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Targeting glioblastoma with NK cells and mAb against NG2/CSPG4 prolongs animal survival.

Poli A, Wang J, Domingues O, Planagumà J, Yan T, Rygh CB, Skaftnesmo KO, Thorsen F, McCormack E, Hentges F, Pedersen PH, Zimmer J, Enger PØ, Chekenya M - Oncotarget (2013)

Bottom Line: There is an urgent, unmet need for novel, effective therapeutic strategies for this devastating disease.Combination treatment with NK+mAb9.2.27 diminished tumor growth that was associated with reduced tumor proliferation, increased cellular apoptosis and prolonged survival compared to vehicle and monotherapy controls.Moreover, mAb9.2.27 reversed tumor-promoting effects of patient-derived tumor-associated macrophage/microglia(TAM) ex vivo.Taken together, these findings indicate thatNK+mAb9.2.27 treatment may be an amenable therapeutic strategy to treat NG2/CSPG4 expressing GBMs. We provide a novel conceptual approach of combination immunotherapy for glioblastoma.

View Article: PubMed Central - PubMed

Affiliation: Translational Cancer Research, Department of Biomedicine, University of Bergen, Norway.

ABSTRACT
Glioblastoma (GBM) is the most malignant brain tumor where patients' survival is only 14.6 months, despite multimodal therapy with debulking surgery, concurrent chemotherapy and radiotherapy. There is an urgent, unmet need for novel, effective therapeutic strategies for this devastating disease. Although several immunotherapies are under development for the treatment of GBM patients, the use of natural killer (NK) cells is still marginal despite this being a promising approach to treat cancer. In regard of our knowledge on the role of NG2/CSPG4 in promoting GBM aggressiveness we investigated the potential of an innovative immunotherapeutic strategy combining mAb9.2.27 against NG2/CSPG4 and NK cells in preclinical animal models of GBM. Multiple immune escape mechanisms maintain the tumor microenvironment in an anti-inflammatory state to promote tumor growth, however, the distinct roles of resident microglia versus recruited macrophages is not elucidated. We hypothesized that exploiting the cytokine release capabilities of activated (NK) cells to reverse the anti-inflammatory axis combined with mAb9.2.27 targeting the NG2/CSPG4 may favor tumor destruction by editing pro-GBM immune responses. Combination treatment with NK+mAb9.2.27 diminished tumor growth that was associated with reduced tumor proliferation, increased cellular apoptosis and prolonged survival compared to vehicle and monotherapy controls. The therapeutic efficacy was mediated by recruitment of CCR2low macrophages into the tumor microenvironment, increased ED1 and MHC class II expression on microglia that might render them competent for GBM antigen presentation, as well as elevated IFN-γ and TNF-α levels in the cerebrospinal fluid compared to controls. Depletion of systemic macrophages by liposome-encapsulated clodronate decreased the CCR2low macrophages recruited to the brain and abolished the beneficial outcomes. Moreover, mAb9.2.27 reversed tumor-promoting effects of patient-derived tumor-associated macrophage/microglia(TAM) ex vivo.Taken together, these findings indicate thatNK+mAb9.2.27 treatment may be an amenable therapeutic strategy to treat NG2/CSPG4 expressing GBMs. We provide a novel conceptual approach of combination immunotherapy for glioblastoma. The results traverse beyond the elucidation of NG2/CSPG4 as a therapeutic target, but demonstrate a proof of concept that this antibody may hold potential for the treatment of GBM by activation of tumor infiltrated microglia/macrophages.

