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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.
Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair.
Objective: To determine the role of the AMPKα2 subunit in vascular repair.
Methods and Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2-/- versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2ΔMC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPKα2ΔMC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPKα2ΔMC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPKα2ΔMC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1α induction was attenuated in AMPKα2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPKα2ΔMC mice. Mechanistically, isocitrate dehydrogenase expression and the production of α-ketoglutarate, which negatively regulate hypoxia-inducible factor-1α stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPKα2ΔMC mice.
Conclusions: AMPKα2 regulates α-ketoglutarate generation, hypoxia-inducible factor-1α stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPKα2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia.