Journal: bioRxiv

Article Title: A genetically encoded fluorescent sensor enables sensitive and specific detection of IDH mutant associated oncometabolite D-2-hydroxyglutarate

doi: 10.1101/2024.09.25.615072

Figure Lengend Snippet: (A) IDH1-R132H catalyzes the conversion of α-ketoglutarate to D-2-HG. L-2-HG is produced by lactate dehydrogenase (LDH) and malate dehydrogenase (MDH). (B) Representative in vitro D-2-HG and L-2-HG binding curves for purified D2HGlo. The average K d ’ ± standard deviation is shown for D-2-HG (blue) and L-2-HG (red) and represents 3-7 independent experiments. (C) The percent increase in FRET ratio above baseline levels in response to D-2-HG and L-2-HG at 1 µM (left) and 10 µM (right). The average ± standard deviation for each condition is shown for three independent experiments. Statistical analysis was performed using an unpaired t-test (****, P < 0.0001). (D) Representative in vitro D-2-HG binding curves for purified D2HGlo in a background of low concentrations of L-2-HG (1.5 – 5.5 µM, top) or high background concentrations of L-2-HG (15 – 55 µM, bottom). (E) The in vitro dynamic range of D2HGlo determined from three independent D-2-HG titrations in the presence of background L-2-HG ranging from 1.5 – 55 µM. (F) The K d ’ of D2HGlo for D-2-HG from three independent experiments performed in the presence of L-2-HG at concentrations ranging from 1.5 – 55 µM.

Article Snippet: U87MG, IDH1 -R132H mutant U87MG, and SVG (ATCC, Manassas, VA, CRL-8621) cells were plated at 10,000 cells per well in an opaque 96-well plate with complete media and incubation conditions as described previously.

Techniques: Produced, In Vitro, Binding Assay, Purification, Standard Deviation

Journal: bioRxiv

Article Title: A genetically encoded fluorescent sensor enables sensitive and specific detection of IDH mutant associated oncometabolite D-2-hydroxyglutarate

doi: 10.1101/2024.09.25.615072

Figure Lengend Snippet: (A) Left : Representative image of a wildtype IDH1 U87MG cell expressing Cyto-D2HGlo. Scale bar is 25 µm. Right : Comparison of the average FRET ratio in wildtype IDH1 and IDH1 -R132H mutant U87MG cells expressing Cyto-D2HGlo. The cells were grown in the presence or absence of the IDH1 -R132H inhibitor AG-120 (3 µM) for 48 hours prior to collecting images. The average ± standard deviation is shown for n = 59 cells obtained from four independent experiments (untreated WT), n = 48 cells obtained from three independent experiments (AG-120-treated WT), n = 61 cells from four independent experiments (untreated R132H) and n = 47 cells from three independent experiments (AG-120-treated R132H). (B) Left : Representative image of a wildtype IDH1 U87MG cell expressing Nuc-D2HGlo. Scale bar is 25 µm. Right : Comparison of the average FRET ratio in wildtype IDH1 and IDH1 -R132H mutant U87MG cells expressing Nuc-D2HGlo. Same treatments as panel A. The average ± standard deviation is shown for n = 59 cells obtained from four independent experiments (untreated WT), n = 45 cells obtained from three independent experiments (AG-120-treated WT), n = 58 cells from four independent experiments (untreated R132H) and n = 45 cells from three independent experiments (AG-120-treated R132H). (C) Left : Representative image of a wildtype IDH1 U87MG cell expressing Mito-D2HGlo. Scale bar is 25 µm. Right : Comparison of the average FRET ratio in wildtype IDH1 and IDH1 -R132H mutant U87MG cells expressing Mito-D2HGlo. Same treatments as panel A. The average ± standard deviation is shown for n = 47 cells obtained from three independent experiments (untreated WT), n = 45 cells obtained from three independent experiments (AG-120-treated WT), n = 48 cells from three independent experiments (untreated R132H) and n = 45 cells from three independent experiments (AG-120-treated R132H). (A-D) Statistical analysis was performed using a one-way ANOVA test with post hoc Tukey (****, P < 0.0001 compared with untreated WT, AG-120-treated WT and AG-120-treated R132H; *, P < 0.05 compared with AG-120-treated WT and AG-120-treated R132H).

