Limits...
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.

Show MeSH

Related in: MedlinePlus

IFN-γ and mAb9.2.27 increase cytotoxicity of microglia against GBM(A) Upper panel: dot plot showing IFN-γ secretion in IL-2 activated NK cells in contact with U87MG cells, Lower panel: quantification of IFN-γ secretion in IL-2 activated NK cells co-cultured with U87MG or HF66 at ratio 2/1 for 18 h. (B) % lysis of U87MG in vitro by microglia from naïve LEWIS rats following activation with IgG2a isotype control, IFN-γ or/and mAb9.2.27. (C) % lysis of U87MG in vitro by NK cells in the presence of IgG2a isotype control or mAb9.2.27. (D-E) Ex vivo purified rat (D) and patient (E) macrophage/microglia from tumor microenvironment were investigated for cytotoxicity against P3-30 tumor pre-incubated or not with mAb9.2.27. Human TAMs were phenotyped for CD45+CD11b+CCR2 expression (E dot plot: CD45+ against CD11b+. E Histogram: isotype control grey histogram, CCR2 black histogram). Lower panel: % lysis of P3-30 tumor by ex vivo TAM from patient GBM following activation with IgG2a isotype control, or mAb9.2.27. (F) Cytotoxicity of rat TAMs was also investigated after 96 h (F) incubation with 100 ng/ml of IFN-γ. Cell viability was determined by flow cytometry using TOPRO-3 as supravital dye. Data are plotted as mean ±SEM, **p<0.01, *p<0.05. (G) Adoptively transferred NK cells as initiators of GBM destruction: schematic representation of the in vivo and in vitro experimental findings.In vivo: combination NK+mAb9.2.27 treatment leads to increased infiltration of the tumor by microglia and macrophages with pro-inflammatory phenotypes, with respectively ED1high ED2low MHC class IIhigh and ED1+ CCR2low molecular expression. This was associated with increase of pro-inflammatory cytokines in the CSF of NK+mAb9.2.27 treated animals, as well as diminution of cellular proliferation and increased tumor cell apoptosis. This resulted in increased animal survival that was abolished by depletion of systemic macrophages by injection of liposome-encapsulated clodronate.In vitro: activated NK cells induced cellular cytotoxicity against GBM (1). The NK cells/GBM interaction led to IFN-γ secretion by NK cells (2). This cytokine inhibited tumor survival promoted by tumor-associated macrophages/microglia (3). NK cells efficiently killed anti-inflammatory M2-like microglia (4). Moreover, mAb9.2.27 inhibited tumor survival mediated by tumor associated macrophages/microglia (5).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3824525&req=5

Figure 5: IFN-γ and mAb9.2.27 increase cytotoxicity of microglia against GBM(A) Upper panel: dot plot showing IFN-γ secretion in IL-2 activated NK cells in contact with U87MG cells, Lower panel: quantification of IFN-γ secretion in IL-2 activated NK cells co-cultured with U87MG or HF66 at ratio 2/1 for 18 h. (B) % lysis of U87MG in vitro by microglia from naïve LEWIS rats following activation with IgG2a isotype control, IFN-γ or/and mAb9.2.27. (C) % lysis of U87MG in vitro by NK cells in the presence of IgG2a isotype control or mAb9.2.27. (D-E) Ex vivo purified rat (D) and patient (E) macrophage/microglia from tumor microenvironment were investigated for cytotoxicity against P3-30 tumor pre-incubated or not with mAb9.2.27. Human TAMs were phenotyped for CD45+CD11b+CCR2 expression (E dot plot: CD45+ against CD11b+. E Histogram: isotype control grey histogram, CCR2 black histogram). Lower panel: % lysis of P3-30 tumor by ex vivo TAM from patient GBM following activation with IgG2a isotype control, or mAb9.2.27. (F) Cytotoxicity of rat TAMs was also investigated after 96 h (F) incubation with 100 ng/ml of IFN-γ. Cell viability was determined by flow cytometry using TOPRO-3 as supravital dye. Data are plotted as mean ±SEM, **p<0.01, *p<0.05. (G) Adoptively transferred NK cells as initiators of GBM destruction: schematic representation of the in vivo and in vitro experimental findings.In vivo: combination NK+mAb9.2.27 treatment leads to increased infiltration of the tumor by microglia and macrophages with pro-inflammatory phenotypes, with respectively ED1high ED2low MHC class IIhigh and ED1+ CCR2low molecular expression. This was associated with increase of pro-inflammatory cytokines in the CSF of NK+mAb9.2.27 treated animals, as well as diminution of cellular proliferation and increased tumor cell apoptosis. This resulted in increased animal survival that was abolished by depletion of systemic macrophages by injection of liposome-encapsulated clodronate.In vitro: activated NK cells induced cellular cytotoxicity against GBM (1). The NK cells/GBM interaction led to IFN-γ secretion by NK cells (2). This cytokine inhibited tumor survival promoted by tumor-associated macrophages/microglia (3). NK cells efficiently killed anti-inflammatory M2-like microglia (4). Moreover, mAb9.2.27 inhibited tumor survival mediated by tumor associated macrophages/microglia (5).

