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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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In vivo depletion of bone marrow derived macrophage abrogates therapeutic benefit from combined NK+mAb9.2.27 treatment(A) Longitudinal axial post-contrast T1-weighted and T2-Weigted MRI images of nude rats bearing P3-30 tumors treated with combination NK+mAb9.2.27, vehicle (PBS/liposomes) control, and NK+mAb9.2.27 treated animals given weekly intraperitoneal clodronate encapsulated liposomes. (B) H&E of representative animals from the vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate (Scale bar 200 μm, Magnification 100 X). (C) Representative Ki67 labeling in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals (Scale bar 100 μm, Magnification 200X). (D) % Ki67 labeling index and (E) quantification of area fraction TUNEL positive apoptotic/necrotic cells. Data in (D) and (E) represent mean ±SEM of all tumors in the groups, **p<0.01 and *p<0.05. (F) Kaplan –Meier survival curves of NK+mAb9.2.27 treated and control tumor with or without macrophage depletion by clodronate, showing surviving fraction, n=8 animals/group.
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Figure 3: In vivo depletion of bone marrow derived macrophage abrogates therapeutic benefit from combined NK+mAb9.2.27 treatment(A) Longitudinal axial post-contrast T1-weighted and T2-Weigted MRI images of nude rats bearing P3-30 tumors treated with combination NK+mAb9.2.27, vehicle (PBS/liposomes) control, and NK+mAb9.2.27 treated animals given weekly intraperitoneal clodronate encapsulated liposomes. (B) H&E of representative animals from the vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate (Scale bar 200 μm, Magnification 100 X). (C) Representative Ki67 labeling in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals (Scale bar 100 μm, Magnification 200X). (D) % Ki67 labeling index and (E) quantification of area fraction TUNEL positive apoptotic/necrotic cells. Data in (D) and (E) represent mean ±SEM of all tumors in the groups, **p<0.01 and *p<0.05. (F) Kaplan –Meier survival curves of NK+mAb9.2.27 treated and control tumor with or without macrophage depletion by clodronate, showing surviving fraction, n=8 animals/group.

Mentions: To investigate further the mechanism leading to tumor regression, we utilized a unique GBM animal model developed in our laboratory where patient GBM biopsy spheroids are serially propagated in vivo over several generations. The resultant xenografts retain the genetic background, cellular heterogeneity and biological features of the original patient tumor [28]. P3-30 GBM cells express elevated levels of NG2/CSPG4 (93.3±3.2 %, n=3), (Supplementary Fig. 1C). As observed with the U87MG tumors, the combination therapy diminished P3-30 lesions volumes including vasogenic edema on T2- and solid tumor sizes on T1-weighted MR imaging, respectively, compared to the vehicle treated control group. The macrophage depletion abrogated this therapeutic effect as indicated by increased lesion volumes including tumor and vasogenic oedema on T2-, as well as solid tumor volume on T1-weighted MR imaging respectively (Fig. 3A). The NK+mAb9.2.27 treated tumors had larger regions with tissue necrosis compared to the control and clodronate treated group that exhibited typical pseudopalisading necrosis and angiogenic vasculature (Fig. 3B). Moreover, the clodronate NK+mAb9.2.27 treated tumors had strikingly numerous mitotic figures and significantly increased tumor proliferation indicated by elevated Ki67 labeling index compared to NK+mAb9.2.27 treated animals (One-Way ANOVA F11.19, df=2, p=0.0011, n=5), (Fig. 3C-D). The macrophage depletion also significantly reduced the fraction of apoptotic/necrotic cells compared to vehicle treated controls, and NK+mAb9.2.27 tumors (One-Way ANOVA F 7.75, p=0.0079, n=4), (Fig. 3B and 3E). The NK+mAb9.2.27 treatment prolonged animal survival compared to control, with median survival of 46 days versus 38.5 days respectively (Log Rank10.06, df=1, p=0.0015, n=7), (Fig. 3F). However, the macrophage depletion abrogated this increase of animal survival, with a median survival of 40.5 days (Log Rank 9.8, df=1, p=0.0017, n=8), (Fig. 3F). These results demonstrated the role of macrophages in therapeutic effect of NK+mAb9.2.27, as their depletion using clodronate diminished the animal survival.


