Biology subjects

Kirkpatrick, J. D.

Publications and source records attributed to Kirkpatrick, J. D..

6 recordsLinked to original sources

bioRxiv2025

Multimodal profiling of proinflammatory protease activity identifies caspase-1 as a target for lung cancer interception

The systemic inhibition of IL-1b, a key mediator of pulmonary inflammation, has been shown to reduce the incidence of lung cancer in patients in years following treatment, but knowledge gaps surrounding its activation and role in the tumor microenvironment are hindering approaches for cancer interception. We developed a suite of activity-based technologies to probe inflammation in early lung cancer and identified a translational target candidate. We designed probes sensitive to various IL-1b-activating proteases and applied them to a murine model of inflammatory lung cancer, Kras/Trp53-mutant with SIINFEKL expression (KPS). Our nanosensors revealed reduced cleavage of a caspase-1 reporter in the lungs of KPS mice treated with IL-1b antibody, as well as elevated caspase-1 expression and activity in naive tumor tissue sections, highlighting the importance of caspase-1 processing of IL-1b during cancer development. We conducted a pre-clinical trial of a novel combination intervention by administering both IL-1b blockade and caspase-1 inhibition shortly after tumor induction. Following treatment, we observed significant reduction in lung cancer formation, including complete ablation of tumor incidence in nearly 20% of KPS mice. Our approach to understand the interplay of protease activity and cytokine activation supports development of new strategies to mitigate inflammation and intercept lung cancer progression.

bioengineering↗

bioRxiv2024

Engineering Multiplexed Synthetic Breath Biomarkers as Diagnostic Probes

Breath biopsy is emerging as a rapid and non-invasive diagnostic tool that links exhaled chemical signatures with specific medical conditions. Despite its potential, clinical translation remains limited by the challenge of reliably detecting endogenous, disease-specific biomarkers in breath. Synthetic biomarkers represent an emerging paradigm for precision diagnostics such that they amplify activity-based biochemical signals associated with disease fingerprints. However, their adaptation to breath biopsy has been constrained by the limited availability of orthogonal volatile reporters that are detectable in exhaled breath. Here, we engineer multiplexed breath biomarkers that couple aberrant protease activities to exogenous volatile reporters. We designed novel intramolecular reactions that leverage protease-mediated aminolysis, enabling the sensing of a broad spectrum of proteases, and that each release a unique reporter in breath. This approach was validated in a mouse model of influenza to establish baseline sensitivity and specificity in a controlled inflammatory setting and subsequently applied to diagnose lung cancer using an autochthonous Alk-mutant model. We show that combining multiplexed reporter signals with machine learning algorithms enables tumor progression tracking, treatment response monitoring, and detection of relapse after 30 minutes. Our multiplexed breath biopsy platform highlights a promising avenue for rapid, point-of-care diagnostics across diverse disease states.

bioengineering↗

bioRxiv2023

Inhalable point-of-care urinary diagnostic platform

The late-stage detection of lung cancer leads to a high global mortality rate. Although low-dose computed tomography screening improves lung cancer survival in at-risk groups, this test still suffers from high rates of false positive results. In addition, inequality remains in the diagnosis of lung cancer as access to medical imaging infrastructure is limited. Here, we designed a needleless and imaging-free platform, termed PATROL (point-of-care aerosolizable nanosensors with tumor-responsive oligonucleotide barcodes), to increase detection accuracy, to reduce resource disparities for early detection of lung cancer, and to enable timely interception. PATROL formulates a set of DNA-barcoded, activity-based nanosensors (ABNs) into inhalable formats that can be delivered using clinical nebulizers or inhalers. Lung cancer-associated proteases in the tumor microenvironment selectively cleave the ABNs, releasing synthetic DNA reporters that are eventually excreted via the urine. The barcoded nanosensor signatures present in urine samples are quantified within 20 minutes using a multiplexable paper-based lateral flow assay at room temperature. PATROL detects early-stage tumors in an autochthonous lung adenocarcinoma mouse model with high sensitivity and specificity. Tailoring the library of ABNs may enable the modular PATROL platform to not only lower the resource thresholds required for early detection of lung cancer, but also enable rapid detection of chronic pulmonary disorders and infections.

bioengineering↗

bioRxiv2021

Spatially regulated protease activity in lymph nodes renders B cell follicles a sanctuary for retention of intact antigens

