Intraoperative Confocal Laser Endomicroscopy Detects Prostate Cancer at the Single-Cell Level with High Specificity and in Real Time: A Preclinical Proof of Concept

Abstract

In prostate cancer (PCa) surgery, precise tumor margin identification remains challenging despite advances in surgical techniques. This study evaluates the combination of tumor-specific near-infrared imaging with the PSMA-targeting molecule PSMA-914 and optical endomicroscopy (NIR-pCLE) for single-cell-level tumor identification in a preclinical proof of concept. Methods: NIR-pCLE imaging of varying PSMA-914 concentrations was performed on PSMA-positive LNCaP and PSMA-negative PC-3 cells using Cellvizio^® 100 with pCLE Confocal Miniprobes™. To identify optimal PSMA-914 dosing for in vivo imaging, different doses (0-10 nmol) were evaluated using NIR-pCLE, Odyssey CLx imaging, and confocal microscopy in an LNCaP tumor-bearing xenograft model. A proof of concept mimicking a clinical workflow was performed using 5 nmol [⁶⁸Ga]Ga-PSMA-914 in LNCaP and PC-3 tumor xenografts, including PET/MRI, in/ex vivo NIR-pCLE imaging, and microscopic/macroscopic imaging. Results: NIR-pCLE detected PSMA-specific fluorescence at concentrations above 30 nM in vitro. The optimal dose was identified as 5 nmol PSMA-914 for NIR-pCLE imaging with cellular resolution in LNCaP xenografts. PET/MRI confirmed high tumor uptake and a favorable distribution profile of PSMA-914. NIR-pCLE imaging enabled real-time, single-cell-level detection of PSMA-positive tissue, visualizing tumor heterogeneity, confirmed by ex vivo microscopy and imaging. Conclusions: This preclinical proof of concept demonstrates the potential of intraoperative PSMA-specific NIR-pCLE imaging to visualize tissue structures in real time at cellular resolution. Clinical implementation could provide surgeons with valuable additional information, potentially advancing PCa patient care through improved surgical precision.

Keywords: PSMA; confocal laser endomicroscopy; guided surgery; intraoperative microscopy; prostate cancer.

Figure 1

Fluorescence imaging of screened PSMA-914 doses in LNCaP tumor tissue 2 h p.i.. Representative images of (A) NIR-pCLE (in vivo, scale bar 20 μm, fluorescence intensity in arbitrary units, gray scale adjusted to min and max counts), (B) Odyssey CLx (ex vivo whole organs, scale bar 5 mm, raw fluorescence data are shown, color-coded by photon counts per pixel), and (C) confocal microscopy (ex vivo tissue cryosections, scale bar 20 μm, raw fluorescence data are shown, color-coded by photon counts per pixel) of 0/0.5/1/2/3/4/5/10 nmol PSMA-914 administered to LNCaP tumor-bearing mice (0 nmol (n = 3), 0.5/1/2/3/4/10 nmol (n = 1), 5 nmol (n = 2)) in the context of the dose finding study.

Figure 2

Heterogeneity in LNCaP tumor tissue as visualized by confocal microscopy. Exemplary confocal images of tumor tissue cryosections from LNCaP tumor-bearing mice, 2 h p.i. of 5 nmol PSMA-914 (n = 5). Scale bar 20 μm. Raw fluorescence data are shown, color-coded by photon counts per pixel.

Figure 3

Small-animal PET/MRI of LNCaP and PC-3 tumor-bearing mice. Exemplary whole-body maximum intensity projections from small-animal PET/MR imaging of 5 nmol ⁶⁸Ga-labeled PSMA-914 administered into the right flank of (A) LNCaP- and (B) PC-3-tumor-bearing BALB/c nu/nu mice 2 h p.i. (n = 3, respectively; corresponding SUV values in Supplemental Table S12).

Figure 4

Multimodal imaging in the context of the in vivo proof-of-concept study. Representative images of (A) PET/MRI (coronal slices, in vivo, scale bar 2.5 mm, color scale in SUV values), (B) NIR-pCLE (in vivo, scale bar 20 μm, fluorescence intensity in arbitrary units, gray scale adjusted to min and max counts), (C) Odyssey CLx (ex vivo whole organs, scale bar 5 mm, raw fluorescence data are shown, color-coded by photon counts per pixel), and (D) confocal microscopy imaging (ex vivo tissue cryosections, scale bar 20 μm, raw fluorescence data are shown, color-coded by photon counts per pixel) of 5 nmol PSMA-914 in tumor, muscle, and kidney tissue of LNCaP and PC-3 xenograft-bearing mice, 2 h p.i. (n = 3, respectively).

Figure 5

Tumor-to-muscle ratio of the fluorescence signal in NIR-pCLE and confocal microscopy. Tumor-to-muscle ratio of the median fluorescence signal (NIR-pCLE, in vivo) or the integrated fluorescence intensity (confocal microscopy, ex vivo tissue cryosections) detected in LNCaP and PC-3 tumor and muscle tissue, 2 h p.i. of 0 (n = 3) or 5 nmol PSMA-914 (n = 5, respectively) with (A,B) NIR-pCLE, using (A) CholangioFlex^TM-C in vivo and (B) GastroFlex^TM UHD-C ex vivo, and (C) confocal microscopy in ex vivo cryosections. Means were compared using Student’s t test. p-Values < 0.05 (*) were considered statistically significant.