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Natural killer (NK) cells inhibit systemic metastasis of glioblastoma cells and have therapeutic effects against glioblastomas in the brain
- Se Jeong Lee†1,
- Won Young Kang†2, 3,
- Yeup Yoon†2, 3,
- Ju Youn Jin2,
- Hye Jin Song1,
- Jung Hyun Her4,
- Sang Mi Kang4,
- Yu Kyeong Hwang4,
- Kyeong Jin Kang1,
- Kyeung Min Joo1, 2, 3, 5Email author and
- Do-Hyun Nam2, 3Email author
© Lee et al. 2015
Received: 18 May 2015
Accepted: 17 December 2015
Published: 24 December 2015
Glioblastoma multiforme (GBM) is characterized by extensive local invasion, which is in contrast with extremely rare systemic metastasis of GBM. Molecular mechanisms inhibiting systemic metastasis of GBM would be a novel therapeutic candidate for GBM in the brain.
Patient-derived GBM cells were primarily cultured from surgical samples of GBM patients and were inoculated into the brains of immune deficient BALB/c-nude or NOD-SCID IL2Rgammanull (NSG) mice. Human NK cells were isolated from peripheral blood mononucleated cells and expanded in vitro.
Patient-derived GBM cells in the brains of NSG mice unexpectedly induced spontaneous lung metastasis although no metastasis was detected in BALB/c-nude mice. Based on the difference of the innate immunity between two mouse strains, NK cell activities of orthotopic GBM xenograft models based on BALB/c-nude mice were inhibited. NK cell inactivation induced spontaneous lung metastasis of GBM cells, which indicated that NK cells inhibit the systemic metastasis. In vitro cytotoxic activities of human NK cells against GBM cells indicated that cytotoxic activity of NK cells against GBM cells prevents systemic metastasis of GBM and that NK cells could be effective cell therapeutics against GBM. Accordingly, NK cells transplanted into orthotopic GBM xenograft models intravenously or intratumorally induced apoptosis of GBM cells in the brain and showed significant therapeutic effects.
Our results suggest that innate NK immunity is responsible for rare systemic metastasis of GBM and that sufficient supplementation of NK cells could be a promising immunotherapeutic strategy for GBM in the brain.
KeywordsGlioblastoma multiforme Natural killer cell Systemic metastasis Orthotopic xenograft model Therapeutic effect
Glioblastoma multiforme (GBM) is the most common primary malignancy of the central nervous system (CNS), and 75 % of affected patients die within two years of their diagnosis [1–3]. GBMs are characterized by their highly infiltrative nature, which causes difficulties in curative surgical resection. Moreover, the resistance of GBM cells to radio- and chemo-therapy provokes a high rate of tumor recurrence [2, 4, 5]. Therefore, unmet medical need new therapeutic modalities with novel treatment mechanisms targeting GBM cells that evade and/or withstand currently available therapies.
Although GBMs are known as highly invasive tumors in the brain, extra-cranial metastasis does rarely occur [6–9]. This clinical characteristic of GBMs could be due to several reasons; short survival length of GBM patients, unique structure of micro-vessels in the CNS, lack of lymphatic systems in the CNS, and immune-privileged microenvironments [10–12]. The underlying mechanisms of the limited systemic metastatic potential of GBM cells are not only interesting from a scientific perspective, but could also provide clues leading to novel therapeutic modalities and unique treatment mechanisms.
Recently, orthotopic GBM xenograft animal models using patient-derived GBM cells have been utilized to test newly developed therapeutic agents of GBMs . The animal models maintain the genetic, molecular, and functional features of the parental tumors to provide reliable preclinical models for GBMs. Since the xenograft models utilize immune deficient mouse strains to avoid graft rejection, immunologic microenvironments of transplanted GBM cells can be specifically modified by choosing a recipient mouse strain with defined immune deficiency status. For example, the BALB/c-nude strain has an innate immune system but no acquired immunity, while both the innate and acquired immune systems of the NOD-scid IL2Rgammanull (NSG) mouse are impaired [14–16].
In this study, we elucidated a possible mechanism regarding the limited systemic metastatic potential of GBM cells in the brain using an orthotopic xenograft animal model in addition to discovering some anti-cancer activities of systemic NK cells. Since the brain microenvironments prevent GBM cells from having direct contact with NK cells, direct or indirect NK cell supplementation to the GBMs demonstrated significant therapeutic effects, in our preclinical model.