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Combination NK+mAb9.2.27 treatment ablates tumor growth, prolongs survival and presented a tropism for cytotoxic cells in combination treated in U87MG bearing rats(A) Longitudinal axial post-contrast T1-weighted images of nude rats bearing U87MG tumors treated with combination NK+mAb9.2.27, NK cell monotherapy, mAb9.2.27 monotherapy, and untreated controls, showing the same animal after 4, 5 weeks and 3 months post NK+mAb9.2.27 treatment. (B), (C) Plot showing the quantification of (B) tumor proliferation by % Ki67 labeling index and (C) cell death by % area fraction TUNEL positive apoptotic/necrotic cells. Data represent mean ± SEM of all tumors in the groups. ***p<0.001 and **p<0.01. (D) Kaplan –Meier survival curves showing surviving fraction, n=7 animals/group. (E) H&E staining of U87MG tumor with necrosis (scale bar 200μm, magnification 100X). Increased myeloperoxidase positive cells in mAb9.2.27 and combination treated tumors compared to control and NK treated tumors (scale bar 100μm, magnification 200X). High granzyme B positivity in combination treated tumors (scale bar 100μm, magnification 400X). Quantification of (F) MPO and (G) granzyme B positive cells. Data in (F) and (G) represent mean SEM from all tumors in the groups. *p<0.05, **p<0.001.
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Figure 1: Combination NK+mAb9.2.27 treatment ablates tumor growth, prolongs survival and presented a tropism for cytotoxic cells in combination treated in U87MG bearing rats(A) Longitudinal axial post-contrast T1-weighted images of nude rats bearing U87MG tumors treated with combination NK+mAb9.2.27, NK cell monotherapy, mAb9.2.27 monotherapy, and untreated controls, showing the same animal after 4, 5 weeks and 3 months post NK+mAb9.2.27 treatment. (B), (C) Plot showing the quantification of (B) tumor proliferation by % Ki67 labeling index and (C) cell death by % area fraction TUNEL positive apoptotic/necrotic cells. Data represent mean ± SEM of all tumors in the groups. ***p<0.001 and **p<0.01. (D) Kaplan –Meier survival curves showing surviving fraction, n=7 animals/group. (E) H&E staining of U87MG tumor with necrosis (scale bar 200μm, magnification 100X). Increased myeloperoxidase positive cells in mAb9.2.27 and combination treated tumors compared to control and NK treated tumors (scale bar 100μm, magnification 200X). High granzyme B positivity in combination treated tumors (scale bar 100μm, magnification 400X). Quantification of (F) MPO and (G) granzyme B positive cells. Data in (F) and (G) represent mean SEM from all tumors in the groups. *p<0.05, **p<0.001.

Mentions: We demonstrated previously that elevated levels of the NG2/CSPG4 proteoglycan on GBM cells and angiogenic vasculature is associated with a more aggressive disease course [6, 8, 12, 13]. We therefore hypothesized that perturbation of NG2/CSPG4 signaling with mAb9.2.27 alone or in combination with adoptively transferred NK cells might have therapeutic benefits for tumor-bearing rats. First we investigated the efficacy of the combination treatment in eradicating U87MG gliomas that are 99.2±0.2 % (n=3) NG2/CSPG4 positive, as recognized by mAb9.2.27 (Supplementary Fig. 1A). Four weeks after treatment, control untreated U87MG tumors were strongly contrast enhancing on T1-weighted MR images indicating increased angiogenesis and rapid growth compared to monotherapy and combination treated animals (Fig. 1A). However, while the monotherapy groups exhibited initial radiological responses of reduced tumor sizes on T1 weighted MRI with contrast, (Fig. 1A), after 5 weeks both monotherapy and control tumors progressed and killed their hosts. The NK+mAb9.2.27 combination treated tumors regressed as indicated by dramatically diminished contrast enhancement in MR images 3 months post-treatment (Fig. 1A). Tumor cell proliferation was significantly attenuated in the combination treatment compared to all other groups (One way ANOVA F7.4, NK p=0.006, n=6; mAb9.2.27 and control p=0.001, n=5), (Fig. 1B). The tumors treated with combined NK+mAb9.2.27 contained significantly larger areas with apoptotic and necrotic tissue compared to all other treatments (One way ANOVA F20, df=3, p=0.0001, n=32), (Fig. 1C). Correspondingly, the combined treatment significantly prolonged the survival of the animals with a median survival time of 91 days compared to 52, 44, and 39 days in the mAb9.2.27, NK cell and control groups, respectively (Log Rank 9.3, df=3, p=0.026, n=7), (Fig. 1D). In NK+mAb9.2.27 group, 60 % of the animals sacrificed for autopsy displayed necrosis with no visible tumor mass.


