br homogeneous staining of healthy
homogeneous staining of healthy human pancreas tissue can be readily observed. In addition, highly selective staining of the ductal adeno-carcinoma versus stroma in the human PDAC pancreas tissue is clear. Two diﬀerent grades of human PDAC pancreas tissue were examined. Fig. 3 shows PanIN tissue, moderately diﬀerentiated tissue, and poorly diﬀerentiated PDAC pancreas tissue stained with 1. The selective ac-cumulation in the ducts versus stroma is observed as the bright white spots (ducts with adenocarcinoma) and relatively dark regions (stroma tissue).
3.2. Staining protocol optimization using mouse tissue
Since the results using human tissue were consistent with those obtained with the KMC mouse model, we utilized mouse frozen sections to optimize the staining procedure. Since H&E staining, the gold stan-dard for FSA pathology, requires ca. 12 min to stain tissue, our aim was to shorten P 22077 1 processing time while maintaining PDAC se-lectivity and optical contrast. In order to evaluate S/B (PDAC/stroma) during optimization, we employed a modified Matlab code used by Barth et al. (2017)  to generate the area under a receiver operating characteristic (ROC) curves, with the AUC used as a metric of diagnostic performance in diﬀerentiating PDAC from stroma. For the KMC tissue used, the ROC curves, the AUC values are reported in Fig. 4 and Table 1, respectively. The z-stat analysis reported the 125 µM, 5 min incubation time AUC value as significantly higher than all other staining conditions (Table S1, Supplementary Information).
The initial fluorophore concentration reported by Wang et al. (2017)
 of 125 µM aﬀorded the highest AUC with a fluorophore incubation time of 5 min (Table 1). A short rinse instead of 5 min washing, plus a
Fig. 4. ROC curves for KMC mouse model ductal adenocarcinoma vs. stroma tissue diﬀerentiation following staining using 1 (125 μM).
Fig. 3. Whole tissue images of frozen sections (top panel) of healthy, PanIN, poorly moderately diﬀerentiated and moderately diﬀerentiated human normal and PDAC pancreas tissue stained with 1. in the bottom panel shows magnified images of healthy ducts (A) and PDAC (B- PanIN (lesions), C- poorly diﬀerentiated and D-moderately diﬀerentiated).
Tissue staining optimization ROC analysis. AUC values are listed for fluor-ophore concentration and incubation time optimization studies with KMC mouse model tissue. ROC AUC values.a
Fluorophore concentration (µM) Incubation time
N/A- not applicable
a ROC AUC values correspond to the area under the ROC curve for each incubation time with 1.
shortened incubation with 2% PFA (paraformaldehyde, 5 min com-pared to 15 min) did not significantly change the AUC (0.9390 vs. 0.9376 for original vs. optimized protocols).
Optimized protocol (15 min processing) results are represented in Fig. 5. The histogram of PDAC pancreas tissue stained with 1 displays a clear resolution of pixel values between the ductal adenocarcinoma and the stroma tissue. This is evidence that when 1 is used in the 15-min protocol it is eﬀective in selectively targeting and imaging PDAC. Conversely, a PDAC negative control was subjected to the same con-ditions except for the addition of 1. It shows an unresolved pixel in-tensity overlay corresponding to PDAC and stroma (Fig. 5, left).
Once the procedure was optimized with mouse tissue, it was applied to human frozen section samples. The optimized protocol was used to stain the four human pancreas tissue samples shown in Fig. 3 (the Fig. 2 images had been obtained using unoptimized conditions), including healthy and pancreatic intraepithelial neoplasia (PanIN, a PDAC pre-cursor lesion of potential utility for early disease detection), as well as moderately- and poorly-diﬀerentiated PDAC tissue. ROC-calculated AUC values were used to monitor the PDAC vs. stroma diagnostic performance (Fig. 6 and Table 2).
The primary aim of this study was to show the utility of an in-herently-targeted fluorophore in the ex vivo staining of human PDAC tissue. Residual tumor during PDAC resection results in low survival rates from PDAC surgery due to locoregional recurrence or follow-up surgeries to ensure clear margins. Intraoperative fluorescence guidance using PDAC-specific contrast agents has the potential to aid the process of margin assessment. Typically, fluorophores are conjugated to tar-geting ligands to increase specificity. Ex vivo staining with fluorophore-
Methods xxx (xxxx) xxx–xxx
Fig. 6. ROC curves for control (black), PanIN (green), high grade (brown) and low grade (red) human ductal adenocarcinoma vs. stroma tissue diﬀerentiation derived from staining with 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)