All Human samples were collected with written informed consent under a protocol approved by the Institutional Review Board of the Samsung Medical Center (2010–04–004, Seoul, Korea). Parts of the surgical samples were enzymatically dissociated, and then red blood cells were removed by percoll gradient centrifugation (Sigma-Aldrich). Dissociated cells were maintained in the ‘NBE’ conditions consisting of Neuro-Basal Media, N2 and B27 supplements (×1/2 each), 2 mM L-glutamine, 100U/ml penicillin and streptomycin (Invitrogen), and human recombinant EGF and bFGF (50 ng/ml each; R&D Systems). The human GBM cell line U-87 MG (ATCC) was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum, 100U/ml penicillin and streptomycin (Invitrogen).
Patient-derived GBM xenograft model
All animal experiments were approved by the Institutional Review Boards of the Samsung Medical Center (Seoul, Korea) and conducted in accord with the ‘Health Guide for the Care and Use of Laboratory Animals’ (NIH publication no. 80–23) and the ARRIVE guidelines for Reporting Animal Research  (Additionanl file 1). For orthotopic GBM xenograft models, anesthetized 6-week-old BALB/c-nude or NOD-SCID IL2Rgammanull (NSG) mice were secured in a rodent stereotactic frame (mice were obtained from Orient Bio Korea). A hollow guide screw was implanted into a small drill hole made at 2 mm left and 1 mm anterior to the bregma, and then 2 × 105 tumor cells in 5 μl HBSS were injected through this guide screw into the white matter at a depth of 2 mm [anterior/posterior (AP) +0.5 mm, medial/lateral (ML) +1.7 mm, dorsal/ventral (DV) -3.2 mm]. Mice with a total body weight reduction >20 % were sacrificed, and their brains and lungs were processed for paraffin sections.
Treatment with anti-asialo GM1 (ASGM1) antibody
Male 6-week-old BALB/c-nude mice were injected intravenously with the ASGM1 antibody (Wako Chemicals) or 1× Phosphate Buffered Saline (PBS, Invitrogen) on Day -1 (40 μl/ea). On Day 0, patient-derived GBM cells (2 × 105) were implanted into the brain as described previously. After the tumor cell inoculation, either ASGM1 antibody or 1× PBS were intravenously injected into the animals twice a week for 4.5 months. Eighteen weeks after the tumor cell inoculation, the mice were sacrificed. Spleens were harvested and measured NK cells activity. Brains and lungs were paraffin-embedded, and then sliced into 4 μm sections for histological analysis.
For murine NK cell activity measurement, the spleens were immersed in Hank's Balanced Salt Solution (HBSS, Invitrogen), and then single cell suspensions were prepared by forcing the spleens through a 70 μm nylon mesh. The resulting cell suspension was placed onto a Ficoll-Paque PLUS (GE Healthcare) and centrifuged for 30 min at 2,000 rpm. Mononuclear cells were isolated, washed, and stained with a PE-Cy7-conjugated anti-mouse CD314 (NKG2D) antibody (eBioscience).
Paraffin-embedded tissue sections were deparaffinized and rehydrated. Heat-induced epitope retrieval was performed using a target retrieval solution (Dako) for 5 min in a microwave. Slides were treated with 3 % hydrogen peroxide for 10 min to inactivate endogenous peroxidase, and then the slides were blocked for 20 min at room temperature in a blocking solution (5 % normal horse serum, 1 % normal goat serum, 0.1 % Triton-X 100 in 1× PBS). After blocking, the slides were incubated in primary antibodies at 4 °C overnight; including mouse monoclonal antibody against human Ki-67 (BD Pharmingen), mouse monoclonal antibody against human cytoplasm (STEM-121, Stem Cells), mouse monoclonal antibody against human nestin (Thermo), mouse monoclonal antibody against human SOX2 (Cell Signaling technology), mouse monoclonal antibody against human GFAP (Sigma), mouse monoclonal antibody against NK1.1 (Novus biological), and rabbit monoclonal antibody against human HLA-A (MHC class I, abcam). Slides were washed and incubated with secondary antibodies for 1 h at room temperature [Avidin-Biotin complex kit (Vector lab) or Alexa Flour 488 or 594 conjugated antibodies (Invitrogen)]. Slides were counterstained with hematoxylin (Sigma-Aldrich) or DAPI (Sigma-Aldrich).