Targeting glioblastoma with NK cells and mAb against NG2/CSPG4 prolongs animal survival.

Poli A, Wang J, Domingues O, Planagumà J, Yan T, Rygh CB, Skaftnesmo KO, Thorsen F, McCormack E, Hentges F, Pedersen PH, Zimmer J, Enger PØ, Chekenya M - Oncotarget (2013)

Combination NK+mAb9.2.27 treatment ablates tumor growth, prolongs survival and presented a tropism for cytotoxic cells in combination treated in U87MG bearing rats(A) Longitudinal axial post-contrast T1-weighted images of nude rats bearing U87MG tumors treated with combination NK+mAb9.2.27, NK cell monotherapy, mAb9.2.27 monotherapy, and untreated controls, showing the same animal after 4, 5 weeks and 3 months post NK+mAb9.2.27 treatment. (B), (C) Plot showing the quantification of (B) tumor proliferation by % Ki67 labeling index and (C) cell death by % area fraction TUNEL positive apoptotic/necrotic cells. Data represent mean ± SEM of all tumors in the groups. ***p<0.001 and **p<0.01. (D) Kaplan –Meier survival curves showing surviving fraction, n=7 animals/group. (E) H&E staining of U87MG tumor with necrosis (scale bar 200μm, magnification 100X). Increased myeloperoxidase positive cells in mAb9.2.27 and combination treated tumors compared to control and NK treated tumors (scale bar 100μm, magnification 200X). High granzyme B positivity in combination treated tumors (scale bar 100μm, magnification 400X). Quantification of (F) MPO and (G) granzyme B positive cells. Data in (F) and (G) represent mean SEM from all tumors in the groups. *p<0.05, **p<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Combination NK+mAb9.2.27 treatment ablates tumor growth, prolongs survival and presented a tropism for cytotoxic cells in combination treated in U87MG bearing rats(A) Longitudinal axial post-contrast T1-weighted images of nude rats bearing U87MG tumors treated with combination NK+mAb9.2.27, NK cell monotherapy, mAb9.2.27 monotherapy, and untreated controls, showing the same animal after 4, 5 weeks and 3 months post NK+mAb9.2.27 treatment. (B), (C) Plot showing the quantification of (B) tumor proliferation by % Ki67 labeling index and (C) cell death by % area fraction TUNEL positive apoptotic/necrotic cells. Data represent mean ± SEM of all tumors in the groups. ***p<0.001 and **p<0.01. (D) Kaplan –Meier survival curves showing surviving fraction, n=7 animals/group. (E) H&E staining of U87MG tumor with necrosis (scale bar 200μm, magnification 100X). Increased myeloperoxidase positive cells in mAb9.2.27 and combination treated tumors compared to control and NK treated tumors (scale bar 100μm, magnification 200X). High granzyme B positivity in combination treated tumors (scale bar 100μm, magnification 400X). Quantification of (F) MPO and (G) granzyme B positive cells. Data in (F) and (G) represent mean SEM from all tumors in the groups. *p<0.05, **p<0.001.
Mentions: We demonstrated previously that elevated levels of the NG2/CSPG4 proteoglycan on GBM cells and angiogenic vasculature is associated with a more aggressive disease course [6, 8, 12, 13]. We therefore hypothesized that perturbation of NG2/CSPG4 signaling with mAb9.2.27 alone or in combination with adoptively transferred NK cells might have therapeutic benefits for tumor-bearing rats. First we investigated the efficacy of the combination treatment in eradicating U87MG gliomas that are 99.2±0.2 % (n=3) NG2/CSPG4 positive, as recognized by mAb9.2.27 (Supplementary Fig. 1A). Four weeks after treatment, control untreated U87MG tumors were strongly contrast enhancing on T1-weighted MR images indicating increased angiogenesis and rapid growth compared to monotherapy and combination treated animals (Fig. 1A). However, while the monotherapy groups exhibited initial radiological responses of reduced tumor sizes on T1 weighted MRI with contrast, (Fig. 1A), after 5 weeks both monotherapy and control tumors progressed and killed their hosts. The NK+mAb9.2.27 combination treated tumors regressed as indicated by dramatically diminished contrast enhancement in MR images 3 months post-treatment (Fig. 1A). Tumor cell proliferation was significantly attenuated in the combination treatment compared to all other groups (One way ANOVA F7.4, NK p=0.006, n=6; mAb9.2.27 and control p=0.001, n=5), (Fig. 1B). The tumors treated with combined NK+mAb9.2.27 contained significantly larger areas with apoptotic and necrotic tissue compared to all other treatments (One way ANOVA F20, df=3, p=0.0001, n=32), (Fig. 1C). Correspondingly, the combined treatment significantly prolonged the survival of the animals with a median survival time of 91 days compared to 52, 44, and 39 days in the mAb9.2.27, NK cell and control groups, respectively (Log Rank 9.3, df=3, p=0.026, n=7), (Fig. 1D). In NK+mAb9.2.27 group, 60 % of the animals sacrificed for autopsy displayed necrosis with no visible tumor mass.