The structural integrity of vaccine antigens is critical, as antigen degradation in vivo could eliminate neutralizing epitopes and create competing B cell responses against irrelevant breakdown products. Using FRET imaging and imaging zymography, we found that protease activity and antigen breakdown are spatially heterogeneous in lymph nodes. Following protein immunization, antigens are rapidly degraded in the subcapsular sinus, paracortex, and interfollicular regions of the tissue. By contrast, the follicles and follicular dendritic cell (FDC) networks exhibit low protease activity and antigen degradation rates. Immunization regimens targeting antigen rapidly to FDCs led to germinal centers (GCs) where responses to intact antigen were highly dominant, while traditional bolus immunizations led to weaker GC responses where more GC B cells bound to breakdown products than intact antigen. Thus, spatially-compartmentalized antigen proteolysis impacts humoral immunity and can be exploited to enhance vaccine-induced production of antibody responses against key pathogen structural epitopes.

immunology↗

bioRxiv2021

Multiscale profiling of enzyme activity in cancer

Diverse processes in cancer are mediated by enzymes, which most proximally exert their function through their activity. Methods to quantify enzyme activity, rather than just expression, are therefore critical to our ability to understand the pathological roles of enzymes in cancer and to harness this class of biomolecules as diagnostic and therapeutic targets. Here we present an integrated set of methods for measuring specific enzyme activities across the organism, tissue, and cellular levels, which we unify into a methodological hierarchy to facilitate biological discovery. We focus on proteases for method development and validate our approach through the study of tumor progression and treatment response in an autochthonous model of Alk-mutant lung cancer. To quantitatively measure activity dynamics over time, we engineered multiplexed, peptide-based nanosensors to query protease activity in vivo. Machine learning analysis of sensor measurements revealed dramatic protease dysregulation in lung cancer, including significantly enhanced proteolytic cleavage of one peptide, S1 (Padj < 0.0001), which returned to healthy levels within three days after initiation of targeted therapy. Next, to link these organism-level observations to the in situ context, we established a multiplexed assay for on-tissue localization of enzyme activity and pinpointed S1 cleavage to endothelial cells and pericytes of the tumor vasculature. Lastly, to directly link enzyme activity measurements to cellular phenotype, we designed a high-throughput method to isolate and characterize proteolytically active cells, uncovering profound upregulation of pro-angiogenic transcriptional programs in S1-positive cells. Together, these methods allowed us to discover that protease production by angiogenic vasculature responds rapidly to targeted therapy against oncogene-addicted tumor cells, identifying a highly dynamic interplay between tumor cells and their microenvironment. This work provides a generalizable framework to functionally characterize enzyme activity in cancer.

bioengineering↗

bioRxiv2020

Activatable zymography probes enable in situ localization of protease dysregulation in cancer

Recent years have seen the emergence of conditionally activated diagnostics and therapeutics that leverage protease-cleavable peptide linkers to enhance their specificity for cancer. However, due to a lack of methods to measure and localize protease activity directly within the tissue microenvironment, the design of protease-activated agents has been necessarily empirical, yielding suboptimal results when translated to patients. To address the need for spatially resolve d protease activity profiling in cancer, we developed a new class of in situ probes that can be applied to fresh-frozen tissue sections in a manner analogous to immunofluorescence staining. These activatable zymography probes (AZPs) detected dysregulated protease activity in human prostate cancer biopsy samples, enabling disease classification. We then leveraged AZPs within a generalizable framework to design conditional cancer diagnostics and therapeutics, and demonstrated the power of this approach in the Hi-Myc mouse model of prostate cancer, which models features of early pathogenesis. Multiplexed screening against barcoded substrates yielded a peptide, S16, that was robustly and specifically cleaved by tumor-associated metalloproteinases in the Hi-Myc model. In situ labeling with an AZP incorporating S16 revealed a potential role of metalloproteinase dysregulation in proliferative, pre-malignant Hi-Myc prostatic glands. Last, we incorporated S16 into an in vivo imaging probe that, after systemic administration, perfectly classified diseased and healthy prostates, supporting the relevance of ex vivo activity assays to in vivo translation. We envision AZPs will enable new insights into the biology of protease dysregulation in cancer and accelerate the development of conditional diagnostics and therapeutics for multiple cancer types.

bioengineering↗