Western blotting and flow cytometry
Cell lysates were prepared using lysis buffer (50 mM HEPES, pH7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 % Triton X-100, 10 % glycerol, and protease/phosphatase inhibitors; Roche). Protein concentrations were determined using a BCA protein assay kit according to the manufacturer's directions (Thermo). Equivalent amounts of proteins were separated by 10 % SDS gel electrophoresis, transferred onto PVDF membranes (Thermo), and immunoblotted with primary antibodies overnight at 4 °C, including a rabbit monoclonal antibody against human HLA-A (MHC class I, abcam) and a rabbit monoclonal antibody against GAPDH (Cell Signaling technology). Antibodies were visualized using a horseradish peroxidase-conjugated anti-rabbit IgG (Thermo) and analyzed using enhanced chemiluminescence western blot detection reagent (GE Healthcare).
For flow cytometry, anti-HLA-ABC-PE (G46-2.6), anti-MIC-A/B-PE (6D4) (BD Biosciences), anti-ULBP-1-PE (170818), anti-ULBP-2-PE (165903) (R&D systems), anti-HLA-E-PE (3D12), anti-CD112-PE (TX31), and anti-CD155-PE (SKII.4) antibody (BioLegend) were utilized. Samples were run on a BD Fortessa (BD Biosciences) and data were analyzed using FlowJo software (TreeStar Inc., OR).
Human NK cell preparation and in vitro expansion
In vitro expansion of human NK cells was conducted as previously described . Briefly, peripheral blood mononucleated cells (PBMCs) were isolated from healthy donors by leukapheresis and CD3+ T cells were depleted by VarioMACS (Miltenyi Biotech). T cell-depleted PBMCs were expanded at a seeding concentration of 2 × 105 cells/ml in CellGro SCGM serum-free medium (CellGenix) with 1 % autoplasma, 1 × 106 cells/ml irradiated (2,000 rad) autologous PBMCs, 10 ng/ml anti-CD3 monoclonal antibody, OKT3 (Orthoclon), and 500 IU/ml IL-2 (Proleukin) in an A-350 N culture bag (NIPRO). On day 5 of culture, NK cells were fed with fresh medium containing 500 IU/ml IL-2 and 1 % autoplasma every two days without removal of preexisting culture medium to maintain a cellular concentration at 1 ~ 2 × 106 cells/ml for 14 days. The viability of expanded NK cells was determined by propidium iodide staining.
In vitro expanded NK cells were stained with primary antibodies and analyzed by a flow cytometry using anti-CD56-PE-Cy5 (B159), anti-CD16-PE (3G8), anti-CD3-FITC (UCHT1), anti-NKp30-PE (P30-15), anti-NKp44-PE (P44-8.1), anti-NKp46-PE (9E2/NKp46), anti-DNAM-1-PE (DX11), anti-CD14-FITC (M5E2), anti-CD19-PE (HIB19) (BD Biosciences), anti-NKG2D-PE (149810) (R&D systems), anti-CD158a-PE (EB6Bf), anti-CD158b-PE (GL183), and anti-CD158e-PE (Z27.3.7) (Beckman Coulter).
51Cr-release cytotoxicity assay
Target cells were labeled with 100 μCi 51 Cr sodium chromate (BMS), and incubated with NK cells in U-bottom 96-well plates (BD falcon) at three different effector:target (E:T) ratios. Spontaneous and maximum releases were determined by incubating target cells without effector cells in the absence or presence of 4 % Triton X-100. Radioactivity was counted using a gamma counter (PerkinElmer), and the percentage of specific lysis was calculated as follows: % specific lysis = [(experimental release–spontaneous release)/(maximum release-spontaneous release)] × 100. The assay was performed in triplicate.
In vivo anti-tumor activities of NK cells
Orthotopic GBM xenograft models were established as described previously, using 2 × 105 U-87 MG cells in 5 μl HBSS were injected in male 6-week-old BALB/c-nude mice brains. Human NK cells were injected intratumorally (1 × 103, 1 × 104, 1 × 105 in 5 μl HBSS) or intravenously (1 × 105, 1 × 106, 1 × 107, in 100 μl HBSS) into animals once a week for 3 weeks or 3 times a week for 1 week. After 28 days, the animals’ brains were harvested and cut into 4–6 mm thick slices.
