokines interleukin (IL)-2, IL-12, IL-15, IL-18, and IL-21 are used to culture and expand NK cells. The selection of the respective cytokine combination is an important factor that directly affects NK cell maturation, proliferation, survival, distribution of NK cell subpopulations, activation, and function in terms of cytokine production and cytotoxic potential. Importantly, cytokines can upregulate the expression of certain activating receptors on NK cells, thereby increasing their responsiveness against tumor cells that express the corresponding ligands. Apart from using cytokines, cocultivation with autologous accessory non-NK cells or addition of growth-inactivated feeder cells are approaches for NK cell cultivation with pronounced effects on NK cell activation and expansion. Furthermore, ex vivo cultivation was reported to prime NK cells for the killing of tumor cells that were previously resistant to NK cell attack. In general, NK cells become frequently dysfunctional in cancer patients, for instance, by downregulation of NK cell activating receptors, disabling them in their antitumor response. In such scenario, ex vivo cultivation can be helpful to arm NK cells with enhanced antitumor properties to overcome immunosuppression. In this review, we summarize the current knowledge on NK cell modulation by different ex vivo cultivation strategies focused on increasing NK cytotoxicity for clinical application in malignant diseases. Moreover, we critically discuss the technical and regulatory aspects and challenges underlying NK cell based therapeutic approaches in the clinics.