Bottom Line: There is an urgent, unmet need for novel, effective therapeutic strategies for this devastating disease.Combination treatment with NK+mAb9.2.27 diminished tumor growth that was associated with reduced tumor proliferation, increased cellular apoptosis and prolonged survival compared to vehicle and monotherapy controls.Moreover, mAb9.2.27 reversed tumor-promoting effects of patient-derived tumor-associated macrophage/microglia(TAM) ex vivo.Taken together, these findings indicate thatNK+mAb9.2.27 treatment may be an amenable therapeutic strategy to treat NG2/CSPG4 expressing GBMs. We provide a novel conceptual approach of combination immunotherapy for glioblastoma.

View Article: PubMed Central - PubMed

Affiliation: Translational Cancer Research, Department of Biomedicine, University of Bergen, Norway.

ABSTRACT
Glioblastoma (GBM) is the most malignant brain tumor where patients' survival is only 14.6 months, despite multimodal therapy with debulking surgery, concurrent chemotherapy and radiotherapy. There is an urgent, unmet need for novel, effective therapeutic strategies for this devastating disease. Although several immunotherapies are under development for the treatment of GBM patients, the use of natural killer (NK) cells is still marginal despite this being a promising approach to treat cancer. In regard of our knowledge on the role of NG2/CSPG4 in promoting GBM aggressiveness we investigated the potential of an innovative immunotherapeutic strategy combining mAb9.2.27 against NG2/CSPG4 and NK cells in preclinical animal models of GBM. Multiple immune escape mechanisms maintain the tumor microenvironment in an anti-inflammatory state to promote tumor growth, however, the distinct roles of resident microglia versus recruited macrophages is not elucidated. We hypothesized that exploiting the cytokine release capabilities of activated (NK) cells to reverse the anti-inflammatory axis combined with mAb9.2.27 targeting the NG2/CSPG4 may favor tumor destruction by editing pro-GBM immune responses. Combination treatment with NK+mAb9.2.27 diminished tumor growth that was associated with reduced tumor proliferation, increased cellular apoptosis and prolonged survival compared to vehicle and monotherapy controls. The therapeutic efficacy was mediated by recruitment of CCR2low macrophages into the tumor microenvironment, increased ED1 and MHC class II expression on microglia that might render them competent for GBM antigen presentation, as well as elevated IFN-γ and TNF-α levels in the cerebrospinal fluid compared to controls. Depletion of systemic macrophages by liposome-encapsulated clodronate decreased the CCR2low macrophages recruited to the brain and abolished the beneficial outcomes. Moreover, mAb9.2.27 reversed tumor-promoting effects of patient-derived tumor-associated macrophage/microglia(TAM) ex vivo.Taken together, these findings indicate thatNK+mAb9.2.27 treatment may be an amenable therapeutic strategy to treat NG2/CSPG4 expressing GBMs. We provide a novel conceptual approach of combination immunotherapy for glioblastoma. The results traverse beyond the elucidation of NG2/CSPG4 as a therapeutic target, but demonstrate a proof of concept that this antibody may hold potential for the treatment of GBM by activation of tumor infiltrated microglia/macrophages.

Show MeSH
Related in: MedlinePlus