Cytotoxicity and infiltration of human NK cells in in vivo-like tumor spheroids.

Ariane Giannattasio,Stephan Kloess,Ernst H K Stelzer,Nariman Ansari,Joachim Koch,Sandra Weil,Adelheid Cerwenka,Ulrike Koehl,Alexander Steinle

doi:10.1186/s12885-015-1321-y

Abstract

BackgroundThe 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.MethodsThree-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.ResultsThe 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.ConclusionTumor 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1321-y) contains supplementary material, which is available to authorized users.

Highlights

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
Generation of tumor spheroid arrays In order to investigate Natural killer (NK) cell cytotoxicity against cervical carcinoma, the cervical carcinoma cell lines CaSki and SiHa were tested for their ability to form solid tumor spheroids (Figure 1B)
We examined the plasma membrane expression of the stress-induced tumor antigens MHC class I chain-related protein A (MICA), MICB, ULBP1, ULBP2 and ULBP3 on the tumor spheroids for comparison with the expression levels found in the corresponding parental monolayer cultures (Figure 2A, Additional file 2: Figure S1, representative histograms Additional file 2: Figure S1B)

Summary

Introduction

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. Natural killer (NK) cells rapidly recognize and destroy malignantly transformed cells [1,2,3]. Human NKG2D recognizes several structurally related ligands (NKG2DLs) on tumor cells from various cytological origin, including the MHC class I chain-related protein A (MICA), MICB, and the UL16-binding proteins (ULBPs) [15,17,18]. Besides their role as tumor antigens, the ectodomains of the NKG2DLs can be shed from the plasma membrane of malignantly transformed cells and subsequently inhibit NKG2D-dependent NK cell cytotoxicity [19,20]. NK cell cytotoxicity might be modulated by cytokines, the cellular crosstalk with tumor-associated cells and tumor immune escape mechanisms within the tumor microenvironment [26]

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Discussion

Conclusion