Intra-Operative Imaging of the Invasive Tumor Border by a Cathepsin-Sensing Near-Infrared Fluorescence Probe in a Syngeneic Breast Cancer Rat Model.
Authors: Mieog S. , van de Velde C. , Hutteman M. , Que I. , Keijzer R. , Kaijzel E. , Dijkstra J. , Kuppen P. , Lowik C. , Vahrmeijer A. 
Published: December 2010

Background: An incomplete tumor resection occurs in 5-45% of the patients undergoing breast-conserving surgery. As the surgeon can only rely on palpation and visual inspection, real-time visualization of cancer cells at the time of surgery is needed to increase the number of complete tumor resections. Near-infrared fluorescence (NIRF) imaging is a promising technique to assess the extent of disease during surgery. This study aimed to validate NIRF imaging in a breast cancer rat model.Methods: The hormone-dependent syngeneic breast cancer rat model EMR86 and its derived cell line MCR86 were used. Tumor cells were detected with the cathepsin-activatable (mainly cathepsin-B) NIRF probe ProSense® (VisEn Medical, USA). Fluorescence imaging of the animals, organs and tissue sections was performed using IVIS Spectrum (Caliper, USA), Odyssey (LI-COR, USA) and SP5 microscope (Leica, Germany). Intra-operative fluorescence imaging was performed using the Fluobeam700 system equipped with a 690 nm class 3B laser and a CCD camera (Fluoptics, France).Results: ProSense was activated by cleavage in MCR86 cells. The signal intensity was linearly correlated with the number of cells, the concentration of ProSense and the incubation time (all R2&gt;0.93, p&lt;0.0001). The influence of ProSense concentration and tumor volume on fluorescent intensity was confirmed in nine rats each bearing four tumors (mean volume=0.38 ±0.36 cm3). In contrast to the in vitro data, no difference in fluorescence intensity was found between 24hr and 48hr after injection of ProSense (paired t=-0.27, p=0.80). Therefore, subsequent experiments were conducted 24hr after injection. Calibration of the intra-operative camera system demonstrated a strong correlation of fluorescence intensity and activated ProSense concentration (R2=0.9903). Using the intra-operative NIRF camera, all 64 mammary tumors (0.01-1.8 cm3) were successfully detected. Histological assessment of residual fluorescent hotspots confirmed the presence of breast cancer cells indicating incomplete resections of the primary tumor (margin positivity). The signal intensity of the tumor was 60-fold higher than the surrounding tissue (p&lt;0.0001). Fluorescence scanning microscopy of 2-mm thick tumor sections revealed a significantly two-fold higher signal intensity at the tumor border compared to the tumor center (t=-8.12, p&lt;0.00001). Fluorescent microscopy of 10 um frozen tissue sections confirmed the higher intensity at the border, which was mainly located in the tumor-associated stroma.Conclusion: We demonstrated that it is possible to detect and resect sub-mm breast tumor depositions under fluorescent guidance in a syngeneic rat model. The higher fluorescent signal at the tumor border likely reflects the extra-cellular matrix degradation induced by increased cathepsin-B activity. Intra-operative imaging of cathepsin activity at the invasive border facilitates clear demarcation of tumor margins. Clinical introduction of this technique has the potential to substantially improve breast-conserving surgery.Acknowledgement: We want to thank Fluoptics for providing us with the Fluobeam700 system to perform the above described experiments.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5007.