Article Snippet: U87MG, IDH1 -R132H mutant U87MG, and SVG (ATCC, Manassas, VA, CRL-8621) cells were plated at 10,000 cells per well in an opaque 96-well plate with complete media and incubation conditions as described previously.

Techniques: Expressing, Comparison, Mutagenesis, Standard Deviation

Journal: bioRxiv

Article Title: A genetically encoded fluorescent sensor enables sensitive and specific detection of IDH mutant associated oncometabolite D-2-hydroxyglutarate

doi: 10.1101/2024.09.25.615072

Figure Lengend Snippet: (A) Comparison of the D-2-HG concentration in supernatants collected from wildtype IDH1 and IDH1 -R132H mutant U87MG cells. Cells were grown in the presence or absence of the IDH1 -R132H inhibitor AG-120 (3 µM) for 48 hours prior to collecting supernatant. (B) Titration of purified D2HGlo with increasing concentrations of D-2-HG (100 nM – 1 mM) spiked into human serum. (C) Average FRET ratio of D2HGlo in buffer, unspiked human serum and serum containing 1 µM, 3.2 µM or 10 µM D-2-HG. (D) Titration of purified D2HGlo with increasing concentrations of D-2-HG (100 nM – 1 mM) spiked into human urine. (E) Average FRET ratio of D2HGlo in buffer, unspiked human urine and urine containing 1 µM, 3.2 µM or 10 µM D-2-HG. (F) Titration of purified D2HGlo with increasing concentrations of D-2-HG (100 nM – 1 mM) spiked into aCSF. (G) Average FRET ratio of D2HGlo in buffer, unspiked aCSF and aCSF containing 1 µM, 3.2 µM or 10 µM D-2-HG. The K d ’ and dynamic range for panels B, D and F are the result of three independent replicates.

Article Snippet: U87MG, IDH1 -R132H mutant U87MG, and SVG (ATCC, Manassas, VA, CRL-8621) cells were plated at 10,000 cells per well in an opaque 96-well plate with complete media and incubation conditions as described previously.

Techniques: Comparison, Concentration Assay, Mutagenesis, Titration, Purification

Journal: bioRxiv

Article Title: A genetically encoded fluorescent sensor enables sensitive and specific detection of IDH mutant associated oncometabolite D-2-hydroxyglutarate

doi: 10.1101/2024.09.25.615072

Figure Lengend Snippet: (A) The D2HGlo FRET ratio is shown for twenty brain tumor samples. 5 samples were derived from IDH1 -R132H mutant individuals, and 15 were derived from wildtype IDH1 samples. A FRET ratio of 1.7 was used as a threshold to predict IDH1 mutational status. (B) The average FRET ratio for wildtype IDH1 compared with IDH1 –R132H mutants. Statistical analysis was performed using an unpaired t-test (****, P < 0.0001). (C) Table showing diagnosis, pathology report, raw FRET ratio, and D-2-HG concentration for all twenty brain tumor samples. This pathology report was provided prior to the release of the fifth edition of the World Health Organization (WHO) Classification of Tumors of the Central Nervous System (CNS5). While samples 2 and 3 are listed as glioblastoma, new classifications place gliomas with IDH mutations in a separate category from GBM .

Article Snippet: U87MG, IDH1 -R132H mutant U87MG, and SVG (ATCC, Manassas, VA, CRL-8621) cells were plated at 10,000 cells per well in an opaque 96-well plate with complete media and incubation conditions as described previously.

