Claudin-4 probe identifies triple negative breast cancer using multispectral optoacoustic tomography

Abstract Background: Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. The cancer is known for the lack of estrogen and her2 receptors. TNBC has a 5-year survival rate of 77% as for the other breast cancers averaging out at 93%. The CL-750 probes are made to identify the claudin-4 that is expressed in TNBC, making it easy to identify the cancer quickly so we can treat quickly. Methods: TNBC, aggressive pancreatic and kidney cancer cell lines were used to assess the expression of claudin-4 via western blot. Based upon the C-terminal fragment of Clostridium perfingens, we synthesized a 30 aa peptide. CL peptide was conjugated to amine reactive HiLite 750 nm fluorescent dye. The CL-750 probes were purified using dialysis to remove unattached dye. MDA-MB-468 (TNBC), MDA-MB-231 (TNBC), S2VP10 (pancreatic), and 293 (kidney) cells were incubated with CL-750 probes for 30 minutes and evaluated with near infrared-fluorescence using Odyssey imaging software. Using the treated cells were then scraped into agar tissue mimicking phantoms, the probes were assessed for optoacoustic activity using the MSOT (Multispectral optoacoustic tomography). Results: Using western blot to show expression of claudin-4 within cell lines the S2VP10 pancreatic (positive control) showed the most expression of claudin-4, while the 293 kidney cells showed a lesser amount. Both MDA-MB-468 and MDA-MB-231 TNBC cells showed the lowest amount of claudin-4 nonetheless both showed enough to continue with experiments. Cell line S2VP10 showed an extreme amount of binding to probe, while as the probe also bound to both smaller amount of claudin positive breast cancer cell line (MDA-MB-231) and larger amount of claudin breast cancer cell line (MDA-MB-468). Both CL-Short-750 and CL-Long-750 showed signal using MSOT. The CL-Long-750 showed 2x as much signal as CL-Short-750 making the Cl-long-750 probe more efficient in identifying claudin positive cell TNBC (p<0.05). Conclusion: Both CL-750-probes have the capability of identifying claudin-4 positive cell lines, but CL-Long-750 was more effective and had greater binding. Citation Format: Laycee Parsons, William M. MacCuaig, Lacey R. McNally. Claudin-4 probe identifies triple negative breast cancer using multispectral optoacoustic tomography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2390.