Fig 2: Target validation. A Hsp90 levels in the whole cell lysates and in CM after 20 h of serum starvation in U87 and MDA-MB-231 cell lines, GAPDH and Actin are used as both loading controls in lysates and as controls for cell contamination in the extracellular fraction, LDH is used as a control for cell death in CM. B Ratio of WB fluorescent signal in CM / total signal in lysates and CM expressed in %. C Flow cytometry analysis of U87 and D MDA-MB-231 cells. A small Hsp90-positive population indicated with a black arrow is observed in both cell lines if data are visualized in a histogram and if dead cells are not excluded from the analysis. Hsp90-positive cells are double positive for DAPI and Annexin V and number of Hsp90-positive cells is proportional to the level of cell death. Samples with 50% dead cells were used for better visual example, however the same is true in samples with low levels of cellular death. E Morphological analysis of different MDA-MB-231 cell populations acquired with imaging modality of BD FACSDiscover S8 and live/dead and Hsp90-positive populations determined using flow cytometry modality. Differences in membrane integrity are clearly visible across populations, with dead and Hsp90 positive cells lacking defined membrane compared to live cells. Fluorescent signal in yellow is located intracellularly in cells lacking defined membrane, with no membrane staining pattern observed in any cells. The statistical analysis was performed using unpaired t test and Welch’s test in GraphPad Prism v. 10 (GraphPad Software)

Fig 3: 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 [11C]HSP990. Data are shown as %applied radioactivity bound to 1 × 105 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 Bmax values were calculated from saturation binding curves obtained by incubating MDA-MB-231 and U87 Hsp90 KD cells with increasing concentrations of [3H]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

Fig 4: [11C]HSP990 biodistribution in subcutaneous MDA-MB-231 and U87 tumour xenograft mice. Ex vivo [11C]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 [11C]HSP990 injection, RBC and WBC stand to the red blood cells and white blood cells, respectively C In vivo [11C]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 [11C]HSP990 injection. Whole-body MIP of 15–90-min averaged dynamic [11C]HSP990 and whole-body image of static 10 min [.18F]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 (****)

Fig 5: Overview of all Hsp90 inhibitors used in this study. IC50 = half maximal inhibitory concentration, Ki = inhibition constant