Mentions: In order to investigate the mechanisms of the synergistic mAb9.2.27 and NK cell cross-talk with microglia, the cytotoxicity of resting microglia and NK cells with and without mAb9.2.27 was tested in NG2/CSPG4 positive GBM cells in vitro. First, we observed that activated NK cells in contact with GBM cells produced IFN-γ, as revealed by intracellular flow cytometry (Two tailed T-test, NK cells alone vs. NK cells+U87MG p=0.0043, n=5 and vs. HF66 p=0.0095, n=4), (Fig. 5A). This was corroborated by increased IFN-γ and TNF-α released into the supernatant culture medium from NK cells in contact with GBM cells compared to supernatants from tumor cells only, (IFN-γ: One-Way ANOVA6.54, p=0.03; n=11; and TNFα: One-Way ANOVA 9.59, p=0.0083; n=11). Correspondingly, the killing capacity of resting microglia against U87MG GBM cells was increased by overnight activation with IFN-γ and against target pre-incubated with mAb9.2.27 compared to the cytotoxicity of resting microglia with IgG2a isotype control (IC) (One-Way ANOVA, p<0.05, n=5 and p<0.05, n=4 respectively), (Fig. 5B). Interestingly, there was no cumulative effect of IFN-γ and the mAb9.2.27 on the cytotoxicity of microglia. Indeed the cytotoxic capacity of microglia following activation with IFN-γ in absence of mAb9.2.27 was not significantly different from their killing capacity in presence of this mAb, with respectively 37.8 ±4.2 % and 43.2±5.9 % U87MG lysis (Fig. 5B). In contrast, there was no significant difference between cytotoxicity of IL-2 activated NK cells in the presence or absence of mAb9.2.27 against all GBM cell lines tested (Fig. 5C). The cytotoxicity of microglia and activated NK cells was also tested against the HF66 GBM cells lines, recognized by mAb9.2.27 at almost 100 %, and the previous results with U87MG were confirmed. Activation of microglia by IFN-γ, overnight or during 5 days rendered them highly cytotoxic against GBM, whereas culture with GBM conditioned medium promoted the survival of HF66 GBM cells, consistent with microglial M1- and M2-like physiology and phenotype, (Supplementary Fig. 5A and B respectively). As was the case for U87MG, the mAb9.2.27 abrogated the survival of HF66 tumor cells, mediated by M2-like differentiated microglia (Supplemental Fig. 5B). Moreover, NK cells preferentially killed the differentiated M2-like microglia, whereas M1-like microglia was more resistant to NK cell lysis (Supplementary Fig. 5C). Collectively, these data indicate that mAb9.2.27 could mediate ADCC by microglia but not by NK cells in vitro.


