B. R. Herring2, W. Jason2, S. Jang2, R. Jaskula-Sztul2, H. Chen2 2University Of Alabama at Birmingham,Department Of Surgery,Birmingham, Alabama, USA
Introduction: Ex vivo patient-derived xenografts have the potential to test personalized therapies prior to their administration to the patient. However, techniques to measure cancer cell proliferation in these models are lacking. In this study, we investigated a novel cancer-specific near-infrared (NIR) fluorescent dye, IR-783. Specifically, we hypothesized that IR-783 accurately measures neuroendocrine (NE) cancer cell proliferation in vitro and in vivo.
Methods: NE cancer cell lines (TT, H727, UMCII, MZ, and QGP1) and non-cancerous control cells (HEK293, WI-38 and 917) were plated in culture slides coated with fibronectin. Cells were then incubated with 20 uM IR-783 before fixation and images were acquired with confocal microscopy. Images of single cells were then obtained with an Imagestream Flow Cytometer, and their signal intensities were measured. Uptake of the dye in 2D culture was measured with an In Vivo Imaging System (IVIS) in 12 well plates containing increasing cell number, and the signal intensities were then compared to the results of the MTT assay. Furthermore, NE cancer cells stably transfected with Luciferase were subcutaneously injected into Nu/Nu mice, excised after 7wks of growth, and implanted into a 3D surrogate Bioreactor system for growth up to 20 days. IR-783 was added to the growth medium. The Bioreactor system was exposed to Luciferin before imaging with an IVIS for Luciferase activity and IR-783 uptake.
Results: IR-783 was retained to a higher degree in NE cancer cells compared to non-cancerous cells as detected by confocal microscopy and flow cytometry. NE cancer cells exhibited a mean maximum pixel intensity (mMPI) of 247 while non-cancerous control cells showed an mMPI of 103 (P=.015). In 2D culture, IR-783 signal intensity increased with increasing cancer cell density. This correlation was also shown in the 3D surrogate Bioreactor system (R2=0.49 and 0.96 for IR-783 signal and Luciferase activity, respectively)
Conclusion: As IR-783 is more avidly internalized by NE cancer cells compared to non-cancerous cells, it is a reliable indicator of changes in NE cancer cell number in both 2D culture and the 3D Bioreactor system. It could serve as a powerful tool for detecting the cytotoxic effects of drug candidates in the 3D Bioreactor system for NE cancer cells derived from patients.
