Fig 2: In vitro evaluation of EpCAM-AF680 as a NIRF imaging probe. In vitro imaging of Ishikawaluc+ cells using BLI and EpCAM-AF680 NIRF demonstrates comparable photonic linearity (A) and increase in signal (B) with higher number of cells (range: 0–106 cells). The lack of significant effects of in vitro incubation with EpCAM-AF680 antibody on proliferation and apoptosis demonstrated by MTS assay (C) and Annexin V/PI staining (D) in various cell lines. Abbreviations: Bioluminescent imaging (BLI), Epithelial cell adhesion molecule (EpCAM), Near-infrared fluorescent (NIRF), and Propium iodide (PI).

Fig 3: Optical imaging of tumor growth in an orthotopic Ishikawaluc+ xenograft model. In vivo BLI and EpCAM-AF680 NIRF imaging of primary tumor growth in mice orthotopically implanted with Ishikawaluc+ cells. Metastatic lesions (arrows) were detected at an earlier time point in EpCAM-AF680 NIRF images. A tumor free mouse was used as control (upper left) (A). Macroscopic images of uterus, pancreas, lungs and abdominal metastases (B, upper panel) and corresponding ex vivo BLI and EpCAM-AF680 NIRF images (B, middle panels). Tumor cells are demonstrated in H&E stained sections (20x magnification) (B, lower panel). Positive EpCAM expression in primary tumor and metastases is demonstrated by IHC (C). Abbreviations: Bioluminescent imaging (BLI), Epithelial cell adhesion molecule (EpCAM), Hematoxylin and eosin (H&E), Immunohistochemistry (IHC), and Near-infrared fluorescence (NIRF).

Fig 4: In vivo imaging of tumor growth in PDX models using EpCAM-AF680 NIRF and 18F-FDG PET/CT. Longitudinal monitoring of uterine tumors of different histologic types in PDX models using EpCAM-AF680 NIRF and 18F-FDG PET/CT imaging. Arrows mark probable uterine tumors in PET/CT images (A–D). H&E staining demonstrating uterine tumor cells, and positive EpCAM IHC staining of uterine tumors from both donor patients and mouse xenografts (20× magnification) (E–H). Large bladder removed from image for visualization purposes. An uncropped version of this image can be found in Figure S2. Abbreviations: Epithelial cell adhesion molecule (EpCAM), Fluorine-18-fluorodeoxyglucose (18FDG), Hematoxylin and eosin (H&E), Near-infrared fluorescence (NIRF), Patient-derived xenograft (PDX), Positron emission tomography/computed tomography (PET/CT), and Standardized uptake value (SUV).

Fig 5: In vivo monitoring of paclitaxel or trastuzumab treatment in an endometrial carcinoma PDX model using EpCAM-AF680 NIRF imaging. EpCAM-AF680 NIRF images of mice treated with control vehicle (n = 8), paclitaxel (n = 8) or trastuzumab (n = 8) prior to (day 26) and after (day 55) treatment. Pre-and post-treatment scans were paired and signal intensity scale synchronized for each individual mouse (A). Representative 18F-FDG PET/CT images (day 47), uterine tumors are indicated by arrows (B). Group means of total fluorescent signal before (day 26) and after (day 55) treatment, n = 8 mice per group (C). Group means of total fluorescent signal after treatment (day 55, n = 8 mice per group) compared to group SUVmean values measured in 18F-FDG PET/CT scans at day 47, control group: n = 6, paclitaxel group: n = 5, trastuzumab: n = 6 (D). Tumor weights after necropsy on day 49 (control group: mouse 1, paclitaxel group: mouse 2), day 51(control group: mice 3 and 8, trastuzumab group: mouse 2) or day 57 (all other mice), n = 8 in all groups (20× magnification) (E,F). Graphs are group means +/- SD. Abbreviations: Epithelial cell adherence molecule (EpCAM), Fluorine-18-fluorodeoxyglucose (18F-FDG), Metabolic tumor volume (MTV), Near infrared fluorescence (NIRF), Positron emission tomography/computed tomography (PET/CT), and Standardized uptake value (SUV).