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

Differential macrophage/microglia phenotypes in response to treatment in U251-NG2 tumours(A) Upper panels, H&E staining showing tumor-brain boarders with varying amounts of large foamy cells (white) in response to mAb9.2.27 monotherapy and NK+mAb9.2.27 combination treatment (scale bar 200 μm, magnification 100 X). (A) Lower panels show higher magnification of the confrontation zone (scale bar 200 μm, magnification 200 X), (B) Upper panel show quantification of area fraction CD8+ cells in control, monotherapy NK cells, mAb9.2.27 as well as in NK+mAb9.2.27 combination treatment, lower panel shows quantification of area fraction ED1+ cells, in the same groups as above. Data represent mean ±SEM from all tumors in the groups. (C) Immunolabeling of CD8 shows abundant recruitment via vascular cuffs and choroids plexus in NK+mAb9.2.27 and mAb9.2.27 treatments, (arrowheads) respectively. Control and NK treated tumors show CD8+ cells that are restricted to tumor/brain periphery scale bar 200 μm, magnification 200X. (D) Double labeling for CD8 (brown) and ED1 (red) show abundant double positive macrophage/microglia in the tumor core (T) as well as those migrating from the brain (B, insert). mAb9.2.27 treated tumors show predominantly ED1 single positive, controls show CD8 single positive, and NK monotherapy shows fewer CD8/ED1 double positive cells that remain at the tumor/brain periphery (scale bar 100 μm, magnification 400 X).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Differential macrophage/microglia phenotypes in response to treatment in U251-NG2 tumours(A) Upper panels, H&E staining showing tumor-brain boarders with varying amounts of large foamy cells (white) in response to mAb9.2.27 monotherapy and NK+mAb9.2.27 combination treatment (scale bar 200 μm, magnification 100 X). (A) Lower panels show higher magnification of the confrontation zone (scale bar 200 μm, magnification 200 X), (B) Upper panel show quantification of area fraction CD8+ cells in control, monotherapy NK cells, mAb9.2.27 as well as in NK+mAb9.2.27 combination treatment, lower panel shows quantification of area fraction ED1+ cells, in the same groups as above. Data represent mean ±SEM from all tumors in the groups. (C) Immunolabeling of CD8 shows abundant recruitment via vascular cuffs and choroids plexus in NK+mAb9.2.27 and mAb9.2.27 treatments, (arrowheads) respectively. Control and NK treated tumors show CD8+ cells that are restricted to tumor/brain periphery scale bar 200 μm, magnification 200X. (D) Double labeling for CD8 (brown) and ED1 (red) show abundant double positive macrophage/microglia in the tumor core (T) as well as those migrating from the brain (B, insert). mAb9.2.27 treated tumors show predominantly ED1 single positive, controls show CD8 single positive, and NK monotherapy shows fewer CD8/ED1 double positive cells that remain at the tumor/brain periphery (scale bar 100 μm, magnification 400 X).

Mentions: Next, we aimed to identify the cell populations implicated and delineate the mechanisms mediating the therapeutic effect of the combination treatment. Histological analysis revealed striking clusters of large foamy cells at the periphery and within the core of the mAb9.2.27 and NK+mAb9.2.27 treated U251-NG2 tumors that were markedly diminished in the control and NK cell treated tumors (Fig. 2A). At perivascular cuffs and choroid plexus, CD8+ cells were most pronounced in the mAb9.2.27 monotherapy and NK+mAb9.2.27 treated tumors compared to the control (One-Way ANOVA F11.0, df=3, p=0.0011, n=5) and NK cell monotherapy tumors (One-Way ANOVA F11.0, p=0.0001, n=5), where the CD8+ cells were restricted to the tumor/brain periphery (Fig. 2B and 2C). No significant difference in the abundance of phagocytic ED1+ cells across the different treatment groups was found in the U251-NG2 tumors (Fig. 2B). However, the majority of CD8+ cells in the NK cell monotherapy and combination therapy tumors co-expressed ED1. Moreover, while CD8+ED1+ cells deeply infiltrated the combination treated tumors, these cells remained at the tumor/brain border in the NK cell monotherapy (Fig. 2D). In contrast, CD8− ED1+ cells densely infiltrated the mAb9.2.27 treated tumors, while the control tumors contained predominantly CD8+ ED1− cells (Fig. 2D). The tropism for CD8+ ED1+ cells in the combination treated tumors was also confirmed in the U87MG tumors (Supplementary Fig. 6A-D). CD8+ cells were more abundant in the NK+mAb9.2.27 treated tumors compared to monotherapy and controls (One-Way ANOVA 19.24, p=0.0002, n=5, (Supplementary Fig 4C). Likewise, ED1+ cells were most abundant after NK+mAb9.2.27 treatment (Two-Way ANOVA t3.928, p=0.0003, n=5), (Supplementary Fig 4D). As CD8 and ED1 markers can be expressed by both microglia and macrophages [27] that are difficult to distinguish by immunohistochemistry, we systemically depleted macrophages by intraperitoneal injection of clodronate once a week for 4 weeks, starting at the day of the treatment.


