Journal: EJNMMI Radiopharmacy and Chemistry
Article Title: [ 11 C]HSP990 PET as a translational tool to investigate the role of Hsp90 in tumours and support the development of Hsp90 therapeutics
doi: 10.1186/s41181-025-00386-z
Figure Lengend Snippet: In vitro cell binding. A MDA-MB-231, U87, LBT005, CME038 and NS/CT-2A cells were pre-incubated with vehicle (baseline) or 100 µM pan-selective Hsp90 inhibitors (block), followed by 250 kBq/mL [ 11 C]HSP990. Data are shown as %applied radioactivity bound to 1 × 10 5 cells B MDA-MB-231 and U87 cells were pre-incubated with 100 µM isoform-selective Hsp90 inhibitors (block): iHsp90α (A1, A2), iHsp90β (B1, B2), iGRP94 (C1, C2, PU-WS-13) and iTRAP1 (D3). Data are normalized to the baseline control conditions. Data in A-B are presented as mean ± SD, with individual values shown as dots C MDA-MB-231 and U87 cells were subjected to the DsiRNA-induced KD of Hsp90α, Hsp90β KD, or both isoforms 48 h before tracer incubation D B max values were calculated from saturation binding curves obtained by incubating MDA-MB-231 and U87 Hsp90 KD cells with increasing concentrations of [ 3 H]HSP990 E Hsp90α/β protein levels following DsiRNA KD were determined by WB and expressed as %KD. Data in C–E are normalized to the negative control and presented as mean ± SD; statistics: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P < 0.0001 (****), in ( A) , ( C), (D), (E) , P < 0.0001 for all treatments compared to control, unless otherwise specified
Article Snippet: The reference compound HSP990, ( R )-2-amino-7-(4-fluoro-2-(6-methoxypyridin-2-yl)phenyl)-4-methyl-7,8-dihydropyrido[4,3- d ]pyrimidin-5( 6H )-one, other Hsp90 inhibitors, including Onalespib, PU-H71, PU-WS13 and TAS-116, and HDAC6 inhibitor ACY-775 were purchased from commercial suppliers (Biorbyt, Selleckchem Chemicals, MedChem Express or CSNpharm), analyzed by liquid chromatography-mass spectrometry (LC–MS) and/or nuclear magnetic resonance (NMR) to confirm their identity and used without further purification.
Techniques: In Vitro, Binding Assay, Incubation, Blocking Assay, Radioactivity, Control, Negative Control
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![In vitro cell binding. A MDA-MB-231, U87, LBT005, CME038 and NS/CT-2A cells were pre-incubated with vehicle (baseline) or 100 µM pan-selective Hsp90 inhibitors (block), followed by 250 kBq/mL [ 11 <t>C]HSP990.</t> Data are shown as %applied radioactivity bound to 1 × 10 5 cells B MDA-MB-231 and U87 cells were pre-incubated with 100 µM isoform-selective Hsp90 inhibitors (block): iHsp90α (A1, A2), iHsp90β (B1, B2), iGRP94 (C1, C2, PU-WS-13) and iTRAP1 (D3). Data are normalized to the baseline control conditions. Data in A-B are presented as mean ± SD, with individual values shown as dots C MDA-MB-231 and U87 cells were subjected to the DsiRNA-induced KD of Hsp90α, Hsp90β KD, or both isoforms 48 h before tracer incubation D B max values were calculated from saturation binding curves obtained by incubating MDA-MB-231 and U87 Hsp90 KD cells with increasing concentrations of [ 3 H]HSP990 E Hsp90α/β protein levels following DsiRNA KD were determined by WB and expressed as %KD. Data in C–E are normalized to the negative control and presented as mean ± SD; statistics: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P < 0.0001 (****), in ( A) , ( C), (D), (E) , P < 0.0001 for all treatments compared to control, unless otherwise specified](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4755/pmc12474755/pmc12474755__41181_2025_386_Fig2_HTML.jpg)
![In vitro [ 11 C]HSP990 autoradiography. Sections were pre-incubated with vehicle (baseline) or 10 µM Hsp90 inhibitors (block) followed by 74 kBq/mL [ 11 C]HSP990 A B16.F10 melanoma, B PC3 prostate tumour, C NS/CT-2A glioma, D U87 glioblastoma, E MDA-MB-231breast tumour and F muscle tissues. Intensity is expressed as DLU/mm 2 ; data are shown as mean ± SD, NP = not performed](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4755/pmc12474755/pmc12474755__41181_2025_386_Fig3_HTML.jpg)
![[ 11 C]HSP990 biodistribution in subcutaneous MDA-MB-231 and U87 tumour xenograft mice. Ex vivo [ 11 C]HSP990 biodistribution in A MDA-MB-231 and B U87 tumour mice 60 min post-injection; mice were pre-treated with vehicle (baseline), or 10 mg/kg Hsp90 inhibitors (block) i.p. 60 min before [ 11 C]HSP990 injection, RBC and WBC stand to the red blood cells and white blood cells, respectively C In vivo [ 11 C]HSP990 µPET in U87 and MDA-MB-231 tumour mice; animals were pre-treated with vehicle (baseline), or 5 mg/kg Hsp90 inhibitors (block) i.p. 60 min before [ 11 C]HSP990 injection. Whole-body MIP of 15–90-min averaged dynamic [ 11 C]HSP990 and whole-body image of static 10 min [. 18 F]FDG scans 60 min p.i. are shown. White arrows indicate tumour location D Corresponding averaged tumour SUV TACs of U87 tumour mice. Data are shown as mean ± SD; significance is reported for block vs baseline condition, P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P < 0.0001 (****)](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4755/pmc12474755/pmc12474755__41181_2025_386_Fig4_HTML.jpg)
![[ 11 C]HSP990 biodistribution in orthotopic NS/CT-2A high grade glioma mice A In vivo [ 11 C]HSP990 µPET imaging studies; animals were pre-treated with vehicle (baseline), or 5 mg/kg HSP990 inhibitor (block) i.p. 60 min before [ 11 C]HSP990 injection; shown as whole-body MIP of 15–90-min averaged dynamic [ 11 C]HSP990 scans B Corresponding averaged brain SUV TACs. No significant difference between right (tumour) and left (healthy) cerebrums C Ex vivo [ 11 C]HSP990 biodistribution 60 min post-injection; mice were pre-treated with vehicle (baseline), or 10 mg/kg HSP990 inhibitor (block) i.p. 60 min before [ 11 C]HSP990 injection. Results are expressed as mean SUV ± SD. Significance is reported for block vs baseline condition, P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P < 0.0001 (****); no significant difference is observed right (tumour) and left (healthy) cerebrums](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4755/pmc12474755/pmc12474755__41181_2025_386_Fig5_HTML.jpg)