Urinary detection of lung cancer in mice via noninvasive pulmonary protease profiling

Abstract

Lung cancer is the leading cause of cancer-related death, and patients most commonly present with incurable advanced-stage disease. U.S. national guidelines recommend screening for high-risk patients with low-dose computed tomography, but this approach has limitations including high false-positive rates. Activity-based nanosensors can detect dysregulated proteases in vivo and release a reporter to provide a urinary readout of disease activity. Here, we demonstrate the translational potential of activity-based nanosensors for lung cancer by coupling nanosensor multiplexing with intrapulmonary delivery and machine learning to detect localized disease in two immunocompetent genetically engineered mouse models. The design of our multiplexed panel of sensors was informed by comparative transcriptomic analysis of human and mouse lung adenocarcinoma datasets and in vitro cleavage assays with recombinant candidate proteases. Intrapulmonary administration of the nanosensors to a Kras- and Trp53-mutant lung adenocarcinoma mouse model confirmed the role of metalloproteases in lung cancer and enabled accurate detection of localized disease, with 100% specificity and 81% sensitivity. Furthermore, this approach generalized to an alternative autochthonous model of lung adenocarcinoma, where it detected cancer with 100% specificity and 95% sensitivity and was not confounded by lipopolysaccharide-driven lung inflammation. These results encourage the clinical development of activity-based nanosensors for the detection of lung cancer.

CRISPR-Cas-amplified urine biomarkers for multiplexed and portable cancer diagnostics

“…Multiplexing also contributed to enhanced the sensitivity of activity-based nanosensors in previous work by our group. Urinary detection of cancers in mice via profiling protease activities by mass spectrometry showed improved sensitivity relative to existing and emerging bloodbased diagnostics 1, 38 . Owing to the inherent programmability of Cas proteins, Cas12a exhibited collateral activity upon multiple 20-mer barcodes tested, which can be extensible to hundreds of orthogonal codes, providing a level of multiplexing that is challenging to attain with commercial isobaric tags for mass-spectrometry 1 .…”

“…S7B, D). To identify peptide substrates specific to the selected proteases, we screened 16 peptide sequences against purified recombinant proteases and identified the top five substrates using a fluorogenic activity assay 29,38 (Fig. S7E).…”

The Tumor Proteolytic Landscape: A Challenging Frontier in Cancer Diagnosis and Therapy

“…The AuNC can be renally excreted after cleavage and enable catalytically amplified readouts [ 292 ]. Importantly, similar protease nanosensors designed in a bottom-up approach for prostate cancer detection could outperform the PSA prostate cancer marker [ 293 ] and a multiplexed substrate panel for lung cancer proteases has demonstrated excellent specificity and sensitivity for disease detection [ 294 ]. These encouraging results suggest that the development of future protease activity sensors will be based on synergies with the fields of nanotechnology and nanomedicine.…”

Activity-based urinary biomarkers of response and resistance to checkpoint blockade immunotherapy

“…Furthermore, by feature importance scores, the three response monitoring probes were among the least important probes for resistance stratification, and vice versa (Fig. 6i) We engineered INSIGHT based on our work on synthetic biomarkers, which are composed of activity-based biomarkers that monitor dysregulated protease activities for early disease detection in bacterial infection 31 , thrombosis 32 , cancer 10,11,33 , and organ transplant rejection 18 . Previous synthetic biomarkers rely on nanoparticle or polymeric carriers to prevent rapid renal clearance of free peptides, but their utility to target specific disease sites has not been explored.…”

“…In addition, T cell-mediated tumor control is primarily carried out by granzymes, which are serine proteases, released by cytotoxic T cells 17 . The ubiquity of protease dysregulation has motivated the development of molecular imaging probes for visualizing tumor or T cell proteases 18–21 , as well as synthetic biomarkers for multiplexed quantification of protease activity from urine 22–27 . Building on these studies, we developed Immune Sensors for monItorinG cHeckpoint blockade Therapy (INSIGHT) to detect tumor and immune proteases during treatment as activity-based biomarkers of response and resistance.…”