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)

Differential macrophage/microglia phenotypes in response to treatment in U251-NG2 tumours(A) Upper panels, H&E staining showing tumor-brain boarders with varying amounts of large foamy cells (white) in response to mAb9.2.27 monotherapy and NK+mAb9.2.27 combination treatment (scale bar 200 μm, magnification 100 X). (A) Lower panels show higher magnification of the confrontation zone (scale bar 200 μm, magnification 200 X), (B) Upper panel show quantification of area fraction CD8+ cells in control, monotherapy NK cells, mAb9.2.27 as well as in NK+mAb9.2.27 combination treatment, lower panel shows quantification of area fraction ED1+ cells, in the same groups as above. Data represent mean ±SEM from all tumors in the groups. (C) Immunolabeling of CD8 shows abundant recruitment via vascular cuffs and choroids plexus in NK+mAb9.2.27 and mAb9.2.27 treatments, (arrowheads) respectively. Control and NK treated tumors show CD8+ cells that are restricted to tumor/brain periphery scale bar 200 μm, magnification 200X. (D) Double labeling for CD8 (brown) and ED1 (red) show abundant double positive macrophage/microglia in the tumor core (T) as well as those migrating from the brain (B, insert). mAb9.2.27 treated tumors show predominantly ED1 single positive, controls show CD8 single positive, and NK monotherapy shows fewer CD8/ED1 double positive cells that remain at the tumor/brain periphery (scale bar 100 μm, magnification 400 X).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Differential macrophage/microglia phenotypes in response to treatment in U251-NG2 tumours(A) Upper panels, H&E staining showing tumor-brain boarders with varying amounts of large foamy cells (white) in response to mAb9.2.27 monotherapy and NK+mAb9.2.27 combination treatment (scale bar 200 μm, magnification 100 X). (A) Lower panels show higher magnification of the confrontation zone (scale bar 200 μm, magnification 200 X), (B) Upper panel show quantification of area fraction CD8+ cells in control, monotherapy NK cells, mAb9.2.27 as well as in NK+mAb9.2.27 combination treatment, lower panel shows quantification of area fraction ED1+ cells, in the same groups as above. Data represent mean ±SEM from all tumors in the groups. (C) Immunolabeling of CD8 shows abundant recruitment via vascular cuffs and choroids plexus in NK+mAb9.2.27 and mAb9.2.27 treatments, (arrowheads) respectively. Control and NK treated tumors show CD8+ cells that are restricted to tumor/brain periphery scale bar 200 μm, magnification 200X. (D) Double labeling for CD8 (brown) and ED1 (red) show abundant double positive macrophage/microglia in the tumor core (T) as well as those migrating from the brain (B, insert). mAb9.2.27 treated tumors show predominantly ED1 single positive, controls show CD8 single positive, and NK monotherapy shows fewer CD8/ED1 double positive cells that remain at the tumor/brain periphery (scale bar 100 μm, magnification 400 X).
Mentions: Next, we aimed to identify the cell populations implicated and delineate the mechanisms mediating the therapeutic effect of the combination treatment. Histological analysis revealed striking clusters of large foamy cells at the periphery and within the core of the mAb9.2.27 and NK+mAb9.2.27 treated U251-NG2 tumors that were markedly diminished in the control and NK cell treated tumors (Fig. 2A). At perivascular cuffs and choroid plexus, CD8+ cells were most pronounced in the mAb9.2.27 monotherapy and NK+mAb9.2.27 treated tumors compared to the control (One-Way ANOVA F11.0, df=3, p=0.0011, n=5) and NK cell monotherapy tumors (One-Way ANOVA F11.0, p=0.0001, n=5), where the CD8+ cells were restricted to the tumor/brain periphery (Fig. 2B and 2C). No significant difference in the abundance of phagocytic ED1+ cells across the different treatment groups was found in the U251-NG2 tumors (Fig. 2B). However, the majority of CD8+ cells in the NK cell monotherapy and combination therapy tumors co-expressed ED1. Moreover, while CD8+ED1+ cells deeply infiltrated the combination treated tumors, these cells remained at the tumor/brain border in the NK cell monotherapy (Fig. 2D). In contrast, CD8− ED1+ cells densely infiltrated the mAb9.2.27 treated tumors, while the control tumors contained predominantly CD8+ ED1− cells (Fig. 2D). The tropism for CD8+ ED1+ cells in the combination treated tumors was also confirmed in the U87MG tumors (Supplementary Fig. 6A-D). CD8+ cells were more abundant in the NK+mAb9.2.27 treated tumors compared to monotherapy and controls (One-Way ANOVA 19.24, p=0.0002, n=5, (Supplementary Fig 4C). Likewise, ED1+ cells were most abundant after NK+mAb9.2.27 treatment (Two-Way ANOVA t3.928, p=0.0003, n=5), (Supplementary Fig 4D). As CD8 and ED1 markers can be expressed by both microglia and macrophages [27] that are difficult to distinguish by immunohistochemistry, we systemically depleted macrophages by intraperitoneal injection of clodronate once a week for 4 weeks, starting at the day of the treatment.

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