Cytoreductive surgery is a major procedure for treating advanced-stage epithelial ovarian cancer by removing tumors and nearby tissues to which the cancer lesions may have spread. However, current intraoperative procedures still rely heavily on the surgeon’s discretion, e.g. naked-eye gross inspection and palpation, which is highly variable. Large tumors can be quickly removed, but smaller lesions could be left behind. Incomplete resection can lead to disease recurrence, while overresection may cause surgical complications. Thus, fluorescence-guided surgery (FGS), an emerging technology utilizing a tumor-specific imaging probe, is actively being tested to augment the efficiency of intraoperative operations. Current tumor imaging probes are mostly delivered through blood vessels and may not reach all areas, resulting in undetected small lesions. Moreover, patients need to be hospitalized for imaging probe injection many hours, or even days before the actual surgical procedure, which extends their time in the hospital and adds additional cost of care. We recently developed a sprayable near-infrared (NIR) pH-responsive fluorogenic probe named CypH-11, which aims to sense viable tumor tissues in real-time for surgical guidance. At neutral pH, CypH-11 (pKa = 6.0) emits almost no fluorescence; however, it fluoresces brightly in an acidic environment which is an omnipresent consequence of cancer cell proliferation. In our previous studies, we demonstrated CypH-11’s feasibility for FGS in subcutaneous and orthotropic mouse models. In this study, for optimal peritoneal cytoreduction, we have improved the stability and signal intensity of CypH-11 by formulating CypH-11 with the nonionic ethoxylated solubilizer,  Kolliphor. CypH-11 in Kolliphor was stable for at least 7 days and its signal intensity was 1.4-fold higher than CypH-11 in PBS on exposure to high-grade serous ovarian cancer (HGSOC) OVSAHO cells. After topical application of CypH-11 in Kolliphor onto subcutaneous ovarian tumor xenografts, dye was absorbed immediately, and its fluorescence signal was developed within a minute. The signal-to-background ratio was 1.9 at 1 min, and remained constant over 10 mins. Thereafter, the use of CypH-11 spray was further validated in an orthotopic ovarian cancer model. After spraying CypH-11 in Kolliphor on the exposed abdomen of HGSOC OVCAR3/RFP-Luc tumor-bearing mice, the peritoneal tumors were not visible by naked eye under white light, but promptly and clearly visualized under Fluobeam 800, a clinically available Food and Drug Administration (FDA)-approved fluorescence imaging system. Impressively, after cytoreductive surgery aided by CypH-11, the NIR signals from the very tiny excised tumors, diameters of less than 2 mm, overlapped with RFP signals emanating from the tumor cells. This convenient fast-acting CypH-11 spray is promising as an effective adjuvant for real time use in FGS to allow the surgeon to see small and occult tumors thereby achieving a more complete resection of peritoneally disseminated tumors, potentially improving patient safety by avoiding unnecessary damage to normal tissue, and increasing survival of patients with HGSOC.

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Instantaneous visualization of peritoneal tumors using a pH-responsive near-infrared (NIR) fluorogenic spray.

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