Techniques: Derivative Assay, Mutagenesis, Concentration Assay

Journal: bioRxiv

Article Title: A genetically encoded fluorescent sensor enables sensitive and specific detection of IDH mutant associated oncometabolite D-2-hydroxyglutarate

doi: 10.1101/2024.09.25.615072

Figure Lengend Snippet: (A) IDH1-R132H catalyzes the conversion of α-ketoglutarate to D-2-HG. L-2-HG is produced by lactate dehydrogenase (LDH) and malate dehydrogenase (MDH). (B) Representative in vitro D-2-HG and L-2-HG binding curves for purified D2HGlo. The average K d ’ ± standard deviation is shown for D-2-HG (blue) and L-2-HG (red) and represents 3-7 independent experiments. (C) The percent increase in FRET ratio above baseline levels in response to D-2-HG and L-2-HG at 1 µM (left) and 10 µM (right). The average ± standard deviation for each condition is shown for three independent experiments. Statistical analysis was performed using an unpaired t-test (****, P < 0.0001). (D) Representative in vitro D-2-HG binding curves for purified D2HGlo in a background of low concentrations of L-2-HG (1.5 – 5.5 µM, top) or high background concentrations of L-2-HG (15 – 55 µM, bottom). (E) The in vitro dynamic range of D2HGlo determined from three independent D-2-HG titrations in the presence of background L-2-HG ranging from 1.5 – 55 µM. (F) The K d ’ of D2HGlo for D-2-HG from three independent experiments performed in the presence of L-2-HG at concentrations ranging from 1.5 – 55 µM.

Article Snippet: IDH1 wildtype U87MG, CRISPR edited IDH1 -R132H Isogenic U87HTB-141G™ (ATCC, Manassas, VA), and HeLa cells (ATCC, Manassas, VA) were cultured in DMEM (Lonza, Portsmouth, NH) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA) and 1% penicillin/streptomycin/amphotericin B (PSA) (Lonza, Portsmouth, NH,) at 37°C and 5% CO 2 .

Techniques: Produced, In Vitro, Binding Assay, Purification, Standard Deviation

Journal: bioRxiv

Article Title: A genetically encoded fluorescent sensor enables sensitive and specific detection of IDH mutant associated oncometabolite D-2-hydroxyglutarate

doi: 10.1101/2024.09.25.615072

Figure Lengend Snippet: (A) Left : Representative image of a wildtype IDH1 U87MG cell expressing Cyto-D2HGlo. Scale bar is 25 µm. Right : Comparison of the average FRET ratio in wildtype IDH1 and IDH1 -R132H mutant U87MG cells expressing Cyto-D2HGlo. The cells were grown in the presence or absence of the IDH1 -R132H inhibitor AG-120 (3 µM) for 48 hours prior to collecting images. The average ± standard deviation is shown for n = 59 cells obtained from four independent experiments (untreated WT), n = 48 cells obtained from three independent experiments (AG-120-treated WT), n = 61 cells from four independent experiments (untreated R132H) and n = 47 cells from three independent experiments (AG-120-treated R132H). (B) Left : Representative image of a wildtype IDH1 U87MG cell expressing Nuc-D2HGlo. Scale bar is 25 µm. Right : Comparison of the average FRET ratio in wildtype IDH1 and IDH1 -R132H mutant U87MG cells expressing Nuc-D2HGlo. Same treatments as panel A. The average ± standard deviation is shown for n = 59 cells obtained from four independent experiments (untreated WT), n = 45 cells obtained from three independent experiments (AG-120-treated WT), n = 58 cells from four independent experiments (untreated R132H) and n = 45 cells from three independent experiments (AG-120-treated R132H). (C) Left : Representative image of a wildtype IDH1 U87MG cell expressing Mito-D2HGlo. Scale bar is 25 µm. Right : Comparison of the average FRET ratio in wildtype IDH1 and IDH1 -R132H mutant U87MG cells expressing Mito-D2HGlo. Same treatments as panel A. The average ± standard deviation is shown for n = 47 cells obtained from three independent experiments (untreated WT), n = 45 cells obtained from three independent experiments (AG-120-treated WT), n = 48 cells from three independent experiments (untreated R132H) and n = 45 cells from three independent experiments (AG-120-treated R132H). (A-D) Statistical analysis was performed using a one-way ANOVA test with post hoc Tukey (****, P < 0.0001 compared with untreated WT, AG-120-treated WT and AG-120-treated R132H; *, P < 0.05 compared with AG-120-treated WT and AG-120-treated R132H).

