Lung cancer is the most fatal form of cancer worldwide and is often only detected in later stages, leading to high mortality rates. However, researchers at the Massachusetts Institute of Technology (MIT) have developed a pioneering approach to identify early-stage lung cancer using a simple urine test. The current screening methods, like low-dose CT scans, aren’t accessible in many developing countries and some parts of the United States. Therefore, MIT researchers aim to provide a resource-efficient and noninvasive early lung cancer detection alternative.
To achieve this goal, the researchers developed activity-based diagnostics (ABDs) that employ catalytic proteases to liberate synthetic barcodes and expand the available analytes for more predictive diagnostics. Point-of-care (POC) tests that provide accurate real-time ABD readouts could reduce the demand for early lung cancer detection tools and improve cost-effectiveness, particularly in resource-poor settings. Lateral flow assays (LFAs) could enable accurate LFA-based readouts of dysregulated proteolysis through fluid biopsy.
A new modular platform called PATROL (point-of-care aerosolized nanosensors with tumor-responsive oligonucleotide barcodes) has been introduced for the early detection of lung cancer. It integrates low-plex nanosensors, a portable inhalation unit, and a paper-based LFA. This noninvasive platform accurately diagnoses early-stage lung cancer without requiring trained medical personnel or centralized diagnostic laboratories.
How It Functions
Coated in DNA barcodes, the polymer nanoparticles are engineered to be targeted by protease enzymes linked to stage I lung adenocarcinoma. Upon contact, the proteases cleave off the barcodes, which enter the bloodstream and are excreted in urine. Similar to a pregnancy test, a test strip can detect these barcodes, providing results within approximately 20 minutes.
Animal Testing Success
The researchers tested the system’s effectiveness in mouse models engineered to develop human-like lung tumors. Using aerosol nebulizers, they delivered 20 sensors to mice with stage I or II cancer. A machine learning algorithm identified the four most accurate sensors, exhibiting 100% specificity and 84.6% sensitivity.
Figure 1: PATROL for early detection of lung adenocarcinoma at the POC.
The Innovative Technology and Its Results
The MIT team developed nanosensors delivered through inhalers or nebulizers. These nanosensors target specific proteins associated with lung cancer and, when detected, produce a signal in the urine. The results are then easily visible on a paper test strip, creating a straightforward and user-friendly diagnostic process.
Figure 2: Engineering inhalable ABNs to noninvasively probe protease dysregulation.
Researchers have developed ABNs to be delivered via inhalation by nebulizers. The ABNs were synthesized by functionalizing a nano scaffold with peptides containing a matrix metalloproteinase substrate and a Cy7-tagged glu-fibrinopeptide B reporter. The aerosols generated from the ABNs were largely unchanged and could be delivered efficiently to the lungs. The researchers also explored the feasibility of formulating nanosensors into portable, handheld devices called DPIs. These showed comparable delivery efficiency and dose uniformity of the ABN-containing microparticles across different inhalers.
Figure 3: Nomination and down-selection of protease substrates specific to early-stage lung cancer for POC adaptability.
Researchers aimed to develop a POC diagnostic tool for the early detection of lung cancer using minimal precision probes that could offer high predictive power via viable screening operations suitable for decentralized settings. They leveraged tumor proteases and established a library of activatable peptide-based probes to detect early-stage lung cancer. The team used DESeq2 to identify differentially expressed proteases in tumors and nontumor adjacent tissues from patients with stage I lung adenocarcinoma in The Cancer Genome Atlas (TCGA) database. From the in vitro screening, the researchers selected 20 peptide sequences with high specificity against a family of proteases and preferential selectivity against proteases of the same subtype. These probes could help inform the early detection and timely lung cancer interception in a low-plex POC assay format.
A panel of 20 candidates was evaluated through in vivo screening in mouse models. The aim was to nominate a small bespoke probe set with low-plex compatibility. The set of 20-plex ABNs was synthesized by conjugating mass-coded tandem peptides to the PEG nano scaffolds. The ABNs were evaluated in an autochthonous lung adenocarcinoma mouse model. The pooled 20 ABNs were instilled in the mice, and the urine produced after ABN administration was collected. The mass-encoded reporters in the urine samples were analyzed using LC-MS/MS. Nine of 20 probes offered significant detection power. The top 5 probes were further identified among those with the greatest fold change and significance. The selected four ABNs were reformulated into a low-plex, inhalable panel to assess its diagnostic performance in vivo to detect early-stage lung adenocarcinoma.
