Extended Data Figure 6: HSP90 is functional and susceptible to exogenous inhibitors in type 2 as well as in type 1 cells, but only inhibition of HSP90 in type 1 cells is toxic to the cell.

a, The response of type 1 and 2 tumours classified by PU-PET avidity, to PU-H71 treatment, is shown. Patients were treated as part of the NCT01393509 clinical study. Each picture is a scan of data taken of an individual patient. PU-PET images were taken at 24 h after ¹²⁴I-PU-H71 tracer administration. Scale bars (bottom of panel); PET window display intensity scales for FDG and PU-PET fusion PET-CT images. Numbers in the scale bars indicate upper and lower SUV thresholds that define pixel intensity on PET images. b, Changes in HSP90 machinery function upon pharmacologic inhibition (PU-H71, 1 μM for 24 h). Inhibition of PI3K/AKT activity was monitored; see p-S6K surrogate for AKT activity in cell lines and p-AKT in primary specimens (below). Data in cell lines were repeated independently twice with representative data shown. For HSP90α/β knockdown data, see Extended Data Fig. 5g. c–f, Treatment schematic and representative examples of primary breast cancer specimens (n = 2) treated ex vivo with PU-H71. c, Workflow for the analysis of the primary specimens. d, Molecular signature of tumour and adjacent normal tissue of the surgical specimen as analysed by native, nanofluidic proteomic assay (NanoPro; native IEF), for HSP90 and HSP70 (gel representation), and AKT (chromatogram representation). e, Molecular response of tumour sections treated for 24 h ex vivo with PU-H71 (1 μM). AKT (an HSP90 client) activity was probed with the indicated antibody. BCL2 was chosen as a loading standard; this protein is insensitive to HSP90 inhibition in the analysed primary breast specimens (native IEF, chromatogram representation). f, Apoptotic response of the indicated tumour specimens to ex vivo treatment with PU-H71 or vehicle. Apoptosis and necrosis of the tumour cells (as percentage) is assessed by reviewing all the haematoxylin and eosin (H&E) slides of the case (controls and treated ones) in toto, blindly, allowing for better estimation of the overall treatment effect to the tumour. Image representative of the entire specimen section. g–j, Response profile of a panel of pancreatic cancer cells to HSP90 inhibition. g, Changes in cell viability following HSP90 pharmacologic inhibition by three chemically distinct agents, as indicated. Mean from two to three technical replicates is shown. Subclassification of the analysed cell lines by PU-FITC binding is shown on the left. h, The effect of PU-H71 on cell growth was measured with an assay that analyses intracellular ATP levels. Cells were treated for 72 h with PU-H71 and the half maximal inhibitory growth concentration (IC₅₀) was determined. Mean ± s.d.; n = 6. i, Representative examples of type 1 and type 2 cells treated for 24 h with the indicated concentrations of PU-H71. Inhibition of HSP90 is demonstrated by a decrease in HSP90 client function (p-S6K and p-ERK) and by HSP70 induction, and evidenced in both type 1 and 2 tumour cells. Induction of apoptosis, as demonstrated by the appearance of cleaved PARP (cPARP), is however, specific to type 1 tumour cells. β-actin, protein loading control. The HSP90 biochemical signature of the select cells is shown on the right. The blue arrows indicate the close relationship between the growth inhibitory IC₅₀ values and HSP90 function inhibition, suggesting that HSP90 inactivation is sufficient to inhibit growth (that is, have a static effect) in both type 1 and 2 tumours. In contrast, substantial induction of apoptosis is specific to type 1 tumours. Thus, HSP90 is functional in type 2 and is engaged by the HSP90 inhibitors—the resistance phenotype of type 2 tumours cannot be explained by an inability of the HSP90 inhibitor to engage HSP90. Data are representative of two independent experiments. For uncropped gel data, see Supplementary Fig. 1.