Article Snippet: IDH1 wildtype U87MG, CRISPR edited IDH1 -R132H Isogenic U87HTB-141G™ (ATCC, Manassas, VA), and HeLa cells (ATCC, Manassas, VA) were cultured in DMEM (Lonza, Portsmouth, NH) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA) and 1% penicillin/streptomycin/amphotericin B (PSA) (Lonza, Portsmouth, NH,) at 37°C and 5% CO 2 .

Techniques: Expressing, Comparison, Mutagenesis, Standard Deviation

Journal: bioRxiv

Article Title: A genetically encoded fluorescent sensor enables sensitive and specific detection of IDH mutant associated oncometabolite D-2-hydroxyglutarate

doi: 10.1101/2024.09.25.615072

Figure Lengend Snippet: (A) Comparison of the D-2-HG concentration in supernatants collected from wildtype IDH1 and IDH1 -R132H mutant U87MG cells. Cells were grown in the presence or absence of the IDH1 -R132H inhibitor AG-120 (3 µM) for 48 hours prior to collecting supernatant. (B) Titration of purified D2HGlo with increasing concentrations of D-2-HG (100 nM – 1 mM) spiked into human serum. (C) Average FRET ratio of D2HGlo in buffer, unspiked human serum and serum containing 1 µM, 3.2 µM or 10 µM D-2-HG. (D) Titration of purified D2HGlo with increasing concentrations of D-2-HG (100 nM – 1 mM) spiked into human urine. (E) Average FRET ratio of D2HGlo in buffer, unspiked human urine and urine containing 1 µM, 3.2 µM or 10 µM D-2-HG. (F) Titration of purified D2HGlo with increasing concentrations of D-2-HG (100 nM – 1 mM) spiked into aCSF. (G) Average FRET ratio of D2HGlo in buffer, unspiked aCSF and aCSF containing 1 µM, 3.2 µM or 10 µM D-2-HG. The K d ’ and dynamic range for panels B, D and F are the result of three independent replicates.

Article Snippet: IDH1 wildtype U87MG, CRISPR edited IDH1 -R132H Isogenic U87HTB-141G™ (ATCC, Manassas, VA), and HeLa cells (ATCC, Manassas, VA) were cultured in DMEM (Lonza, Portsmouth, NH) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA) and 1% penicillin/streptomycin/amphotericin B (PSA) (Lonza, Portsmouth, NH,) at 37°C and 5% CO 2 .

Techniques: Comparison, Concentration Assay, Mutagenesis, Titration, Purification

Journal: bioRxiv

Article Title: A genetically encoded fluorescent sensor enables sensitive and specific detection of IDH mutant associated oncometabolite D-2-hydroxyglutarate

doi: 10.1101/2024.09.25.615072

Figure Lengend Snippet: (A) The D2HGlo FRET ratio is shown for twenty brain tumor samples. 5 samples were derived from IDH1 -R132H mutant individuals, and 15 were derived from wildtype IDH1 samples. A FRET ratio of 1.7 was used as a threshold to predict IDH1 mutational status. (B) The average FRET ratio for wildtype IDH1 compared with IDH1 –R132H mutants. Statistical analysis was performed using an unpaired t-test (****, P < 0.0001). (C) Table showing diagnosis, pathology report, raw FRET ratio, and D-2-HG concentration for all twenty brain tumor samples. This pathology report was provided prior to the release of the fifth edition of the World Health Organization (WHO) Classification of Tumors of the Central Nervous System (CNS5). While samples 2 and 3 are listed as glioblastoma, new classifications place gliomas with IDH mutations in a separate category from GBM .

