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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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mIDH1-GL261 cells release 2HG in vitro and are tumorigenic. a RT-PCR analysis shows that IDH1 expression was significantly higher in mIDH1-GL261 compared to control cells. b Western blot analysis of IDH1-R132H expression in transfected GL261 cells after 15, 18 and 24 days of selection. Compared to empty (control cells), the levels of IDH1-R132H are significantly increased. Vinculin was used as housekeeping protein. c GL261 cells over-expressing IDH1-R132H selected for 24 days in the presence of G418 changed morphology compared to control cells. d Proliferation of pGL261 and mIDHI-GL261 as shown by WST proliferation assay performed at four different time points. e mIDH1-GL261 released 2HG in the cell supernatant as evaluated by LC-MS/MS. f mIDH1-GL261 when injected in vivo grew slower than control, however in the later stages the survival curves overlapped (n = 8 mice/group, p = 0.36). g, h, i MRI T2-weighted images confirmed the slower engraftment of mIDH1-GL261 glioma-bearing mice compared with controls at three different times. l RT-PCR performed on explanted gliomas shows that IDH1 overexpression was higher that controls at day 20 and begins to decrease 30 days after tumor implantation. m, n Representative IHC shows the expression of IDH1-R132H in gliomas from mIDH1-GL261 compared to control pGL261-gliomas where expression of IDH1 mutation was totally absent.
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Fig1: mIDH1-GL261 cells release 2HG in vitro and are tumorigenic. a RT-PCR analysis shows that IDH1 expression was significantly higher in mIDH1-GL261 compared to control cells. b Western blot analysis of IDH1-R132H expression in transfected GL261 cells after 15, 18 and 24 days of selection. Compared to empty (control cells), the levels of IDH1-R132H are significantly increased. Vinculin was used as housekeeping protein. c GL261 cells over-expressing IDH1-R132H selected for 24 days in the presence of G418 changed morphology compared to control cells. d Proliferation of pGL261 and mIDHI-GL261 as shown by WST proliferation assay performed at four different time points. e mIDH1-GL261 released 2HG in the cell supernatant as evaluated by LC-MS/MS. f mIDH1-GL261 when injected in vivo grew slower than control, however in the later stages the survival curves overlapped (n = 8 mice/group, p = 0.36). g, h, i MRI T2-weighted images confirmed the slower engraftment of mIDH1-GL261 glioma-bearing mice compared with controls at three different times. l RT-PCR performed on explanted gliomas shows that IDH1 overexpression was higher that controls at day 20 and begins to decrease 30 days after tumor implantation. m, n Representative IHC shows the expression of IDH1-R132H in gliomas from mIDH1-GL261 compared to control pGL261-gliomas where expression of IDH1 mutation was totally absent.

Mentions: To verify the potential effects of IDH1 mutation on tumor progression, we over-expressed IDH1-R132H in GL261 cells creating mIDH1-GL261. Expression of mIDH1 mRNA was detectable by RT-PCR in mIDH1-GL261 15 days after selection with G418, and highly increased on day 24 after transfection (Figure 1a). Accordingly, expression of the mutant protein was clearly observed from day 15 by Western blot with R132H mutation specific antibodies (Figure 1b). At the same time point mIDH1-GL261 cells appeared smaller that pGL261 and with fibroblast-like features (Figure 1c). No significant difference in proliferation was visible in vitro, as evaluated at four time points (24, 48, 72 and 96 h, Figure 1d). mIDH1-GL261 released 2HG in micromolar amounts in the culture medium, as measured by LC-MS/MS (Figure 1e).Figure 1


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)

mIDH1-GL261 cells release 2HG in vitro and are tumorigenic. a RT-PCR analysis shows that IDH1 expression was significantly higher in mIDH1-GL261 compared to control cells. b Western blot analysis of IDH1-R132H expression in transfected GL261 cells after 15, 18 and 24 days of selection. Compared to empty (control cells), the levels of IDH1-R132H are significantly increased. Vinculin was used as housekeeping protein. c GL261 cells over-expressing IDH1-R132H selected for 24 days in the presence of G418 changed morphology compared to control cells. d Proliferation of pGL261 and mIDHI-GL261 as shown by WST proliferation assay performed at four different time points. e mIDH1-GL261 released 2HG in the cell supernatant as evaluated by LC-MS/MS. f mIDH1-GL261 when injected in vivo grew slower than control, however in the later stages the survival curves overlapped (n = 8 mice/group, p = 0.36). g, h, i MRI T2-weighted images confirmed the slower engraftment of mIDH1-GL261 glioma-bearing mice compared with controls at three different times. l RT-PCR performed on explanted gliomas shows that IDH1 overexpression was higher that controls at day 20 and begins to decrease 30 days after tumor implantation. m, n Representative IHC shows the expression of IDH1-R132H in gliomas from mIDH1-GL261 compared to control pGL261-gliomas where expression of IDH1 mutation was totally absent.
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Fig1: mIDH1-GL261 cells release 2HG in vitro and are tumorigenic. a RT-PCR analysis shows that IDH1 expression was significantly higher in mIDH1-GL261 compared to control cells. b Western blot analysis of IDH1-R132H expression in transfected GL261 cells after 15, 18 and 24 days of selection. Compared to empty (control cells), the levels of IDH1-R132H are significantly increased. Vinculin was used as housekeeping protein. c GL261 cells over-expressing IDH1-R132H selected for 24 days in the presence of G418 changed morphology compared to control cells. d Proliferation of pGL261 and mIDHI-GL261 as shown by WST proliferation assay performed at four different time points. e mIDH1-GL261 released 2HG in the cell supernatant as evaluated by LC-MS/MS. f mIDH1-GL261 when injected in vivo grew slower than control, however in the later stages the survival curves overlapped (n = 8 mice/group, p = 0.36). g, h, i MRI T2-weighted images confirmed the slower engraftment of mIDH1-GL261 glioma-bearing mice compared with controls at three different times. l RT-PCR performed on explanted gliomas shows that IDH1 overexpression was higher that controls at day 20 and begins to decrease 30 days after tumor implantation. m, n Representative IHC shows the expression of IDH1-R132H in gliomas from mIDH1-GL261 compared to control pGL261-gliomas where expression of IDH1 mutation was totally absent.
Mentions: To verify the potential effects of IDH1 mutation on tumor progression, we over-expressed IDH1-R132H in GL261 cells creating mIDH1-GL261. Expression of mIDH1 mRNA was detectable by RT-PCR in mIDH1-GL261 15 days after selection with G418, and highly increased on day 24 after transfection (Figure 1a). Accordingly, expression of the mutant protein was clearly observed from day 15 by Western blot with R132H mutation specific antibodies (Figure 1b). At the same time point mIDH1-GL261 cells appeared smaller that pGL261 and with fibroblast-like features (Figure 1c). No significant difference in proliferation was visible in vitro, as evaluated at four time points (24, 48, 72 and 96 h, Figure 1d). mIDH1-GL261 released 2HG in micromolar amounts in the culture medium, as measured by LC-MS/MS (Figure 1e).Figure 1

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