NAALADL1 modulates cellular resistance to Tumor Treating Fields in colorectal cancer

Colorectal cancer (CRC), the third most common malignancy worldwide, remains difficult to treat in advanced stages. Tumor Treating Fields (TTFields) are a non-invasive therapy that disrupts mitosis via intermediate frequency alternating electric fields, already clinically approved and widely implemented in the treatment of glioblastoma. This study is the first to systematically evaluate the therapeutic efficacy of TTFields in a large panel of low-passage CRC cell lines, representing diverse biological backgrounds. Our results demonstrate that TTFields exert antitumor effects through multiple mechanisms, including microtubule disruption, metabolic stress induction, mitotic aberrations, DNA damage, and activation of apoptotic pathways. Transcriptomic profiling, targeted sequencing, and weighted gene co-expression network analysis identified N-acetylated alpha-linked acidic dipeptidase-like 1 (NAALADL1) as a resistance-associated gene. NAALADL1 proved significantly upregulated in resistant lines, and its knockdown via shRNA sensitized cells to TTFields. Mechanistically, NAALADL1 depletion destabilized microtubules and induced G2/M arrest, enhancing TTFields efficacy. Virtual screening further identified Lumacaftor and Bestatin as candidate NAALADL1 inhibitors, which synergized with TTFields to suppress CRC cell growth. These findings highlight significant heterogeneity in CRC cell line responses to TTFields and identify NAALADL1 as a key modulator of resistance, suggesting its potential as a target for improving TTFields-based therapies in CRC.