Haspels Heleen Neeltje, Rahman Mohummad Aminur, Joseph Justin Vareecal, Gras Navarro Andrea, Chekenya Martha
Department of Biomedicine, University of Bergen, Bergen, Norway.
Front Immunol. 2018 Jun 18;9:1345. doi: 10.3389/fimmu.2018.01345. eCollection 2018.
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 = 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 = 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 αβ 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.
胶质母细胞瘤（GBM）是成人中最具侵袭性的脑恶性肿瘤，患者的生存期约为14.6个月。目前迫切需要新的治疗方法，免疫疗法为实体瘤的治疗带来了新的曙光。自然杀伤（NK）细胞可能是针对异质性GBM的合适治疗效应细胞，因为它们也不需要共刺激和抗原特异性。然而，尚不清楚临床前研究中常规使用的培养基如何影响GBM细胞对NK介导的细胞毒性的反应。我们假设培养基会影响GBM细胞的表型、增殖以及对NK细胞毒性的反应。我们进行了配对分析，研究了6例源自患者的原发性GBM细胞在干细胞培养基或含血清培养基中培养后的形态、增殖情况，以及对NK细胞溶解的敏感性，并将其与表面和细胞内谱系标志物的表达以及NK细胞激活和抑制受体的配体相关联。我们对GBM细胞进行了人类白细胞抗原（HLA）基因分型，以及用作效应细胞的6例同种异体NK细胞的杀伤免疫球蛋白样受体（KIR）基因分型。在含血清培养基中培养会导致GBM细胞形态从悬浮神经球转变为贴壁上皮-间充质样表型，这种转变部分是可逆的。分化后的细胞中巢蛋白、CD133（prominin-1）和A2B5等假定的胶质瘤干细胞标志物的表达减少，生长减缓，激活NK细胞受体的配体表达减少，而NK细胞抑制受体的I类HLA配体表达上调。当在含血清培养基中培养时，与在干细胞培养基中培养的细胞相比，更少的GBM细胞表达细胞间黏附分子-1（ICAM-1），并且对表达αβ整合素受体（LFA-1）的NK细胞介导的裂解更不敏感，这是由抑制性KIR-HLA配体错配和激活受体-配体相互作用减少共同介导的。我们得出结论，针对GBM的临床前免疫治疗策略的开发不应使用在含血清培养基中培养的细胞，以避免对潜在治疗反应的误解。
