Petrillo, Gianluca (2025) Selection of aptamers targeting ALK+ NSCLC exosomes: innovative tools for cancer diagnosis and therapy. [Tesi di dottorato]

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Abstract

Among Non-Small Cell Lung Cancers (NSCLC), Anaplastic Lymphoma Kinase (ALK) gene rearrangements occur in 3-7% of cases. Although effective ALK inhibitor drugs (ALKi) are now available, often patients relapse after the initial response. Moreover, actual diagnostic technologies fail to detect the tumor in the early stages of the disease. To address the limits of tissue-based diagnosis, liquid biopsy has drawn attention. In my thesis project I focused on Extracellular vesicles (EV), lipidic vesicles secreted into body fluids by cancer cells since the first stages of tumorigenesis, thus playing an important role as potential cancer biomarkers. EV, through their content, may also participate in the mechanisms of tumor progression by several means. Thus, in this study we aimed at developing new strategies to target NSCLC exosomes for both therapy and diagnosis. To this end, we exploited aptamers, an innovative nucleic acid-based tool selected by an in vitro combinatorial chemistry approach, named Systematic Evolution of Ligands by Exponential Enrichment (SELEX). To identify aptamers binding ALK+ NSCLC exosomes, we used a 2-step cell SELEX (Systematic Evolution of Ligands by EXponential Enrichment). We first enriched a starting random RNA library for aptamers discriminating between NSCLC (A549) and normal lung epithelial (BEAS-2B) cells. Then, to select the sequences of the pool specifically recognizing tumor-associated antigens typical of ALK+ NSCLC, we switched A549 as counterselection steps and introduced in selection an isogenic A549 cell line CRISPR modified to express EML4-ALK rearrangement (EML4-ALK Fusion-A549), so that the two cell lines only differ for proteins whose expression is driven by the ALK oncogene. The final aptamer pools were tested and showed high affinity and specificity for ALK+ NSCLC cells and exosomes. Then, the pools were sequenced to study their aptamers composition, and eleven sequences emerged as the most enriched. In conclusion, we predict that the identified aptamers will represent key molecular tools to improve the clinical management of ALK+ NSCLC patients.

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