2ⁿᵈ Edition of the Cancer R&D World Conference 2026

Abstract

Introduction: Identifying lung cancer at an early stage has been correlated with a higher 5-year survival rate due to timely intervention. Current diagnostic regimes require extensive resources, which can be inaccessible especially in geographic regions with escalating cancer incidences and limited infrastructure. Furthermore, the recommended screening for high-risk patients is low-dose computed tomography, which suffers from high false positive rates. Our previous work has demonstrated lung cancer can be detected using imaging-free sensing of aberrant protease activity in the tumor microenvironment via urinary reporters shed from activity-based nanosensors (ABNs). Further engineering of ABNs for clinical implementation at the point-of-care could significantly improve resource inequalities in the detection of lung cancer. In this work, we designed a needle- and imaging-free platform — Point-of-care Aerosolizable nanosensors with Tumor-Responsive Oligonucleotides (PATROL) — to screen for lung cancer at early stages.

Materials and Methods: PATROL integrates 3 modules: a set of multiplexed ABNs, a portable inhalation device, and a paper-based, multiplexable lateral flow assay kit. The ABNs were conjugated with pre-designed DNA barcodes via protease-activatable peptides. ABNs were then reformulated into an aerosolizable format.  Using a mouse model of autochthonous lung adenocarcinoma (LUAD) at an early stage, we delivered ABN aerosols to the tumor-bearing lungs, where the selected substrates were cleaved in the tumor microenvironment to shed DNA barcodes concentrate in urine for detection. The urinary output was obtained using a paper-based, multiplexable lateral flow assay. Unsupervised learning model was used to differentiate lung adenocarcinoma from healthy counterparts.

Results and Discussion: We used transcriptomic and proteomic analyses to nominate protease substrates specific to early-stage LUAD and constructed a corresponding set of DNA-coded ABNs to probe tumor-associated proteolytic signatures. Through in vivo screening, we further narrowed down a small library of 4 DNA-barcoded ABNs that not only retain adequate diagnostic power to detect unique proteolytic activity patterns but also are adapted to low resource requirements in point-of-care assays.  We demonstrated the inhaled ABNs possessed similar diagnostic capability as those administered via invasive intratracheal instillation. We validated that the rationally designed single strip lateral flow assay can read out the unique, multiplexed DNA signatures in the urine at room temperature in only 20 minutes, with comparable sensitivity and specificity to that via liquid chromatography-mass spectrometry in centralized diagnostic laboratories. Collectively, we used an autochthonous mouse model of LUAD and validated that PATROL differentiated Grade I/II tumors with high specificity and sensitivity.

Conclusions: In summary, we engineered PATROL as a detection paradigm to screen for lung cancer using point-of-care-relevant technology. PATROL integrates inhalation technology, synthetic activity-based biomarkers, and multiplexable lateral flow assays, and has been validated as a non-invasive, imaging-free approach for early screening of lung cancer in a preclinical mouse model. PATROL also holds the clinical potential to enable a larger, high-risk population to gain access to periodic screening of lung cancer in all settings. We envision that this modular platform could be applied to point-of-care detection and therapeutic response assessment of other lung diseases.