Tumor Paint: A Chlorotoxin:Cy5.5 Bioconjugate for Intraoperative Visualization of Cancer Foci Mandana Veiseh, 1 Patrik Gabikian, 2 S-Bahram Bahrami, 1 Omid Veiseh, 3 Miqin Zhang, 2,3 Robert C. Hackman, 1,4 Ali C. Ravanpay, 1,8 Mark R. Stroud, 1 Yumiko Kusuma, 1 Stacey J. Hansen, 1 DeborahKwok, 1 NinaM.Munoz, 1 RaymondW.Sze, 5 WilliamM.Grady, 6,10,11 NormanM.Greenberg, 1 Richard G. Ellenbogen, 2,9 and James M. Olson 1,4,7,8,9 1 Clinical Research Division, Fred Hutchinson Cancer Research Center; Departments of 2 Neurosurgery, 3 Material Science, 4 Pathology, 5 Radiology, 6 Medicine, and 7 Pediatrics, and 8 Program in Neurobiology and Behavior, University of Washington; 9 Children’s Hospital and Regional Medical Center; and 10 Puget Sound Health Care System, Seattle, Washington; and 11 Cancer Biology Department, Vanderbilt University Medical School, Nashville, Tennessee Abstract Toward the goal of developing an optical imaging contrast agent that will enable surgeons to intraoperatively distinguish cancer foci from adjacent normal tissue, we developed a chlorotoxin:Cy5.5 (CTX:Cy5.5) bioconjugate that emits near-IR fluorescent signal. The probe delineates malignant glioma, medulloblastoma, prostate cancer, intestinal cancer, and sarcoma from adjacent non-neoplastic tissue in mouse models. Metastatic cancer foci as small as a few hundred cells were detected in lymph channels. Specific binding to cancer cells is facilitated by matrix metalloproteinase-2 (MMP-2) as evidenced by reduction of CTX:Cy5.5 binding in vitro and in vivo by a pharmacologic blocker of MMP-2 and induction of CTX:Cy5.5 binding in MCF-7 cells following transfection with a plasmid encoding MMP-2. Mouse studies revealed that CTX:Cy5.5 has favorable biodistribution and toxicity profiles. These studies show that CTX:Cy5.5 has the potential to fundamentally improve intraoperative detection and resection of malignancies. [Cancer Res 2007;67(14):6882–8] Introduction For many types of cancer, the precision of surgical resection directly influences patient prognosis. Unfortunately, intraoperative identification of tumor margins or small foci of cancer cells remains imprecise or depends on surgical judgment. Thus, the extent of surgical resection is constrained by the requirement to avoid harming vital healthy structures. Nowhere is this more problematic than in the brain, where >80% of malignant cancers recur at the surgical margin (1). Despite advances in intraoperative monitoring and image guidance, postoperative scans sometimes reveal bulky residual tumor that may have been resected safely if the surgeon had improved tools to distinguish tumor tissue from normal brain (Supplementary Fig. S1). Recent advances in molecular biology, genomics, and proteomics have yielded information about molecules that are preferentially expressed in malignant cells compared with normal tissue. This information has the potential to fundamentally transform surgical oncology if used to specifically illuminate tumor cells with targeted molecular beacons. We developed and conducted preclinical evaluation of a molecular imaging bioconjugate composed of chlorotoxin (CTX) and Cy5.5. CTX is a 36 amino acid peptide with four disulfide bridges. CTX is thought to bind to a lipid raft-anchored complex that contains matrix metalloproteinase-2 (MMP-2), membrane type-I MMP, and transmembrane inhibitor of metalloproteinase-2 (TIMP2), as well as ClC-3 chloride ion channels, and other proteins (2, 3). Upon CTX binding, the complex is postulated to be internalized into the cell, eliminating the functional chloride ion channel (2, 3). Several studies showed that CTX binds preferentially to glioma cells compared with non-neoplastic cells or normal brain (2, 4, 5). A radiopharmaceutical bound to synthetic chlorotoxin, 131 I-TM-601, showed safety in phase I/II clinical trials for human brain cancer therapy based on an acceptable safety profile in preclinical studies (6). Cy5.5 is a fluorescent molecular beacon that emits photons in the near-IR (NIR) spectrum. Because photons of this wavelength are minimally absorbed by water or hemoglobin, NIR beacons are well suited for intraoperative imaging (7, 8). Previous attempts to image brain tumors by NIR have focused on targeting the probe to inflammatory microglia around the tumor or using probes that require proteolytic cleavage for activation (9, 10). The former approach is challenging because the presence of microglia correlates poorly, if at all, with margins of many brain tumors. Furthermore, to reduce perioperative brain edema, patients are treated with dexamethasone, which is a potent inhibitor of microglial activation. These factors, coupled with preference for a probe that does not require enzymatic cleavage, prompted us to focus on targeting Cy5.5 directly to cancer cells. This paper reports the development of CTX:Cy5.5 bioconjugate and the efficacy of this imaging agent in mouse models of glioma, medulloblastoma, prostate cancer, intestinal cancer, and sarcoma. We addressed the sensitivity of CTX:Cy5.5 for detecting cancer foci and metastases noninvasively and under simulated surgical operating conditions and conducted murine biodistribution and toxicity studies. We evaluated the role of MMP-2 in CTX:Cy5.5 binding to cancer cells. Materials and Methods Probe synthesis and analysis. Probe was synthesized using a mixture of CTX [Alomone Labs, 2 mg/mL in bicarbonate buffer (pH, 8.5)] and Cy5.5- NHS ester (Invitrogen, 10 mg/mL in anhydrous dimethyl formamide) at the molar ratio of 3:1 (dye/CTX). Conjugation was done in the dark at room temperature for 1 h. Unconjugated dye was removed by dialysis against PBS Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). M. Veiseh and P. Gabikian contributed equally. Requests for reprints: James M. Olson, Fred Hutchinson Cancer Research Center, Mailstop D4-100, 1100 Fairview Avenue N., Seattle, WA 98109. Phone: 206-667-7955; Fax: 206-667-2917; E-mail: jolson@fhcrc.org. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-3948 Cancer Res 2007; 67: (14). July 15, 2007 6882 www.aacrjournals.org Research Article Research. on February 12, 2016. © 2007 American Association for Cancer cancerres.aacrjournals.org Downloaded from