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)

IFN-γ and mAb9.2.27 increase cytotoxicity of microglia against GBM(A) Upper panel: dot plot showing IFN-γ secretion in IL-2 activated NK cells in contact with U87MG cells, Lower panel: quantification of IFN-γ secretion in IL-2 activated NK cells co-cultured with U87MG or HF66 at ratio 2/1 for 18 h. (B) % lysis of U87MG in vitro by microglia from naïve LEWIS rats following activation with IgG2a isotype control, IFN-γ or/and mAb9.2.27. (C) % lysis of U87MG in vitro by NK cells in the presence of IgG2a isotype control or mAb9.2.27. (D-E) Ex vivo purified rat (D) and patient (E) macrophage/microglia from tumor microenvironment were investigated for cytotoxicity against P3-30 tumor pre-incubated or not with mAb9.2.27. Human TAMs were phenotyped for CD45+CD11b+CCR2 expression (E dot plot: CD45+ against CD11b+. E Histogram: isotype control grey histogram, CCR2 black histogram). Lower panel: % lysis of P3-30 tumor by ex vivo TAM from patient GBM following activation with IgG2a isotype control, or mAb9.2.27. (F) Cytotoxicity of rat TAMs was also investigated after 96 h (F) incubation with 100 ng/ml of IFN-γ. Cell viability was determined by flow cytometry using TOPRO-3 as supravital dye. Data are plotted as mean ±SEM, **p<0.01, *p<0.05. (G) Adoptively transferred NK cells as initiators of GBM destruction: schematic representation of the in vivo and in vitro experimental findings.In vivo: combination NK+mAb9.2.27 treatment leads to increased infiltration of the tumor by microglia and macrophages with pro-inflammatory phenotypes, with respectively ED1high ED2low MHC class IIhigh and ED1+ CCR2low molecular expression. This was associated with increase of pro-inflammatory cytokines in the CSF of NK+mAb9.2.27 treated animals, as well as diminution of cellular proliferation and increased tumor cell apoptosis. This resulted in increased animal survival that was abolished by depletion of systemic macrophages by injection of liposome-encapsulated clodronate.In vitro: activated NK cells induced cellular cytotoxicity against GBM (1). The NK cells/GBM interaction led to IFN-γ secretion by NK cells (2). This cytokine inhibited tumor survival promoted by tumor-associated macrophages/microglia (3). NK cells efficiently killed anti-inflammatory M2-like microglia (4). Moreover, mAb9.2.27 inhibited tumor survival mediated by tumor associated macrophages/microglia (5).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: IFN-γ and mAb9.2.27 increase cytotoxicity of microglia against GBM(A) Upper panel: dot plot showing IFN-γ secretion in IL-2 activated NK cells in contact with U87MG cells, Lower panel: quantification of IFN-γ secretion in IL-2 activated NK cells co-cultured with U87MG or HF66 at ratio 2/1 for 18 h. (B) % lysis of U87MG in vitro by microglia from naïve LEWIS rats following activation with IgG2a isotype control, IFN-γ or/and mAb9.2.27. (C) % lysis of U87MG in vitro by NK cells in the presence of IgG2a isotype control or mAb9.2.27. (D-E) Ex vivo purified rat (D) and patient (E) macrophage/microglia from tumor microenvironment were investigated for cytotoxicity against P3-30 tumor pre-incubated or not with mAb9.2.27. Human TAMs were phenotyped for CD45+CD11b+CCR2 expression (E dot plot: CD45+ against CD11b+. E Histogram: isotype control grey histogram, CCR2 black histogram). Lower panel: % lysis of P3-30 tumor by ex vivo TAM from patient GBM following activation with IgG2a isotype control, or mAb9.2.27. (F) Cytotoxicity of rat TAMs was also investigated after 96 h (F) incubation with 100 ng/ml of IFN-γ. Cell viability was determined by flow cytometry using TOPRO-3 as supravital dye. Data are plotted as mean ±SEM, **p<0.01, *p<0.05. (G) Adoptively