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)

In vivo depletion of bone marrow derived macrophage abrogates therapeutic benefit from combined NK+mAb9.2.27 treatment(A) Longitudinal axial post-contrast T1-weighted and T2-Weigted MRI images of nude rats bearing P3-30 tumors treated with combination NK+mAb9.2.27, vehicle (PBS/liposomes) control, and NK+mAb9.2.27 treated animals given weekly intraperitoneal clodronate encapsulated liposomes. (B) H&E of representative animals from the vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate (Scale bar 200 μm, Magnification 100 X). (C) Representative Ki67 labeling in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals (Scale bar 100 μm, Magnification 200X). (D) % Ki67 labeling index and (E) quantification of area fraction TUNEL positive apoptotic/necrotic cells. Data in (D) and (E) represent mean ±SEM of all tumors in the groups, **p<0.01 and *p<0.05. (F) Kaplan –Meier survival curves of NK+mAb9.2.27 treated and control tumor with or without macrophage depletion by clodronate, showing surviving fraction, n=8 animals/group.
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Related In: Results  -  Collection

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Figure 3: In vivo depletion of bone marrow derived macrophage abrogates therapeutic benefit from combined NK+mAb9.2.27 treatment(A) Longitudinal axial post-contrast T1-weighted and T2-Weigted MRI images of nude rats bearing P3-30 tumors treated with combination NK+mAb9.2.27, vehicle (PBS/liposomes) control, and NK+mAb9.2.27 treated animals given weekly intraperitoneal clodronate encapsulated liposomes. (B) H&E of representative animals from the vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate (Scale bar 200 μm, Magnification 100 X). (C) Representative Ki67 labeling in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals (Scale bar 100 μm, Magnification 200X). (D) % Ki67 labeling index and (E) quantification of area fraction TUNEL positive apoptotic/necrotic cells. Data in (D) and (E) represent mean ±SEM of all tumors in the groups, **p<0.01 and *p<0.05. (F) Kaplan –Meier survival curves of NK+mAb9.2.27 treated and control tumor with or without macrophage depletion by clodronate, showing surviving fraction, n=8 animals/group.
Mentions: To investigate further the mechanism leading to tumor regression, we utilized a unique GBM animal model developed in our laboratory where patient GBM biopsy spheroids are serially propagated in vivo over several generations. The resultant xenografts retain the genetic background, cellular heterogeneity and biological features of the original patient tumor [28]. P3-30 GBM cells express elevated levels of NG2/CSPG4 (93.3±3.2 %, n=3), (Supplementary Fig. 1C). As observed with the U87MG tumors, the combination therapy diminished P3-30 lesions volumes including vasogenic edema on T2- and solid tumor sizes on T1-weighted MR imaging, respectively, compared to the vehicle treated control group. The macrophage depletion abrogated this therapeutic effect as indicated by increased lesion volumes including tumor and vasogenic oedema on T2-, as well as solid tumor volume on T1-weighted MR imaging respectively (Fig. 3A). The NK+mAb9.2.27 treated tumors had larger regions with tissue necrosis compared to the control and clodronate treated group that exhibited typical pseudopalisading necrosis and angiogenic vasculature (Fig. 3B). Moreover, the clodronate NK+mAb9.2.27 treated tumors had strikingly numerous mitotic figures and significantly increased tumor proliferation indicated by elevated Ki67 labeling index compared to NK+mAb9.2.27 treated animals (One-Way ANOVA F11.19, df=2, p=0.0011, n=5), (Fig. 3C-D). The macrophage depletion also significantly reduced the fraction of apoptotic/necrotic cells compared to vehicle treated controls, and NK+mAb9.2.27 tumors (One-Way ANOVA F 7.75, p=0.0079, n=4), (Fig. 3B and 3E). The NK+mAb9.2.27 treatment prolonged animal survival compared to control, with median survival of 46 days versus 38.5 days respectively (Log Rank10.06, df=1, p=0.0015, n=7), (Fig. 3F). However, the macrophage depletion abrogated this increase of animal survival, with a median survival of 40.5 days (Log Rank 9.8, df=1, p=0.0017, n=8), (Fig. 3F). These results demonstrated the role of macrophages in therapeutic effect of NK+mAb9.2.27, as their depletion using clodronate diminished the animal survival.

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