Development of Protein Biosensors Targeting Sialic Acid Variants to Detect Cancer — 81a — Owen Growney, Hala Bayoumi, Dr. Rachel Willand-Charnley

Cancers utilize the sugar sialic acid (Sia) to engage in immune evasion, multidrug resistance, and alter pro-inflammatory pathways, enhancing their survival. Notably, three non-canonical forms of Sia exist that are found on cancer’s cell surface: N-acetylneuraminic acid (Neu5Ac), 7,9-O-acetylated Neu5Ac, and 5-N-glycolylneuraminic acid (Neu5Gc). These variations are particularly overexpressed in lung and colon cancers, leading to the suppression of cytotoxicity of natural killer cells. The exact levels of the non-canonical Sias necessary to initiate these cancer mechanisms, however, are not well understood. This gap underscores the urgent need for specialized sensors that can identify and quantify each Sia form on cancer cells. Our project aims to develop a comprehensive set of sensors to quantify each cancer-associated Sia variant, ultimately defining the thresholds at which tumor mechanisms are activated or inhibited. By leveraging Sia-binding proteins, we constructed sensors capable of distinguishing the cancer-relevant forms of Sia. We achieved our objective by developing Sia sensors—SiaP, NanS, and SubB—each tagged with an enhanced green fluorescent protein (eGFP) which bind specifically to Neu5Ac, 7,9-O-Acetylated Neu5Ac, and Neu5Gc, respectively. We then quantified the Sia variants on the surface of lung cancer cells, which were prepared in collaboration with Dr. Willand-Charnley’s lab at SDSU, using our developed sensors. The binding of each sensor was measured using flow cytometric analysis, and the results were promising. SiaP_wt demonstrated significantly high selectivity towards cells expressing Neu5Ac after 2 trials. NanS_S19A showed significantly high selectivity for cells expressing 7,9-O-Acetylated Neu5Ac after 1 trial. A cell line expressing Neu5Gc was not created, but will be cultured in the future to determine selectivity of SubB. This collaborative effort highlights the importance of interdisciplinary research in overcoming the challenges posed by cancer’s complexity.

Black Hills State University
Dr. Yun-Seok Choi