An ideal biomarker should be secreted at high levels relative to native background, remain stable or persistent in circulation until detection, be readily accessible from compositionally simple host fluids and discriminate disease with high sensitivity and specificity. In practice, these parameters are often difficult to improve or control for naturally occurring biomarkers and consequently, many promising biomarkers fail during rigorous evaluation for clinical translation. Here, we devised a system of synthetic biomarkers with the capacity to (i) amplify biomarker levels through substrate turnover by targeting aberrant protease activities, (ii) release stable, disomer enriched mass reporters designed to appear within a narrow mass window free of host molecules, (iii) trigger reporter clearance from blood into urine to reduce matrix complexity and facilitate facile extraction and (iv) simultaneously monitor libraries of candidate synthetic biomarkers in vivo to identify and validate lead biomarkers.
An enabling feature of our platform is the use of a nanoscale scaffold to direct the traffic of peptides in vivo
. Although free peptides are typically cleared rapidly from circulation via urinary secretion, we showed that NW-conjugated peptides are endowed with long-circulation times to allow transport into diseased tissues across porous vasculature and appear in urine only after release from NWs by disease-associated proteases. Several reports have highlighted the potential of applying peptide substrates to patient serum samples followed by MS profiling to uncover disease-specific activity signatures4,29,30,41
. However, without a delivery mechanism, in vitro
serum analysis cannot sample proteases expressed on the membrane of cells residing in the disease microenvironment (e.g. MMP9 expression by liver-resident macrophages in fibrosis). Similar to blood biomarkers, secreted proteases are significantly diluted in circulation and are often challenging to detect above highly-abundant plasma proteins5
, potent proteolytic cascades activated during sample collection (e.g. coagulation) and pan protease inhibitors in plasma (e.g. α2
-macroglobulin). Here, we chose NWs as chaperones because iron oxide nanoparticles are safe for use in humans, but a broad range of nontoxic scaffolds including proteins and sugars (e.g. albumin and dextran respectively) would also be amenable for peptide delivery. Given the cumulative wealth of nanomaterials, targeting ligands and enhanced delivery strategies available in nanomedicine, we expect this work to be transferrable to many additional formulations to gain access to different organs, types of vasculature and tissue depths42,43
Our library of isobaric mass tags to track the response of 10 peptides in vivo
provides a level of multiplexing that is currently challenging to attain with molecular and activity-based imaging probes11–13
. The vast majority of these approaches make use of modified protease substrates that emit fluorescent signals following proteolytic cleavage. Consequently, substrate multiplexing is limited by emission overlap as well as the need to emit in the near infrared window (600–900 nm) to minimize signal attenuation from tissue absorption, constraining most of these studies to single probes. Conversely, our work demonstrates the generation of a synthetic biomarker library that is 5–10× more densely multiplexed than existing state-of-the-art activity-based probes, compares favorably with commercial isobaric tags (e.g. 8-plex iTRAQ) and with additional parent peptides, is extensible to hundreds of orthogonal mass codes.
In addition to its invasiveness, a major limitation of the core biopsy for liver fibrosis is that tissue specimens are only representative of ~1/50,000th the size of an adult liver leading to sampling variation that can result in inaccurate diagnosis or staging and repeat biopsies. Here, we show how nanoparticles accumulate uniformly in the liver, penetrating without bias into regions of active fibrosis to release urinary biomarkers as integrated measures of disease burden. Our work in two models of fibrosis with different mechanisms of induction (i.e. xenobiotic vs. genetic) indicates the value of biomarker G7 for monitoring fibrosis. These results are reflective of fibrosis as a conserved tissue response to diverse chronic liver diseases (e.g. viral hepatitis, alcohol abuse, fatty liver disease, etc.) and suggest that biomarker G7 could be useful for monitoring fibrosis stemming from distinct underlying pathologies. Moving forward, an important area for future study is elucidating the biological mechanisms that are ultimately responsible for the release of individual reporters. This could be accomplished, for example, by comparing urinary signatures from animals lacking specific proteases (e.g. MMP9−/−) to their wild type counterpart or the use of clodronate liposomes to deplete liver macrophages to identify reporters that track with cellular inflammation.
A major factor preventing early detection of cancer is the tremendous dilution biomarkers experience upon release from tumor cells into systemic circulation. Recent computational estimates by Gambhir and colleagues18
revealed that solid tumors could potentially remain undetectable for 10–12 years and reach spherical diameters > 2.5 cm before biomarker levels becomes sufficiently elevated to indicate disease. Here, the advantage of our system is the ability to amplify tumor responses by leveraging enzymatic turnover (i.e. a single copy of a protease can cleave 100's of peptide substrates per hour) and the renal system's natural capacity to remove and concentrate plasma peptides into urine (i.e. from ~5 L of blood to 300 ml void volume). Our study shows that the combined effects of protease amplification and renal concentration can lead to promising results such as the detection of small tumors that CEA failed to discriminate even at the highest tumor burdens allowable in our animal models. Since many tumors do not secrete biomarkers at sufficient rates (or at all) for detection, targeting tumor proteases should allow a broader range of cancers to be discovered at an early stage because proteases are uniformly implicated during tumor invasion and metastasis. Extension of this platform to multiple types of cancers would benefit from the development of cancer-type specific tests that could be accomplished by identifying unique biomarker panels for each cancer44
. Conversely, a highly-sensitive, pan-cancer test comprised of a single set of diverse probes would be useful in clinical settings when the primary tumor is already known, such as monitoring for recurrence or metastases following surgical resection of primary tumors.
The successful translation of this platform to humans would require further confirmation of our lead biomarkers in patients as well as quantifying the potential benefits of monitoring biomarker panels versus single markers. A crucial limitation of existing single biomarker assays is their relatively poor disease specificity (e.g. CEA is elevated in smokers). These assays can be improved via multiplexing (e.g. prenatal triple screening) or specifying their use in well-defined clinical contexts (e.g. PSA is now recommended for recurrence monitoring but not screening). Similarly, the ability of this platform to differentiate protease-driven diseases (e.g. inflammation vs. cancer) would benefit from multiplexing and serial measurements in high-risk populations.
Lastly, a general concern with rodent studies is the relatively small number of animals used for hypotheses testing and validation. In this study, the number of animals selected per experimental condition (n = 5–10) allowed reasonable estimation of the mean and variance based on a normal distribution. The statistical power of our study was further bolstered by the prominent effect sizes observed (e.g. large AUCs) in both diseases studied as well as concordant biomarker responses across distinct models. Nonetheless, looking forward and in light of recent expert recommendations regarding biomarker qualification37
, the results of this study will require further confirmation and rigorous evaluation in humans.
In summary, we believe this study provides a framework for engineering diagnostic agents that can exploit fundamental features of human disease and physiology for noninvasive urinary monitoring. Future expansion and inclusion of additional enzymatic families (e.g. lipases, nucleases, glycosidases), organ-specific delivery strategies and broader multiplexing capabilities would provide opportunities for systems-level monitoring of disease and elucidating multi-enzymatic networks in health and disease.