Brain slices were fixed in 4 % paraformaldehyde, embedded in paraffin, sectioned into 4 μm coronal sections using a microtome, and stained with hematoxylin and eosin (Sigma). The tumor volume was calculated by measuring the section with the largest tumor portion and applying the formula: (width)2 × length × 0.5. The DeadEnd™ Colorimetric TUNEL System (Promega) was used to assay apoptosis. For human NK cell detection, immunohistochemistry was performed using a mouse monoclonal antibody against CD56 (Dako). Numbers of human NK cells or TUNEL-positive cells were counted in 5 randomly selected fields for each mouse.
Data are presented as mean + standard deviation (SD) or standard error (SE). Statistical comparisons of groups were performed using the Student’s t test. Values of P <0.05 were considered to be statistically significant.
Spontaneous lung metastasis of patient-derived GBM cells in orthotopic xenograft animal models using NSG mice
In vivo tumor formation rate and median survival length of orthotopic GBM xenograft animal models
Mouse number (Tumor formation/Total)
Median survival day
4/5 (80 %)
0/5 (0 %)
4/5 (80 %)
2/5 (40 %)
4/5 (80 %)
0/5 (0 %)
3/5 (60 %)
4/5 (80 %)
3/5 (60 %)
0/5 (0 %)
3/5 (60 %)
2/5 (40 %)
4/5 (80 %)
0/5 (0 %)
4/5 (80 %)
3/5 (60 %)
Unexpectedly, we detected abnormal respiratory movements in the NSG groups. Although extra-cranial metastasis of GBMs is extremely rare in GBM patients [7–9] the abnormal behavior observed suggested a spontaneous lung metastasis of orthotopically-implanted patient-derived GBM cells. When the lungs of both the BALB/c-nude and NSG groups were harvested, multiple visible lung nodules were observed only in the NSG groups (Table 1, arrowheads in Fig. 1a). The oncologic origin of the nodules was confirmed by the pathology and immunohistochemistry against Ki-67 (Fig. 1a). The lung nodules in the NSG mice were consistently detected in all four kinds of patient-derived GBM cells (Fig. 1b). Compared with the BALB/c-nude mouse, the NSG mouse strain has severe impairments, not only in adaptive T- and B-cell activities but also in innate immunity, involving macrophages and natural killer (NK) cells. Since metastasizing GBM cells meet circulating immune cells in the blood stream, we hypothesized that presence of NK cells in the circulating system might cause those sharp differences in the extra-cranial metastatic potential of implanted patient-derived GBM cells.
Spontaneous extra-cranial metastasis of GBM cells provoked by NK cell-inactivation in BALB/c-nude mice
NK cell distribution in orthotopic GBM xenografts and MHC class I molecule expression of patient-derived GBM cells
The absence of NK cells in brain tumor and heterogeneous MHC class I molecule expression of GBM cells suggest followings. First, engraftment of patient-derived GBM cells could be more successful in the brain than other organs that have reactive NK cells. Second, circulating NK cells could target metastasizing GBM cells in extra-cranial sites. Previously, invasive GBM cells were observed to lose expression of MHC class I molecules , which would increase the possibility that NK cells might recognize invasive and/or migrating GBM cells.
Applicability of NK cell therapy to GBM
The absence of NK cells in brain tumors and the anti-cancer effects of NK cells inversely indicate that supplementation of reactive NK cells to GBMs could have therapeutic effects. To evaluate the possibility, we transferred NK cells to an orthotopic xenograft GBM animal model and its examined therapeutic effects. In the clinic, mouse NK cells could not be hired to treat human GBMs. To address this issue, human NK cells were utilized for the experiments.
Large-scale expansion of human NK cells
In vitro GBM cell lysis effects of expanded NK cells
In vivo therapeutic effects of NK cells against orthotopic GBM xenograft tumors
Implications of treatment schedule in the NK cell therapy against GBM
In this study, we observed the spontaneous systemic metastasis of patient-derived GBM cells that were inoculated into the brains of NSG mice. Since the systemic metastasis was not induced in the BALB/c-nude mice, we hypothesized that the difference originates from the innate immune system such as NK cells, macrophages, and the complement system [14, 16, 22]. When we administrated murine NK cell inhibition antibody to BALB/c-nude orthotopic GBM animal models, extra-cranial metastases were induced. These results suggest that NK cells play an important inhibitory role in the systemic metastasis of GBMs and that GBM cells are sensitive to the cytotoxic activities of NK cells.
