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.
Highlights
Glioblastoma (GBM) is the most frequent malignant brain tumor in adults [1]
natural killer (NK) cells express an exquisite repertoire of inhibitory killer immunoglobulin-like receptors (KIR) for major histocompatibility complex (MHC)/ human leukocyte antigen (HLA), that when ligated to cognate self-HLA, dampen their effector functions to maintain selftolerance and avoid autoreactivity, while simultaneously killing targets lacking the self-molecules [14, 15]
NK cells hold great promise as immunotherapy effectors against GBM, since they broadly recognize transformed and stressed cells in a manner that is unrestricted by HLA bound tumor neoantigens, while sparing healthy cells
Summary
Glioblastoma (GBM) is the most frequent malignant brain tumor in adults [1]. the age-adjusted incidence is relatively low at 3.2/100,000 people in Western countries including United States [2], patients’ median survival rate is only 14.6 months, notwithstanding aggressive multimodal treatment consisting of surgery, radiotherapy, and temozolomide (TMZ) chemotherapy [3]. Immunotherapy has emerged as a successful treatment of choice for several cancers, with the advent of checkpoint blockade and T cell receptor engineering [5,6,7] This treatment modality has yet to reap benefit for brain tumor patients, largely due to challenges posed by the blood–brain barrier, tumor heterogeneity, and the highly immunosuppressive solid tumor contexture [8,9,10,11]. Among cytotoxic lymphocytes, natural killer (NK) cells may be more suitable as therapeutic effectors against highly heterogeneous tumors such as GBM, since unlike T and B lymphocytes, they do not possess rearranged V(D)J receptors and are not restricted by major histocompatibility complex (MHC)-bound antigen presentation. NK cells express an exquisite repertoire of inhibitory killer immunoglobulin-like receptors (KIR) for MHC/ human leukocyte antigen (HLA), that when ligated to cognate self-HLA, dampen their effector functions to maintain selftolerance and avoid autoreactivity, while simultaneously killing targets lacking the self-molecules [14, 15]
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