Targeted elastin-like polypeptide fusion protein for near-infrared imaging of human and canine urothelial carcinoma
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Aayush Aayush1,3,*, Saloni Darji1,3,*, Deepika Dhawan2,3, Alexander Enstrom2, Meaghan M. Broman3,4, Muhammad T. Idrees5, Hristos Kaimakliotis6, Timothy Ratliff3,4, Deborah Knapp2,3 and David Thompson1,3
1 Department of Chemistry, Purdue University, Bindley Bioscience Center, West Lafayette, IN 47907, USA
2 Department of Veterinary Clinical Sciences, Purdue University, West Lafayette, IN 47907, USA
3 Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA
4 Department of Comparative Pathobiology, Purdue University, West Lafayette, IN 47907, USA
5 Department of Pathology and Laboratory Medicine, Indiana University, Indianapolis, IN 46202, USA
6 Department of Urology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
* These authors contributed equally to this work
Keywords: bladder cancer; elastin-like polypeptide; NIR imaging; epidermal growth factor receptor (EGFR); translational studies
Received: June 28, 2022 Accepted: August 06, 2022 Published: September 06, 2022
Cystoscopic visualization of bladder cancer is an essential method for initial bladder cancer detection and diagnosis, transurethral resection, and monitoring for recurrence. We sought to develop a new intravesical imaging agent that is more specific and sensitive using a polypeptide based NIR (near-infrared) probe designed to detect cells bearing epidermal growth factor receptors (EGFR) that are overexpressed in 80% of urothelial carcinoma (UC) cases. The NIR imaging agent consisted of an elastin like polypeptide (ELP) fused with epidermal growth factor (EGF) and conjugated to Cy5.5 to give Cy5.5-N24-EGF as a NIR contrast agent. In addition to evaluation in human cells and tissues, the agent was tested in canine cell lines and tissue samples with naturally occurring invasive UC. Flow cytometry and confocal microscopy were used to test cell-associated fluorescence of the probe in T24 human UC cells, and in K9TCC-SH (high EGFR expression) and K9TCC-Original (low EGF expression) canine cell lines. The probe specifically engages these cells through EGFR within 15 min of incubation and reached saturation within a clinically relevant 1 h timeframe. Furthermore, ex vivo studies with resected canine and human bladder tissues showed minimal signal from normal adjacent tissue and significant NIR fluorescence labeling of tumor tissue, in good agreement with our in vitro findings. Differential expression of EGFR ex vivo was revealed by our probe and confirmed by anti-EGFR immunohistochemical staining. Taken together, our data suggests Cy5.5-ELP-EGF is a NIR probe with improved sensitivity and selectivity towards BC that shows excellent potential for clinical translation.
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