A nanotherapeutic strategy to target drug-tolerant cells and overcome EGFR tyrosine kinase inhibitor resistance in lung cancer

Introduction:: For epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC), EGFR tyrosine kinase inhibitors (TKIs) are the first-line treatment. Despite initial responses to EGFR TKIs, patients inevitably experience tumor progression due to acquired resistance. Resistance arises when a subset of cancer cells, i.e., drug tolerance cells (DTCs) undergo epithelial-mesenchymal transition (EMT), survive TKI treatment, evolve and lead to various resistance mechanisms (Fig. 1a). To address this clinical challenge, we developed a novel liposomal nanodrug Axl-LP-VD-CTA091 to inhibit DTCs, thus targeting the origin of diverse resistance mechanisms. In this nanodrug, Axl aptamers enabled targeting of nanodrug to DTCs, and 1,25-dihydroxyvitamin D3 (VD) and CTA091 suppressed EMT. Axl-LP-VD-CTA091 showed strong therapeutic efficacy in osimertinib tolerant H1975OR cells-based in vitro and in vivo models.

Materials and Methods:: The Axl aptamers conjugated, VD and CTA091 co-encapsulated liposomes (Axl-LP-VD-CTA091) were prepared and characterized for their size, zeta potential, encapsulation efficiency and morphology. Cellular uptake efficiency in H1975OR cells was measured by flow cytometry and confocal microscopy. Effects of Axl-LP-VD-CTA091 treatment on VD targets and EMT-related markers were determined by qRT-PCR and western blotting. The ability of Axl-LP-VD-CTA091 to alter the sensitivity of H1975OR cells to osimertinib was tested using SRB assay. A new orthotopic model was established by ultrasound-guided trans-pleural injection of H1975OR cells into the lungs of mice. The biodistribution study was assessed using IVIS imaging system. The in vivo efficacy and safety of Axl-LP-VD-CTA091 were evaluated through measurement of tumor volume using magnetic resonance imaging (MRI) and analysis of immunohistochemistry (IHC) and serum calcium.


Results, Conclusions, and Discussions:: Axl-LP-VD-CTA091 possessed a multilamellar structure with a size of 106 nm and a zeta potential of +30.9 mV. The encapsulation efficiency of VD and CTA091 was 75%. Axl-LP-VD-CTA091 was efficiently taken up by H1975OR cells, promoted VD signaling by enhancing VD target gene CYP24A1 expression, decreased EMT features by increasing epithelial marker CDH1 levels and decreasing mesenchymal marker MMP2 levels, and thus increased sensitivity of H1975OR cells to osimertinib (Fig. 1b). H1975OR orthotopic model with maintained osimertinib tolerance was successfully established. The biodistribution study showed preferential localization and retention of Axl-LP-Cy5-ODN in the tumor-bearing lungs (Fig. 1c). The in vivo safety and efficacy tests showed that Axl-LP-VD-CTA091 reduced tumor burden (Fig. 1d) and promoted an epithelial phenotype with no significant tissue toxicity. Based on the results, we contend that Axl-LP-VD-CTA091 is a novel strategy to inhibit DTCs and enhance EGFR-TKI sensitivity.


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