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Effective immuno-targeting of the IDH1 mutation R132H in a murine model of intracranial glioma.

Pellegatta S, Valletta L, Corbetta C, Patanè M, Zucca I, Riccardi Sirtori F, Bruzzone MG, Fogliatto G, Isacchi A, Pollo B, Finocchiaro G - Acta Neuropathol Commun (2015)

Bottom Line: Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant.In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261).Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF.

View Article: PubMed Central - PubMed

ABSTRACT
The R132H mutation of cytosolic isocitrate dehydrogenase (IDH1) is present in the majority of low grade gliomas.Immunotherapy in these tumors has an interesting, still unexploited, therapeutic potential, as they are less immunosuppressive than glioblastomas. Using site-directed mutagenesis we introduced the R132H mutation into the murine glioma cell line GL261,creating mIDH1-GL261. Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant. In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261). After intracranial injection, MRI suggested that the initial growth rate was slower in mIDH1-GL261 than p-GL261 gliomas but overall survival was similar. mIDH1-GL261 gliomas showed evidence of R132H expression and of intratumoral 2HG production (evaluated by MRS and LC-MS/MS). Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF. Control mice were injected with four ovalbumin peptides or vehicle. Mice with mIDH1-GL261 but not p-GL261 gliomas treated with mIDH1 peptides survived longer than controls; 25% of them were cured. Immunized mice showed higher amounts of peripheral CD8+ T cells, higher production of IFN-γ, and evidence of anti-mIDH1 antibodies.Immunizations led to intratumoral up-regulation of IFN-γ, granzyme-b and perforin-1 and down-regulation of TGF-β2 and IL-10. These results support the translational potential of immunotherapeutic targeting of gliomas carrying IDH1 mutations.

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Immunization with peptides targets IDH1-R132H and modulates the tumor microenvironment. a, b H&E of representative mIDH1-GL261 gliomas from vehicle and immunized mice confirms a strong vascularization an a higher presence of blood vessels in gliomas from mice treated with vehicle. c Gliomas from immunized mice show a lower cell density and absence of mIDH1 expression. d RT-PCR shows IDH1 expression in gliomas from control mice and a significant reduced expression in immunized mice. The relative expression of IDH1 was normalized with that detected in gliomas from pGL261. e, f RT-PCR on explanted gliomas shows that IFN-γ, granzyme (gzm)-b and perforin (prf)-1 were expressed at higher levels, TGF-β2 and IL-10 expression at lower levels in gliomas from immunized mice than controls. Data reported as peps are mean ± SD of the two groups of immunized mice.
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Fig4: Immunization with peptides targets IDH1-R132H and modulates the tumor microenvironment. a, b H&E of representative mIDH1-GL261 gliomas from vehicle and immunized mice confirms a strong vascularization an a higher presence of blood vessels in gliomas from mice treated with vehicle. c Gliomas from immunized mice show a lower cell density and absence of mIDH1 expression. d RT-PCR shows IDH1 expression in gliomas from control mice and a significant reduced expression in immunized mice. The relative expression of IDH1 was normalized with that detected in gliomas from pGL261. e, f RT-PCR on explanted gliomas shows that IFN-γ, granzyme (gzm)-b and perforin (prf)-1 were expressed at higher levels, TGF-β2 and IL-10 expression at lower levels in gliomas from immunized mice than controls. Data reported as peps are mean ± SD of the two groups of immunized mice.

Mentions: Gliomas from mice treated with vehicle maintained highly vascularized features showing large and medium size-vessels. In gliomas from immunized mice the number of blood vessels was reduced (Figure 4a, b) and was similar to pGL261-gliomas (Additional file 2: Figure S1d). HIF-1α and VEGF expression were significantly reduced in gliomas from immunized mice compared with controls (Additional file 2: Figure S3).Figure 4


Effective immuno-targeting of the IDH1 mutation R132H in a murine model of intracranial glioma.

Pellegatta S, Valletta L, Corbetta C, Patanè M, Zucca I, Riccardi Sirtori F, Bruzzone MG, Fogliatto G, Isacchi A, Pollo B, Finocchiaro G - Acta Neuropathol Commun (2015)

Immunization with peptides targets IDH1-R132H and modulates the tumor microenvironment. a, b H&E of representative mIDH1-GL261 gliomas from vehicle and immunized mice confirms a strong vascularization an a higher presence of blood vessels in gliomas from mice treated with vehicle. c Gliomas from immunized mice show a lower cell density and absence of mIDH1 expression. d RT-PCR shows IDH1 expression in gliomas from control mice and a significant reduced expression in immunized mice. The relative expression of IDH1 was normalized with that detected in gliomas from pGL261. e, f RT-PCR on explanted gliomas shows that IFN-γ, granzyme (gzm)-b and perforin (prf)-1 were expressed at higher levels, TGF-β2 and IL-10 expression at lower levels in gliomas from immunized mice than controls. Data reported as peps are mean ± SD of the two groups of immunized mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359524&req=5

Fig4: Immunization with peptides targets IDH1-R132H and modulates the tumor microenvironment. a, b H&E of representative mIDH1-GL261 gliomas from vehicle and immunized mice confirms a strong vascularization an a higher presence of blood vessels in gliomas from mice treated with vehicle. c Gliomas from immunized mice show a lower cell density and absence of mIDH1 expression. d RT-PCR shows IDH1 expression in gliomas from control mice and a significant reduced expression in immunized mice. The relative expression of IDH1 was normalized with that detected in gliomas from pGL261. e, f RT-PCR on explanted gliomas shows that IFN-γ, granzyme (gzm)-b and perforin (prf)-1 were expressed at higher levels, TGF-β2 and IL-10 expression at lower levels in gliomas from immunized mice than controls. Data reported as peps are mean ± SD of the two groups of immunized mice.
Mentions: Gliomas from mice treated with vehicle maintained highly vascularized features showing large and medium size-vessels. In gliomas from immunized mice the number of blood vessels was reduced (Figure 4a, b) and was similar to pGL261-gliomas (Additional file 2: Figure S1d). HIF-1α and VEGF expression were significantly reduced in gliomas from immunized mice compared with controls (Additional file 2: Figure S3).Figure 4

Bottom Line: Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant.In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261).Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF.

View Article: PubMed Central - PubMed

ABSTRACT
The R132H mutation of cytosolic isocitrate dehydrogenase (IDH1) is present in the majority of low grade gliomas.Immunotherapy in these tumors has an interesting, still unexploited, therapeutic potential, as they are less immunosuppressive than glioblastomas. Using site-directed mutagenesis we introduced the R132H mutation into the murine glioma cell line GL261,creating mIDH1-GL261. Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant. In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261). After intracranial injection, MRI suggested that the initial growth rate was slower in mIDH1-GL261 than p-GL261 gliomas but overall survival was similar. mIDH1-GL261 gliomas showed evidence of R132H expression and of intratumoral 2HG production (evaluated by MRS and LC-MS/MS). Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF. Control mice were injected with four ovalbumin peptides or vehicle. Mice with mIDH1-GL261 but not p-GL261 gliomas treated with mIDH1 peptides survived longer than controls; 25% of them were cured. Immunized mice showed higher amounts of peripheral CD8+ T cells, higher production of IFN-γ, and evidence of anti-mIDH1 antibodies.Immunizations led to intratumoral up-regulation of IFN-γ, granzyme-b and perforin-1 and down-regulation of TGF-β2 and IL-10. These results support the translational potential of immunotherapeutic targeting of gliomas carrying IDH1 mutations.

Show MeSH
Related in: MedlinePlus