The pathological types of esophageal cancer are mainly divided into squamous cell carcinoma and adenocarcinoma, and esophageal squamous cell carcinoma (ESCC) is still dominant worldwide, accounting for more than 85 % of esophageal cancers and remaining a major cause of cancer-related death (Hori et al., 2024; Simba et al., 2023; Thrift, 2021). Owing to its atypical early symptoms and a lack of specific tumor markers, approximately 70 %–80 % of patients are diagnosed in the middle or late stage, at which point the tumor cannot be surgically removed, and the 5-year survival rate is only 15 %–25 % (Ajani et al., 2019; Dong et al., 2019; Ohashi et al., 2015). Therefore, it is necessary to explore specific targets or tools for ESCC early detection and early interventions.

Nucleic acid aptamers are short fragments of RNA or single-stranded DNA (ssDNA) that can specifically recognize and bind corresponding targets. These aptamers are typically obtained from oligonucleotide libraries containing many random sequences based on iterative selection using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method (Ellington and Szostak, 1990; Tuerk and Gold, 1990). Owing to their high specificity and affinity for different targets, including cells, proteins, bacteria, toxins, and parasites, aptamers are emerging as promising molecular tools for disease diagnosis and treatment (Hong et al., 2019; Juliano, 2016; Li et al., 2023a; Xiao et al., 2024; Zahedi et al., 2025). Cell-based SELEX (cell-SELEX) technology can improve the potential for the successful application of selected aptamers in vivo and can help to explore new biomarkers (Catuogno and Esposito, 2017; Li et al., 2023b; Manfredini et al., 2023; Sefah et al., 2010; Shangguan et al., 2006; Wong et al., 2025). In addition, it is desirable for the aptamer to be internalized immediately after binding to a receptor or other target on the surfaces of target cells, thereby allowing the aptamer or its loaded drug molecules to function in the cytoplasm or nucleus. For this purpose, researchers developed the ‘Cell-internalization SELEX’ method (Magalhaes et al., 2012; Thiel et al., 2012a; Thiel et al., 2012b; Thiel et al., 2015; Zhang et al., 2022a). The cellular-internalization properties of aptamers have been demonstrated to enhance their potential for translational applications, particularly in drug delivery (Agnello et al., 2021; Gopinath et al., 2016; He et al., 2020; He et al., 2021; Iaboni et al., 2016; Moosavian and Sahebkar, 2019; Pleiko et al., 2023; Thiel et al., 2015). Currently, research indicates that the aptamer-internalization mechanism mainly involves receptor-mediated endocytosis and micropinocytosis (Alamudi et al., 2021; Tawiah et al., 2017; Wan et al., 2019). The former is further divided into clathrin- and caveolin-mediated endocytosis, whereas the latter mechanism has only been confirmed during the internalization process of nucleolin aptamer AS1411 (Wan et al., 2019). Although the internalization mechanism of most aptamers is currently receptor-mediated endocytosis, some researchers believe that it is important to screen aptamers internalized through macropinocytosis because aptamers internalized via this pathway can freely shuttle between the nucleus and cytoplasm, avoiding the inefficient escape of aptamers from the endosomal compartment (Wan et al., 2019). Notably, aptamers targeting different cell types may have unique internalization mechanisms, providing an important theoretical basis for optimizing their functions (Li et al., 2018; Magalhaes et al., 2012; Wan et al., 2019; Yan and Levy, 2014). However, to date, no reports exist on the development of specific aptamers that can be internalized for ESCC. Therefore, the development of aptamers that are internalized by ESCC cells will contribute to the discovery of ESCC tumor markers, and this will also be helpful for targeted drug delivery. The KYSE450 cell line is highly invasive and prone to tumor formation in xenograft-model mice (Li et al., 2020; Liu et al., 2013). Therefore, here we used KYSE450 as target cells for aptamer screening. Using the cell-internalization selection strategy described, as in previous studies (Iaboni et al., 2016; Magalhaes et al., 2012; Thiel et al., 2012a, 2012b), we aimed to obtain a specific and internalizing aptamer targeting ESCC, enabling the targeted delivery of therapeutic agents to the cytoplasm. This will facilitate precision treatment for ESCC.