Article Snippet: IDH1 wildtype U87MG, CRISPR edited IDH1 -R132H Isogenic U87HTB-141G™ (ATCC, Manassas, VA), and HeLa cells (ATCC, Manassas, VA) were cultured in DMEM (Lonza, Portsmouth, NH) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA) and 1% penicillin/streptomycin/amphotericin B (PSA) (Lonza, Portsmouth, NH,) at 37°C and 5% CO 2 .

Techniques: Derivative Assay, Mutagenesis, Concentration Assay

Journal: Acta Neuropathologica Communications

Article Title: A patient-derived cell model for malignant transformation in IDH-mutant glioma

doi: 10.1186/s40478-024-01860-6

Figure Lengend Snippet: Case presentation. A Left panel: patient’s MRI obtained before (pre-op) and after (post-op) resection #1, when the tumor was low-grade astrocytoma (403L). Right panel: The same patient’s MRI obtained before (pre-op) and after (post-op) resection #3, when the tumor was transformed to high-grade astrocytoma (403H). B H&E staining showing tumor pathology and IHC analysis demonstrating Ki-67 expression, IDH1 R132H mutation, and ATRX loss (10 × magnification) in 403L and 403H patient’s tissues following 1st and 3rd resections, respectively. C . Timeline of disease diagnosis and treatment. Red arrows indicate that 403L and 403H patient-derived cell lines were cultured from the freshly resected tumors at resections #1 and #3, respectively

Article Snippet: The following antibodies were used: IDH1 R132H (Millipore Sigma, clone HMab-1, #MABC171), SOX2 (Cell Signaling, #3579), GFAP (Cell Signaling, #3670), OLIG2 (Abcam, #ab109186), NESTIN (Millipore Sigma, #MAB5326), beta 3-Tubulin (Cell Signaling, #5568), LDHA (Cell Signaling, #3582), LDHB (Santa-Cruz Biotechnology, sc-100775), and ACTIN (Cell Signaling, #4970).

Techniques: Transformation Assay, Staining, Expressing, Mutagenesis, Derivative Assay, Cell Culture

Journal: Acta Neuropathologica Communications

Article Title: A patient-derived cell model for malignant transformation in IDH-mutant glioma

doi: 10.1186/s40478-024-01860-6

Figure Lengend Snippet: In vitro characterization of the established LGG and HGG models. A 403L and 403H cell growth in 2D monolayer and 3D spheroid cultures (scale bar 400 µm). B 3D spheroid invasion assay in 403L and 403H measured by Celigo Image Cytometer over 24 h of 3D culture (scale bar 500 µm). C Invasion area measured by ImageJ in 403L and 403H at 0 h and 24 h. D Western blot analysis of 403L and 403H spheroids showing expression of IDH1 R132H, stem cell and lineage markers. E Immunofluorescent analysis of beta3-Tubulin (neuronal), O4 (oligodendroglial), and GFAP (astrocytic) lineage markers in 403L and 403H cells cultured as 2D monolayers in the culture media containing 1% FBS for 3 days. F MRI of the 403H orthotopic mouse model suggesting tumor growth. G H&E staining of the 403H murine tumors displaying tumor infiltration (left; scale bar 200 µm), polymorphic and bizarre cells (middle; scale bar 100 µm) as well as multiple atypic mitoses (right; scale bar 50 µm). H H&E (left), IHC staining of IDH1 R132H (middle) and ATRX (right) in 403H xenografts (top—scale bar 100 µm; bottom—scale bar 50 µm)

Article Snippet: The following antibodies were used: IDH1 R132H (Millipore Sigma, clone HMab-1, #MABC171), SOX2 (Cell Signaling, #3579), GFAP (Cell Signaling, #3670), OLIG2 (Abcam, #ab109186), NESTIN (Millipore Sigma, #MAB5326), beta 3-Tubulin (Cell Signaling, #5568), LDHA (Cell Signaling, #3582), LDHB (Santa-Cruz Biotechnology, sc-100775), and ACTIN (Cell Signaling, #4970).

Techniques: In Vitro, Invasion Assay, Cytometry, Western Blot, Expressing, Cell Culture, Staining, Immunohistochemistry