Figure 4: Inhalable ABNs enabled classification of early lung adenocarcinoma.
Researchers tested nebulized ABN formulations to detect lung cancer in mice. They found that nebulized ABNs resulted in a more even distribution throughout the lung periphery. The nebulized nanosensors were found to perform statistically equivalent to the intratracheally instilled down-selected library, with a sensitivity of 84.6% and 100% specificity. The study shows that inhalable low-level multiplexing of rationally selected ABNs can effectively detect mouse autochthonous lung adenocarcinoma early.
Figure 5: Lateral flow assay (LFA) for the direct multiplexed detection of urinary DNA barcodes at room temperature.
Researchers have developed a POC detection method using ssDNA as barcodes for ABNs. They designed a multiplexable LFA based on DNA hybridization on a single strip. The optimized LFA is highly sensitive and takes about 20 minutes to complete at room temperature. The assay can quantify subnanomolar concentrations of the target and has an operating linear range from 0 to 100 nM of DNA1, DNA2, DNA3, and DNA4. The customized LFA can simultaneously capture quantifiable amounts of multiplexed DNA barcodes in urine with high sensitivity and specificity.
Figure 6: PATROL enabled classification of lung adenocarcinoma at an early stage.
A diagnostic platform, PATROL, was developed by integrating three modules into a single low-resource capable system. The system used DNA-coded ABNs that were synthesized by coupling biotinylated DNA barcodes to PEG scaffolds via peptide substrates. The ABNs were approximately 15 nm in diameter and highly negatively charged. The researchers validated urinary DNA reporter detection via the LFA after administering nebulizer-delivered ABNs in the KP lung adenocarcinoma mouse model. The diagnostic capacity of the system was retained, and the DNA reporters detected by the LFA had a sensitivity of 75.2% and were comparable to the sensitivity of micro-CT.
Figure 7: Nebulized nanosensors show no obvious preclinical toxicity or immunogenicity.
In a preclinical study, the safety profile of a high dose of inhalable ABNs with DNA barcodes was examined, which was threefold higher than the doses tested previously. Wild-type mice were administered with a single dose of ABNs delivered via nebulization, and no general toxicity or clogging of vasculature was observed in major organs even after seven days. Histological assessment of principal tissues and weight monitoring indicated the same. The immunogenicity of the administered substance was also evaluated by measuring interferon-γ (IFN-γ) secretion, a major inflammatory cytokine produced by activated immune cells. No IFN-γ was secreted by circulating lymphocytes at any tested time points, indicating that each module of the inhaled ABNs, including peptide substrates, PEG scaffolds, and DNA barcodes, was nonimmunogenic.
Potential Applications and Future Challenges
The technology’s applications extend beyond lung cancer, with the MIT team exploring its use in diagnosing liver cancer and nonalcoholic steatohepatitis in a phase 1 clinical trial. Additionally, the team is investigating its potential to distinguish between viral, bacterial, and fungal pneumonia and diagnose other lung conditions like asthma and chronic obstructive pulmonary disease.
While the technology showcases innovation, challenges exist in translating it to human subjects. Factors such as diet, hydration, drug interference, renal function, and certain chronic diseases could impact the effectiveness of urine-based detection. Furthermore, the heterogeneity of human cancer cells poses a challenge, prompting ongoing analysis of human biopsy samples to validate the technology’s efficacy in real-world scenarios.
In the hands of MIT researchers, this inhalable sensor technology not only represents a leap forward in cancer diagnostics but also opens doors to diverse applications in the realm of respiratory health. As they continue to refine and validate their approach, the prospect of an accessible and efficient lung cancer screening method brings hope for improved outcomes and early interventions in the fight against this deadly disease.
Reference
Zhong Q, Edward, Martin-Alonso C, et al. Inhalable point-of-care urinary diagnostic platform. Science Advances. 2024 Jan 5;10(1). DOI: 10.1126/sciadv.adj9591
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