PARP1-targeted fluorescence molecular endoscopy as novel tool for early detection of esophageal dysplasia and adenocarcinoma

Abstract

Background: Esophageal cancer is one of the 10 most common cancers worldwide and its incidence is dramatically increasing. Despite some improvements, the current surveillance protocol with white light endoscopy and random untargeted biopsies collection (Seattle protocol) fails to diagnose dysplastic and cancerous lesions in up to 50% of patients. Therefore, new endoscopic imaging technologies in combination with tumor-specific molecular probes are needed to improve early detection. Herein, we investigated the use of the fluorescent Poly (ADP-ribose) Polymerase 1 (PARP1)-inhibitor PARPi-FL for early detection of dysplastic lesions in patient-derived organoids and transgenic mouse models, which closely mimic the transformation from non-malignant Barrett's Esophagus (BE) to invasive esophageal adenocarcinoma (EAC).

Methods: We determined PARP1 expression via immunohistochemistry (IHC) in human biospecimens and mouse tissues. We also assessed PARPi-FL uptake in patient- and mouse-derived organoids. Following intravenous injection of 75 nmol PARPi-FL/mouse in L2-IL1B (n = 4) and L2-IL1B/IL8Tg mice (n = 12), we conducted fluorescence molecular endoscopy (FME) and/or imaged whole excised stomachs to assess PARPi-FL accumulation in dysplastic lesions. L2-IL1B/IL8Tg mice (n = 3) and wild-type (WT) mice (n = 2) without PARPi-FL injection served as controls. The imaging results were validated by confocal microscopy and IHC of excised tissues.

Results: IHC on patient and murine tissue revealed similar patterns of increasing PARP1 expression in presence of dysplasia and cancer. In human and murine organoids, PARPi-FL localized to PARP1-expressing epithelial cell nuclei after 10 min of incubation. Injection of PARPi-FL in transgenic mouse models of BE resulted in the successful detection of lesions via FME, with a mean target-to-background ratio > 2 independently from the disease stage. The localization of PARPi-FL in the lesions was confirmed by imaging of the excised stomachs and confocal microscopy. Without PARPi-FL injection, identification of lesions via FME in transgenic mice was not possible.

Conclusion: PARPi-FL imaging is a promising approach for clinically needed improved detection of dysplastic and malignant EAC lesions in patients with BE. Since PARPi-FL is currently evaluated in a phase 2 clinical trial for oral cancer detection after topical application, clinical translation for early detection of dysplasia and EAC in BE patients via FME screening appears feasible.

Keywords: Animal Models; Dysplasia; Esophageal Adenocarcinoma; Fluorescence Imaging; Fluorescence Molecular Endoscopy; PARP1.

Fig. 1

PARP1 expression along the BE to EAC transition in patient samples and in the L2-IL1B mouse model. A Human endoscopic submucosal resection samples (n = 14) were annotated according to their histopathology stage on HE-stained sections and PARP1 expression was analyzed within each category. In the displayed example, normal squamous epithelium (green), BE without high-grade Intraepithelial Neoplasia/IEN (white), BE with IEN (blue) and EAC (yellow) were present. B Quantification of the % PARP1 positive tissue area (pta) of all recorded 20 × fields-of-view and the mean value per category. * p < 0.05, ** p < 0.01, **** p < 0.0001 (Kruskal–Wallis-Test with Dunn’s correction for multiple comparisons). C Representative IHC images from the squamocolumnar junction (SCJ) of WT and L2-IL1B mice. In L2-IL1B mice, epithelial dysplastic cells (blue arrows) and inflammatory lymphocytes (orange arrows) were positive for PARP1 compared with WT mice, which did not present dysplastic epithelium and/or inflammation. D Quantification of PARP1 expression in murine SCJ tissues as % pta. PARP1 was significantly more expressed in dysplasia lesions graded 2 (p < 0.0001) and 3 (p = 0.0006) than normal areas without dysplasia (grade 0). Data are represented as single individually plotted values per field (3 WT mice and 2 to 4 L2-IL1B mice per group) and mean ± SEM. *** p < 0.001 and **** p < 0.0001 by one-way ANOVA followed by posthoc Tukey’s test

