EpCAM as multi-tumour target for near-infrared fluorescence guided surgery

Abstract

Background: Evaluation of resection margins during cancer surgery can be challenging, often resulting in incomplete tumour removal. Fluorescence-guided surgery (FGS) aims to aid the surgeon to visualize tumours and resection margins during surgery. FGS relies on a clinically applicable imaging system in combination with a specific tumour-targeting contrast agent. In this study EpCAM (epithelial cell adhesion molecule) is evaluated as target for FGS in combination with the novel Artemis imaging system.

Methods: The NIR fluorophore IRDye800CW was conjugated to the well-established EpCAM specific monoclonal antibody 323/A3 and an isotype IgG1 as control. The anti-EpCAM/800CW conjugate was stable in serum and showed preserved binding capacity as evaluated on EpCAM positive and negative cell lines, using flow cytometry and cell-based plate assays. Four clinically relevant orthotopic tumour models, i.e. colorectal cancer, breast cancer, head and neck cancer, and peritonitis carcinomatosa, were used to evaluate the performance of the anti-EpCAM agent with the clinically validated Artemis imaging system. The Pearl Impulse small animal imaging system was used as reference. The specificity of the NIRF signal was confirmed using bioluminescence imaging and green-fluorescent protein.

Results: All tumour types could clearly be delineated and resected 72 h after injection of the imaging agent. Using NIRF imaging millimetre sized tumour nodules were detected that were invisible for the naked eye. Fluorescence microscopy demonstrated the distribution and tumour specificity of the anti-EpCAM agent.

Conclusions: This study shows the potential of an EpCAM specific NIR-fluorescent agent in combination with a clinically validated intraoperative imaging system to visualize various tumours during surgery.

Keywords: Epithelial cell adhesion molecule; Image-guided surgery; Imaging agent; Near-infrared fluorescence; Optical imaging.

Fig. 1

EpCAM expression: EpCAM overexpression was present in all cell lines except in COLO320 cells. Expression of EpCAM was analyzed in human colon (a HT29-luc2, COLO320), breast (b MCF-7, MDA-MB231) and head & neck (c OSC-19, FaDu-luc2) cancer. Cells were incubated with an anti-EpCAM antibody (323/A3) or an isotype normal mouse IgG (MOPC21). After washing, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG antibody. The anti-mouse antibody was solely used as a control (conjugate)

Fig. 2

Conjugation, serum stability and EpCAM specificity: Conjugation of IRDye 800CW to 323/A3 and MOPC21 was done through NHS ester conjugation. A mean labeling ratio of 2.6 ± 0.98 and 2.8 ± 0.99 was obtained for 323/A3 and MOPC21 respectively a. EpCAM specificity of 323/A3 was confirmed after conjugation of IRDye 800CW on various cell types, corrected for cell number using the 800/700 nm ratio b. Experiments were done in human colon (HT29-luc2, COLO320), breast (MCF-7-luc2-cGFP, MDA-MB231) and head & neck (OSC-19-luc2-cGFP, FaDu-luc2) cancer. Cells were incubated with different concentrations of 323/A3-800CW or an isotype normal mouse IgG (MOPC21) conjugated to 800CW. To correct for the number of cells, the ratio of NIR fluorescence and the fluorescence intensity of TO-PRO-3 was plotted. 323/A3-800CW showed to be stable in human serum with more than 60% of the conjugate still free to bind EpCAM after 96 h with the remaining 40% aggregated or bound to albumin c

Fig. 3

EpCAM specificity and intra-operative tumor-to-background ratios (TBR’s): Competition with 1 mg of cold 323/A3 antibody significantly decreased the percentage of injected dose 323/A3-800CW per gram tumor (%ID/g) indicating in vivo tumor specificity a. After development of subcutaneous HT29-luc2 tumors, 1 nmol (150ug) of 323/A3-800CW, MOPC21-800CW and IRDye 800CW was injected intravenously. TBR’s were calculated from 4 h to 120 h after injection b. Subsequently, 1 nmol (150ug) 323/A3-800CW and MOPC21-800CW was intravenously injected in mice bearing orthotopic colon (HT29-luc2), breast (MCF-7-luc2-cGFP) and tongue (OSC-19-luc2-cGFP) tumors c. TBR’s were calculated 72 h post injection. TBR’s of EpCAM specific antibody 323/A3-800CW in colon, breast and head & neck cancer are significantly higher compared to those of the control antibody MOPC21-800CW

Fig. 4

Intra-operative NIR fluorescence delineation of colon, breast and Head & Neck cancer: Colon a, b breast c and head & neck tumors d could clearly be visualized during operation using EpCAM specific 323/A3-800CW (TBR colon 13.5, breast 6.7 and Head-and-neck 4.9) and the Artemis imaging system. 323/A3-800CW (A, C, D, 1 nmol) and the non-specific antibody MOPC21-800CW (B, 1 nmol) were intravenously injected. After an incubation of 72 h NIR fluorescence imaging was performed. Depicted images are: bright light, fluorescence, overlay (fluorescence and bright light), ex vivo fluorescence and ex vivo overlay. Scale bars are 5 mm. Artemis fluorescence intensities were intra-operatively optimized for optimal contrast. Intensities can only be compared within images

Fig. 5

Visualization of submillimeter sized intra-peritoneal metastases using EpCAM specific 323/A3-800CW in combination with the Artemis system: a MCF-7-luc2-cGFP cells were injected intraperitoneal in mice. After the development of multiple tumors, 1 nmol (150ug) 323/A3-800CW was injected intravenously. NIR fluorescence imaging was performed after 72 h of incubation using the Pearl and Artemis system. Confirmation of tumor tissue was performed by Green Fluorescent Protein (GFP) fluorescence imaging or Bioluminescence Imaging (BLI) using the IVIS spectrum. Micrometastases (arrow) of the colon could be discovered by NIR fluorescence imaging after resection of the peritoneum. b ROC curve analysis of TBR of fluorescent spots in the peritoneum. True positive rate was plotted versus false-positive rate using ascending positive cutoff values. B = bladder. Scale bars are 5 mm. Artemis fluorescence intensities were intra-operatively optimized for optimal contrast. Intensities can only be compared within images

Fig. 6

Histology: 323/A3-800CW is specifically located in head-and-neck a, b, breast c, and colon d tumors. Tumor tissue was sectioned 72 h after injection of 323/A3-800CW, MOPC21-800CW or IRDye 800CW. Shown are anti-GFP (brown, head-and-neck cancer, breast cancer) or cytokeratin (brown, colon cancer) immunohistochemistry stainings and overlays of NIR fluorescence (red) and anti-GFP/cytokeratin stainings. In all tissue a clear overlap is seen between NIR fluorescence from 323/A3-800CW and anti-GFP immunohistochemistry staining (indicating tumor). NIR fluorescence is mainly located in the border of tumors and even small tumor islands (*) are NIR fluorescent. A low, non specific fluorescence signal was observed in sections with MOPC21-800CW and IRDye 800CW (B). T = tumor; N = normal tissue; C = center tumor; B = tumor border. A and B 1× zoom; C and D 10× zoom