Glioblastoma Stem-Like Cells Are More Susceptible Than Differentiated Cells to Natural Killer Cell Lysis Mediated Through Killer Immunoglobulin-Like Receptors–Human Leukocyte Antigen Ligand Mismatch and Activation Receptor–Ligand Interactions

Abstract

Glioblastoma (GBM) is the most aggressive brain malignancy in adults, where survival is approximately 14.6 months. Novel therapies are urgently needed and immunotherapy has hailed a new dawn for treatment of solid tumors. Natural killer (NK) cells may be amenable therapeutic effectors against heterogeneous GBM, since they also do not require co-stimulation and antigen specificity. However, it is unclear how culture media routinely used in pre-clinical studies affect GBM cell responses to NK-mediated cytotoxicity. We hypothesized that the culture medium would affect GBM cell phenotype, proliferation, and responses to NK cytotoxicity. We investigated in paired analyses n = 6 patient-derived primary GBM cells propagated in stem cell or serum-containing medium for morphology, proliferation, as well as susceptibility to NK cytolysis and related this to expression of surface and intracellular lineage markers, as well as ligands for NK cell activating and inhibitory receptors. We genotyped the GBM cells for human leukocyte antigen (HLA) as well as the killer immunoglobulin-like receptors (KIR) of the n = 6 allogeneic NK cells used as effector cells. Culture in serum-containing medium induced a switch in GBM cell morphology from suspension neuropsheres to adherent epithelial–mesenchymal-like phenotypes, which was partially reversible. The differentiated cells diminished expression of nestin, CD133 (prominin-1), and A2B5 putative glioma stem-cell markers, attenuated growth, diminished expression of ligands for activating NK cell receptors, while upregulating class I HLA ligands for NK cell inhibitory receptors. When maintained in serum-containing medium, fewer GBM cells expressed intercellular cell adhesion molecule-1 (ICAM-1) and were less susceptible to lysis by NK cells expressing αLβ2 integrin receptor (LFA-1), mediated through combination of inhibitory KIR–HLA ligand mismatch and diminished activation receptor–ligand interactions compared to cells maintained in stem cell media. We conclude that development of preclinical immunotherapy strategies against GBM should not use cells propagated in serum-containing media to avoid misinterpretation of potential therapeutic responses.

Pharmacologic inhibition of lysine specific demethylase-1 (LSD1) as a therapeutic and immune-sensitization strategy in diffuse intrinsic pontine glioma (DIPG)

“…NK cells have been shown to specifically target glioblastoma stem cells 38 and can circumvent antigen loss seen with CAR-T-based modalities 39 . A clinical trial of NK cell infusion for pediatric patients with CNS tumors is already in-progress 40 and preliminary results indicate an excellent safety profile.…”

Non-Genetically Improving the Natural Cytotoxicity of Natural Killer (NK) Cells

“…Moreover, the results using NK cell therapy in animal models of solid tumors or in clinical trials are disappointing, even if NK cells can eliminate the engrafted cell type or the primary tumor cells in vitro (11)(12)(13). In this context, it should be noted that different culture media affect tumor recognition by NK cells (40). In summary, there is not any expansion protocol that produces allogeneic NK cells able to efficiently eliminate solid tumor cells in vivo.…”

“…Why NK cells destroy most targets in vitro but not in vivo is unknown. Tumor cells strongly modify the expression of ligands, which are recognized by NK cell activating or inhibiting receptors when cultured in vitro (40). This could lead to the mistrust that those specific tumor cells would be NK sensitive or resistant in vivo.…”

Natural killer cells in patients with hematologic malignancies, solid tumors and in recipients of hematopoietic stem cell transplantation

“…Studies have shown that CAR-engineered and PD-1 inhibited NK cells exhibit antitumor effects and can induce apoptosis of GB cells [273][274][275][276]. GB stem-like cells are more susceptible to lysis by NK cells than differentiated GB cells [277]. In animal models, pretreatment of GB with bortezomib has been shown to promote NK cell cytotoxicity and to inhibit tumor growth ultimately leading to prolonged survival [278].…”