HSP90-Specific nIR Probe Identifies Aggressive Prostate Cancers: Translation from Preclinical Models to a Human Phase I Study

Abstract

A noninvasive test to discriminate indolent prostate cancers from lethal ones would focus treatment where necessary while reducing overtreatment. We exploited the known activity of heat shock protein 90 (Hsp90) as a chaperone critical for the function of numerous oncogenic drivers, including the androgen receptor and its variants, to detect aggressive prostate cancer. We linked a near-infrared fluorescing molecule to an HSP90 binding drug and demonstrated that this probe (designated HS196) was highly sensitive and specific for detecting implanted prostate cancer cell lines with greater uptake by more aggressive subtypes. In a phase I human study, systemically administered HS196 could be detected in malignant nodules within prostatectomy specimens. Single-cell RNA sequencing identified uptake of HS196 by malignant prostate epithelium from the peripheral zone (AMACR⁺ERG⁺EPCAM⁺ cells), including SYP⁺ neuroendocrine cells that are associated with therapeutic resistance and metastatic progression. A theranostic version of this molecule is under clinical testing.

Figure 1.

Structure and characteristics of nIR dye–tethered Hsp90 inhibitor HS196. A, Chemical structures of HS196 and HS199. B, Absorption and emission peak of HS196. C, Fluorescence microscope analysis of HS196 uptake by PC-3 prostate cancer cells in vitro. PC-3 cells cultured in glass-bottomed dish were incubated with HS196 or HS199 (10 μmol/L in medium) for 1 hour, washed with the medium, and then fixed with 5% formalin for 30 minutes. WGA Alexa Fluor 488 membrane dye was applied to the dishes to stain cell membrane. Images were acquired using Zeiss LCM880 Confocal Laser Microscope. Membrane: green, nIR: red. Scale bar: 10 μm. D, Detection of the nIR signal of HS196-labeled cancer cells by flow cytometry. PC-3 cells were labeled with HS196 at 0, 1, 3, 10, 30, or 100 μmol/L for 30 minutes, and then washed with PBS three times. Cells were acquired by LSRII flow cytometry machine. Percentage of nIR-positive cells is shown in each histogram and median fluorescence intensity in the graph (n = 4 for each concentration). Red laser (633 nm) and 780/60 detector were used. E, nIR signal detection of HS196-labeled PC-3 cells in vivo. PC-3 cells were labeled with HS196 (10 μmol/L) for 30 minutes, and then washed with PBS three times. Different number (10M, 3M, 1M, 0.3M, 0.1M, and 0.03M) of HS196-labeled PC-3 cells or nonlabeled PC-3 cells was resuspended in 100 μL saline and subcutaneously injected to the flank of SCID-beige mice (yellow arrow: HS196-labeled PC-3, green arrow: nonlabeled PC-3). nIR signals were assessed for 3 injection sites of HS196-labeled PC-3 cells for each cell numbers by LI-COR Pearl (800-nm channel). Mean fluorescence signals are shown on the right. Error bar: SD.

Figure 2.

