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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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Increased recruitment of ED1+ activated macrophage/microglia in NK+mAb9.2.27 treated tumors(A) ED1 labeling in P3-30 bearing animals in the tumor core vs. periphery in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals, (scale bar 100 μm, magnification 200 X). (B) The quantification of positive area fraction for ED1 and Iba1 in tumor core and periphery in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals. (C) Quantification of CD8+ area fraction in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals (D) Flow cytometric representative dot plot showing the gating of cell populations from rat brain cell suspension expressing CD11b/c and CD45, permitting the differentiation of macrophages (CD45highCD11b/c+) and microglia (CD45lowCD11b/c+). (E) Upper panel, % microglia cells expressing MHC class II, ED1 and ED2. Lower panels, mean fluorescence intensity histograms as % of Max of data in (E). (F) Percentages of CCR2+ macrophages (right) and mean fluorescence intensity as % of max for CCR2 (left). The data in (B), (C), (E) and (F) represent mean ±SEM from all tumors in the groups, ***p<0.001, **p<0.01 and *p<0.05.
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Figure 4: Increased recruitment of ED1+ activated macrophage/microglia in NK+mAb9.2.27 treated tumors(A) ED1 labeling in P3-30 bearing animals in the tumor core vs. periphery in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals, (scale bar 100 μm, magnification 200 X). (B) The quantification of positive area fraction for ED1 and Iba1 in tumor core and periphery in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals. (C) Quantification of CD8+ area fraction in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals (D) Flow cytometric representative dot plot showing the gating of cell populations from rat brain cell suspension expressing CD11b/c and CD45, permitting the differentiation of macrophages (CD45highCD11b/c+) and microglia (CD45lowCD11b/c+). (E) Upper panel, % microglia cells expressing MHC class II, ED1 and ED2. Lower panels, mean fluorescence intensity histograms as % of Max of data in (E). (F) Percentages of CCR2+ macrophages (right) and mean fluorescence intensity as % of max for CCR2 (left). The data in (B), (C), (E) and (F) represent mean ±SEM from all tumors in the groups, ***p<0.001, **p<0.01 and *p<0.05.

Mentions: The NK+mAb9.2.27 treatment increased the tropism of ED1+ cells into the tumor core compared to vehicle treated controls (Two-Way ANOVA t3.991, p=0.013, n=4) and macrophage depleted NK+mAb9.2.27 treated tumors (Two-Way ANOVA t4.276, p=0.01, n=4), (Fig. 4A and 4B). Iba1, the calcium binding protein expressed by activated and phagocytic macrophage/microglia was also elevated in the core of the combination treated tumors compared to vehicle treated control (Two-Way ANOVA t4.633, p=0.01, n=4), and macrophage depleted tumors (Two-Way ANOVA t3.235, p=0.0243, n=4), (Fig. 4B). Significant numbers of CD8+ cells were recruited into the tumor microenvironment after the NK+mAb9.2.27 treatment (One-Way ANOVA t7.482, p=0.0001, n=5), (Fig. 4C), as was the case for U87MG and U251-NG2 tumors. Remarkably, macrophage depletion by clodronate injection abrogated this ED1 recruitment compared to NK+mAb9.2.27 treated animals (Two-Way ANOVA t34.276, p=0.013, n=4). The CD8+ cells recruited to the NK+mAb9.2.27 treated tumor were significantly elevated compared to controls and NK+mAb9.2.27 with macrophage depletion (One-Way ANOVA 18.5, p=0.0001, n=5), (Fig. 4A-C).


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)

Increased recruitment of ED1+ activated macrophage/microglia in NK+mAb9.2.27 treated tumors(A) ED1 labeling in P3-30 bearing animals in the tumor core vs. periphery in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals, (scale bar 100 μm, magnification 200 X). (B) The quantification of positive area fraction for ED1 and Iba1 in tumor core and periphery in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals. (C) Quantification of CD8+ area fraction in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals (D) Flow cytometric representative dot plot showing the gating of cell populations from rat brain cell suspension expressing CD11b/c and CD45, permitting the differentiation of macrophages (CD45highCD11b/c+) and microglia (CD45lowCD11b/c+). (E) Upper panel, % microglia cells expressing MHC class II, ED1 and ED2. Lower panels, mean fluorescence intensity histograms as % of Max of data in (E). (F) Percentages of CCR2+ macrophages (right) and mean fluorescence intensity as % of max for CCR2 (left). The data in (B), (C), (E) and (F) represent mean ±SEM from all tumors in the groups, ***p<0.001, **p<0.01 and *p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3824525&req=5

Figure 4: Increased recruitment of ED1+ activated macrophage/microglia in NK+mAb9.2.27 treated tumors(A) ED1 labeling in P3-30 bearing animals in the tumor core vs. periphery in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals, (scale bar 100 μm, magnification 200 X). (B) The quantification of positive area fraction for ED1 and Iba1 in tumor core and periphery in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals. (C) Quantification of CD8+ area fraction in vehicle control, NK+mAb9.2.27 combination and NK+mAb9.2.27+clodronate treated animals (D) Flow cytometric representative dot plot showing the gating of cell populations from rat brain cell suspension expressing CD11b/c and CD45, permitting the differentiation of macrophages (CD45highCD11b/c+) and microglia (CD45lowCD11b/c+). (E) Upper panel, % microglia cells expressing MHC class II, ED1 and ED2. Lower panels, mean fluorescence intensity histograms as % of Max of data in (E). (F) Percentages of CCR2+ macrophages (right) and mean fluorescence intensity as % of max for CCR2 (left). The data in (B), (C), (E) and (F) represent mean ±SEM from all tumors in the groups, ***p<0.001, **p<0.01 and *p<0.05.
Mentions: The NK+mAb9.2.27 treatment increased the tropism of ED1+ cells into the tumor core compared to vehicle treated controls (Two-Way ANOVA t3.991, p=0.013, n=4) and macrophage depleted NK+mAb9.2.27 treated tumors (Two-Way ANOVA t4.276, p=0.01, n=4), (Fig. 4A and 4B). Iba1, the calcium binding protein expressed by activated and phagocytic macrophage/microglia was also elevated in the core of the combination treated tumors compared to vehicle treated control (Two-Way ANOVA t4.633, p=0.01, n=4), and macrophage depleted tumors (Two-Way ANOVA t3.235, p=0.0243, n=4), (Fig. 4B). Significant numbers of CD8+ cells were recruited into the tumor microenvironment after the NK+mAb9.2.27 treatment (One-Way ANOVA t7.482, p=0.0001, n=5), (Fig. 4C), as was the case for U87MG and U251-NG2 tumors. Remarkably, macrophage depletion by clodronate injection abrogated this ED1 recruitment compared to NK+mAb9.2.27 treated animals (Two-Way ANOVA t34.276, p=0.013, n=4). The CD8+ cells recruited to the NK+mAb9.2.27 treated tumor were significantly elevated compared to controls and NK+mAb9.2.27 with macrophage depletion (One-Way ANOVA 18.5, p=0.0001, n=5), (Fig. 4A-C).

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