Traditionally, the brain has been regarded as an immunologically privileged site [2, 4, 5, 27, 28]. In orthotopic GBM xenograft tumors, NK cells were not observed by immunohistochemistry in this study. NK cells identify and eliminate cells lacking MHC class I molecules, which are dependent on the activation status of NK cell-activating and inhibitory receptors [29–33]. We observed that the expression of MHC class I molecules in patient-derived GBM cells is heterogeneous. However, ly49 receptors of mouse NK cells could not recognize human MHC class I molecules [34–37]. Therefore, NK cells of BALB/c-nude mice would target human patient-derived GBM cells, while human patient-derived GBM cells in the brain parenchyma could not have direct contacts with NK cells.
In vitro expanded human NK cells showed cytotoxic effects against human U-87 MG GBM cells in vitro and in vivo, although U-87 MG cells expressed NK cell-inhibiting MHC class I molecules (HLA-ABC). These unexpected results might originate from the expression of NK cell-activating ligands of U-87 MG cells such as ULBP-2, CD112, and CD155. Cytotoxic effects of NK cells are determined by the balance between NK cell-activating and inhibiting signals; when the activating signals are dominant, NK cells have cytotoxic effects [30, 33, 38]. Since human leukemia K562 cells do not express MHC class I molecules, NK cells showed superior killing effects to K562 cells compared with U-87 MG cells in this study.
Moreover, distribution of NK cells in vivo could be an important determinant of their therapeutic effects. In this study, we observed that the treatment efficacy of intratumoral injection of NK cells is better than intravenous injection; 1 × 104 NK cells transplanted intratumorally showed similar therapeutic effects to 1 × 107 intravenously transplanted NK cells. When we compared NK cell distribution in the orthotopic GBM xenograft tumors, similar densities of NK cells were observed in the 1 × 104 intratumor and 1 × 107 intravenous groups. Ratios of TUNEL-positive apoptotic cells to CD56 positive NK cells in the tumor (2 ~ 3:1) was also similar in those groups. Since NK cells do not distribute in the brain, techniques that increase the NK cell number in the GBM would increase the therapeutic effects of NK cells against the intracranial tumor. Accordingly, when NK cells were intravenously transplanted more intensively to increase the NK cell/tumor cell ratio, the treatment effects of NK cells were significantly potentiated in this study.
Immunotherapies such as dendritic cells, lymphocytes, engineered T-cells, and cancer vaccines, have shown treatment effects on patients with various metastatic solid tumors [39–42]. Cellular immunotherapeutic strategies directly target metastatic cancer cells combined with cytotoxic agents or ionizing radiation [43–46]. Moreover, tumor initiating/cancer stem cells with antigenic heterogeneity were suggested to be recognized by immune cells [47, 48]. Given that extremely rare extracranial metastasis of GBM  and sparse distribution of NK cells in the brain, inhibitory effects of NK cells on systemic metastasis of GBM cells in vivo in this study might be mediated by direct interaction between NK cells and GBM cells in the extracranial sites where NK cells monitor abnormal cells.
Recent successes of immunotherapy for solid tumors have generated a resurgence of interest in immunological therapeutic approaches to GBMs [23, 27, 38, 50–54]. Many immune cell-based therapies have been tried for the GBM, including dendritic cells, lymphokine-activated killer cells, NK cells, and cytotoxic T lymphocytes [32, 55]. Among them, NK cells have the strongest cytotoxic activities against malignant tumor cells and play important roles in the initiation of subsequent adaptive immunity [35, 36, 56]. In this study, we generated sufficient human NK cells in vitro and confirmed their direct-tumor killing effects against GBM cells both in vitro and in vivo. Moreover, other immune cells, such as mouse macrophages, could be recruited to the GBM by the NK cell therapy, which would provoke further immunological reactions to GBM cells.
In summary, our results suggest that NK cells play an important inhibitory role in the extra-cranial metastasis of the GBM and that GBM cells are susceptible to NK cells. Therefore, supplementation of NK cells is a promising immunotherapeutic strategy against GBM, which could be potentiated by techniques that augment direct cell-to-cell contact between GBM and NK cells.
We thank Green Cross Lab Cell, Mi-Young Cho, Dong Hyun Lee, Jin Wung Choi for their technical support with the experiments. This work was supported by the Korea Health technology R&D project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C3418) and the Samsung Biomedical Research Institute grant, no. SBRI C-B0-218-3 (D.H.Nam), no. SMX-1131281 (K.M.Joo). This study was supported by grants from Mogam Biotechnology Institute.
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