Fig. 2

PARP1 expression and PARPi-FL binding in human and murine organoids. A Schematic of procedure of the isolation of organoids from patient biopsies and staining with PARPi-FL for confocal microscopy. B Representative PARP1 IHC of human BE organoids. Consecutive z-stack images (step size 0.6 µm) of a representative human (C) or murine (D) organoid incubated with 0.5 µM PARPi-FL for 10 min. Green fluorescence = PARPi-FL

Fig. 3

Ex vivo wide-field imaging showed PARPi-FL accumulation in dysplastic lesions at the squamocolumnar junction (SCJ) of L2-IL1B mice. A Representative white light and fluorescence ex vivo images of the whole excised stomach of a PBS-injected control (WT mouse) and two PARPi-FL injected L2-IL1B mice and confocal microscopy of dysplastic lesions at the SCJ. Black arrows point to macroscopically visible lesions in the L2-IL1B mice. White dotted lines outline the SCJ and white squares are enlarged in the close-up image. B Quantification of PARPi-FL-related fluorescence of all lesions at the SCJ and background fluorescence intensity of each PARPi-FL-injected L2-IL1B mouse (single lesions and mean plotted). C Tumor-to-background ratio (TBR) of individual lesions in PARPi-FL-injected animals. Data are represented as single plotted values of each lesion ROI and mean ± SEM per mouse. D An incidental lesion at the esophagus showed similar PARPi-FL uptake (MFI ~ 15,810) to the SCJ

Fig. 4

PARPi-FL enabled lesion detection in L2-IL1B/IL8Tg mice with dysplastic lesions by fluorescence molecular endoscopy (FME). A Representative endoscopy frames of PARPi-FL injected and non-injected mice of different scores_comb. Gray-scale and intensity-coded false color images of selected frames are displayed. Displayed gray-scale images were normalized to the background (BG) by subtracting the average BG value from each whole image. Individual lesions are highlighted (orange dotted line). B Quantification of TBRs of all individually located lesions in PARPi-FL-injected mice by FME with mean ± SEM. C Quantification of the mean TBR of all lesions per mouse in PARPi-FL-injected mice with mean ± SEM. D Comparison of mean fluorescence intensity (MFI) measured in non-injected mice (n = 2) and the detected lesions in PARPi-FL-injected mice (n = 12). One non-injected mouse was excluded from the endoscopy quantification due to errors in setting acquisition parameters. In non-injected mice, the regions with the highest fluorescence detected by the endoscopy system were considered as a comparison. The quantification of the fluorescence was performed blindly. Significance levels were determined by one-way ANOVA followed by Dunnett’s multiple comparison test. * p < 0.05, ** p < 0.01 and **** p < 0.0001

Fig. 5

Ex vivo imaging following FME indicated that lesion detection during endoscopy in L2-IL1B/IL8Tg mice was PARPi-FL specific. A Ex vivo wide-field fluorescence images of excised stomachs from PARPi-FL-injected and non-injected mice that underwent FME. The SCJ is marked by dotted lines, while black arrows indicate the lesions. Only in PARPi-FL-injected mice, lesions in the brightfield (BF) were also visible in the FITC channel (arrows and close up images). B Confocal microscopy of the SCJ of the same mice confirmed nuclear PARPi-FL uptake. C Quantification of single lesions plotted per Tukey’s method with outliers and line at the median (left) and mean TBR per mouse (center) in PARPi-FL-injected mice by ex vivo wide-field imaging with mean ± SEM. ** p < 0.01 and **** p < 0.0001 by one-way ANOVA followed by Tukey’s test. D Mean fluorescence intensities (MFI) in macroscopically visible lesions measured in ex vivo wide-field imaging comparing non-injected with PARPi-FL injected mice. The horizontal line represented the mean MFI of PBS-injected mice. * p < 0.05 and **** p < 0.0001 by one way ANOVA followed by Tukey’s test. E Quantification of confocal microscopy analysis of sections at the SCJ of PARPi-FL-injected mice according to the dysplasia grade. No significant difference in PARPi-FL signal between dysplasia grade 2 and 3 was found (p = 0.149). Data are plotted as single values per each field and mean ± SEM (grade 0–1 = 1 mouse; grade 2 = 4 mice; grade 3 = 5 mice)