Imaging of human prostate cancer xenograft PC-3 in SCID-mice with Hsp90-targeted Probe HS196. A, SCID-beige male mice were subcutaneously implanted with PC-3 cells (1 × 10E6 cells/mouse) to the flank. When tumor size reached 8–10 mm in diameter, mice were administered HS196 or HS199 (10 nmol/inj) via tail-vein injection. Temporal dynamics of nIR signal from PC-3 tumor was analyzed using LI-COR Pearl Imager (800 nm channel). Six mice were tested for each probe. Representative 3 mice for each probe are shown. B, MFI of the background area was subtracted from MFI of tumor area and plotted for each group (N = 6 mice for each group). Error bar: SE. C,Ex vivo images of PC-3 tumors. Tumors were excised from mice 24 hours after the administration of HS196/HS199 (10 nmol/mouse) or no HS injection (untreated control). N = 4 tumors for each group. Error bar: SD. One-way ANOVA test was performed with Tukey multiple comparison adjustment. ****, P < 0.0001. D, Confocal microscope images of PC-3 tumors excised from mice 6 hours after the administration of HS196 or HS199 (10 nmol/mouse) via tail-vein injection. Membrane: green, nIR: red. Scale bar: 10 μm. E, Flow cytometry of single-cell digests. Six hours after the administration of HS196 or HS199 (10 nmol/mouse, n = 3 mice for each), mice were euthanized and harvested tumors were minced and digested with collagenase III/hyaluronidase/DNase for 1 hour at 37°C, and washed with PBS. Isolated cells were labeled with live/dead dye, followed by FITC-conjugated anti-HLA class 1 mAb. Cells were acquired using LSRII flow cytometer, and nIR signals in alive HLA-positive PC-3 cells were analyzed using red laser, APC-Cy7 channel with 780/60 filter. Representative histograms for each probe are shown, and percentages of nIR-positive cells among PC-3 tumor cells are shown in the right graph. Untreated tumor sample (tumor from a mouse without probe injection) is shown as a reference. Error bar: SD. Student t test was performed between HS196 and HS199 groups. ****, P < 0.0001. F, Comparison of in vivo HS196 uptake by 4 different prostate cancer lines with different metastatic capacities. One million cells of PC-3, DU145, 22Rv1, or LNCaP cell line were resuspended in 50% Matrigel/50% saline and subcutaneously implanted to the flank of male SCID-beige mice (PC-3: 6 mice, DU145: 8 mice, 22Rv1: 5 mice, LNCaP: 3 mice). When tumor size reached approximately 10 mm in diameter, HS196 (10 nmol/mouse) were administered via tail-vein injection. nIR signals were analyzed as described above until 24 hours. Representative whole body images of 3 mice for each prostate cancer xenograft at 6 hour time point are shown. Mean fluorescence intensities of tumor areas are plotted for each prostate tumor line. Error bar: SE.

Figure 3.

Preclinical PK-PD analysis. 1 mg/kg of HS196 was intravenously injected to tumor-bearing SCID-beige mice. Blood and organs/tissues were collected from 3 mice for each time point (5 minutes, 15 minutes, 30 minutes, 1.5 hours, 3 hours, 8 hours , 24hours , 72 hours, and 168 hours). Plasma was isolated and stored at −80^oC until the analysis. Organs/tissues were cryopulverized and homogenized and then stored at −80^oC. Plasma and tissue homogenate were analyzed by LC/MS/MS for HS196 level.

Figure 4.

Preoperative MR images and ex vivo imaging of resected prostate tissue. Preoperative axial MR images of the prostate (T2-weighted and diffusion-weighted), gross images, and nIR images of resected prostate tissues analyzed by LI-COR Odyssey imager are shown. In MR images, T2-weighted and diffusion-weighted hypointense prostate cancer lesions are marked with asterisks at the center and blue arrow heads at the margin (left 2 columns). Resected prostate tissues were cut, and gross images and nIR images of cut surfaces, analyzed using LI-COR Odyssey 800 nm channel, are shown (right 2 columns). Prostate cancer lesions are marked with asterisks and arrow heads.

Figure 5.

Single-cell RNA-seq and confocal microscopy of prostate cancer specimen. A, Process of sample preparation for single-cell RNA-seq of prostate cancer specimen. HS196⁺ and HS196⁻ cell populations were isolated from enzymatically digested prostate specimen by flow-based sorting, and single-cell cDNA libraries were made and sequenced. B,EPCAM⁺ epithelial cells were reclustered and shown in UMAP plot. Six different clusters were identified. C, Cells in peripheral zone and in transition/central zone were identified based on gene expression of KLK3, KRT3, and SCGB1A1. Clusters 1 and 4 consist the peripheral zone, whereas other clusters are in the transition/central zone. D, Percentages of each cluster (clusters 1–6) in EPCAM⁺ cells in HS196⁺ and HS196⁻ samples are shown. E, Expression of known prostate tumor markers by each cell/cluster is shown in the heatmap. Cells in the peripheral zone (clusters 1 and 4) are on the left side of the heatmap, and cells in the transition/central zone (clusters 2, 3, 5, and 6) are on the right side. AMACR expression is highlighted. F, HS196 positivity of AMACR⁺ prostate cancer cells (total 81 cells among 984 EPCAM⁺ cells) is shown. G, Serial frozen sections of the prostate specimen were used for H&E staining and RNA FISH. H&E pathology was coregistered with HS196 signal and an mRNA FISH image of AMACR. H, In the area of invasive prostate cancer, the presence of HS196-positive (magenta) and AMACR-expressing (green) prostate cancer cells was confirmed.