Abstract
Glioblastoma is the leading malignant glioma with a poor prognosis. This study aimed to investigate the antitumor effects of natural killer cells in combination with temozolomide as the standard chemotherapeutic agent for glioblastoma. Using a simple, feeder-less, and chemically defined culture method, we expanded human peripheral blood mononuclear cells and assessed the receptor expression, natural killer cell activity, and regulatory T cell frequency in expanded cells. Next, using the standard human glioblastoma cell lines (temozolomide-sensitive U87MG, temozolomide-resistant T98G, and LN-18), we assessed the ligand expressions of receptors on natural killer cells. Furthermore, the antitumor effects of the combination of the expanded natural killer cells and temozolomide were assessed using growth inhibition assays, apoptosis detection assays, and senescence-associated β-galactosidase activity assays in the glioblastoma cell lines. Novel culture systems were sufficient to attain highly purified (>98%), expanded (>440-fold) CD3−/CD56+ peripheral blood-derived natural killer cells. We designated the expanded population as genuine induced natural killer cells. Genuine induced natural killer cells exhibited a high natural killer activity and low regulatory T cell frequency compared with lymphokine-activated killer cells. Growth inhibition assays revealed that genuine induced natural killer cells inhibited the glioblastoma cell line growth but enhanced temozolomide-induced inhibition effects in U87MG. Apoptosis detection assays revealed that genuine induced natural killer cells induced apoptosis in the glioblastoma cell lines. Furthermore, senescence-associated β-galactosidase activity assays revealed that temozolomide induced senescence in U87MG. Genuine induced natural killer cells induce apoptosis in temozolomide-sensitive and temozolomide-resistant glioblastoma cells and enhances temozolomide-induced antitumor effects in different mechanisms. Hence, the combination of genuine induced natural killer cells and temozolomide may prove to be a promising immunochemotherapeutic approach in patients with glioblastoma if the antitumor effects in vivo can be demonstrated.
Highlights
Glioblastoma (GBM) is the most lethal malignant tumor of the brain
This study aimed to (a) develop highly purified human Natural killer (NK) cells with robust cytotoxic activity derived from peripheral blood mononuclear cells (PBMCs) using a simple, feeder-less method, such as cancer cells; (b) investigate the cellular characteristics of NK cells, including receptor expression, NK activity, and frequency of Tregs in the expanded populations; and (c) investigate the antitumor effects of the expanded NK cells in combination with TMZ, which is the standard chemotherapy agent for GBM, and the mechanisms of the cytotoxicity against GBM in vitro
The findings indicated the superiority of the newly established NK cell culture method over the lymphokine-activated killer cells (LAK) expansion method (Fig 1A and 1B)
Summary
The current standard therapy combines maximal surgical tumor resection with adjuvant therapy, comprising temozolomide (TMZ) chemotherapy, and multifractionated radiation (total dose: 60 Gy) [1]. NK cells exhibit potent cytotoxic activity against tumor cells via apoptosis [4] and can remove abnormal cells including tumor and virus-infected cells as the innate immune system [5,6]. These cells recognize tumor cells by forming a synapse with the tumor cells and induce apoptosis by releasing cytotoxic molecules such as perforin and granzyme against the tumor cells [7]. Ligands of NK inhibitory receptors, such as NKG2A and KIR2DL, are associated with NK cell cytotoxicity against tumor cells [14,15]
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