transferred NK cells as initiators of GBM destruction: schematic representation of the in vivo and in vitro experimental findings.In vivo: combination NK+mAb9.2.27 treatment leads to increased infiltration of the tumor by microglia and macrophages with pro-inflammatory phenotypes, with respectively ED1high ED2low MHC class IIhigh and ED1+ CCR2low molecular expression. This was associated with increase of pro-inflammatory cytokines in the CSF of NK+mAb9.2.27 treated animals, as well as diminution of cellular proliferation and increased tumor cell apoptosis. This resulted in increased animal survival that was abolished by depletion of systemic macrophages by injection of liposome-encapsulated clodronate.In vitro: activated NK cells induced cellular cytotoxicity against GBM (1). The NK cells/GBM interaction led to IFN-γ secretion by NK cells (2). This cytokine inhibited tumor survival promoted by tumor-associated macrophages/microglia (3). NK cells efficiently killed anti-inflammatory M2-like microglia (4). Moreover, mAb9.2.27 inhibited tumor survival mediated by tumor associated macrophages/microglia (5).
Mentions: In order to investigate the mechanisms of the synergistic mAb9.2.27 and NK cell cross-talk with microglia, the cytotoxicity of resting microglia and NK cells with and without mAb9.2.27 was tested in NG2/CSPG4 positive GBM cells in vitro. First, we observed that activated NK cells in contact with GBM cells produced IFN-γ, as revealed by intracellular flow cytometry (Two tailed T-test, NK cells alone vs. NK cells+U87MG p=0.0043, n=5 and vs. HF66 p=0.0095, n=4), (Fig. 5A). This was corroborated by increased IFN-γ and TNF-α released into the supernatant culture medium from NK cells in contact with GBM cells compared to supernatants from tumor cells only, (IFN-γ: One-Way ANOVA6.54, p=0.03; n=11; and TNFα: One-Way ANOVA 9.59, p=0.0083; n=11). Correspondingly, the killing capacity of resting microglia against U87MG GBM cells was increased by overnight activation with IFN-γ and against target pre-incubated with mAb9.2.27 compared to the cytotoxicity of resting microglia with IgG2a isotype control (IC) (One-Way ANOVA, p<0.05, n=5 and p<0.05, n=4 respectively), (Fig. 5B). Interestingly, there was no cumulative effect of IFN-γ and the mAb9.2.27 on the cytotoxicity of microglia. Indeed the cytotoxic capacity of microglia following activation with IFN-γ in absence of mAb9.2.27 was not significantly different from their killing capacity in presence of this mAb, with respectively 37.8 ±4.2 % and 43.2±5.9 % U87MG lysis (Fig. 5B). In contrast, there was no significant difference between cytotoxicity of IL-2 activated NK cells in the presence or absence of mAb9.2.27 against all GBM cell lines tested (Fig. 5C). The cytotoxicity of microglia and activated NK cells was also tested against the HF66 GBM cells lines, recognized by mAb9.2.27 at almost 100 %, and the previous results with U87MG were confirmed. Activation of microglia by IFN-γ, overnight or during 5 days rendered them highly cytotoxic against GBM, whereas culture with GBM conditioned medium promoted the survival of HF66 GBM cells, consistent with microglial M1- and M2-like physiology and phenotype, (Supplementary Fig. 5A and B respectively). As was the case for U87MG, the mAb9.2.27 abrogated the survival of HF66 tumor cells, mediated by M2-like differentiated microglia (Supplemental Fig. 5B). Moreover, NK cells preferentially killed the differentiated M2-like microglia, whereas M1-like microglia was more resistant to NK cell lysis (Supplementary Fig. 5C). Collectively, these data indicate that mAb9.2.27 could mediate ADCC by microglia but not by NK cells in vitro.

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