Clostridium perfringens enterotoxin C-terminal domain labeled to fluorescent dyes for in vivo visualization of micrometastatic chemotherapy-resistant ovarian cancer

Abstract

Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin-3- and -4-overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy-naïve and chemotherapy-resistant human ovarian cancer xenografts or patient-derived xenografts (PDXs) were treated with the carboxyl-terminal binding domain of the Clostridium perfringens enterotoxin (c-CPE) conjugated to FITC (FITC-c-CPE) or the near-infrared (NIR) fluorescent tag IRDye CW800 (CW800-c-CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC-c-CPE and the CW800-c-CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800-c-CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor-to-background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c-CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c-CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real-time detection of micrometastatic ovarian disease overexpressing the Claudin-3 and -4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment. What's new? Intraoperative detection of residual tumor is an emerging technique aimed at improving the prognosis of women with ovarian cancer. The authors developed a new system using the carboxy-terminal fragment of Clostridium Perfringens Enterotoxin (c-CPE) conjugated to fluorescent fluorophores to successfully mark microscopic tumor deposits in mouse models of chemotherapy-naïve and chemotherapy-resistant ovarian cancer. C-CPE binds to claudin 3/4 receptors, which the authors showed earlier are highly overexpressed on most types of ovarian cancer, thus providing a new tumor-specific tool to minimize micrometastic disease in patients with ovarian cancer.