Activatable Zymography Probes Enable <i>In Situ</i> Localization of Protease Dysregulation in Cancer.

Abstract

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 resolved protease activity profiling in cancer, we developed a new class of <i>in situ</i> probes that can be applied to fresh-frozen tissue sections in a manner analogous to immunofluorescence staining. These activatable zymography probes (AZP) detected dysregulated protease activity in human prostate cancer biopsy samples, enabling disease classification. AZPs were leveraged within a generalizable framework to design conditional cancer diagnostics and therapeutics and showcased 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. <i>In situ</i> labeling with an AZP incorporating S16 revealed a potential role of metalloproteinase dysregulation in proliferative, premalignant Hi-Myc prostatic glands. Systemic administration of an <i>in vivo</i> imaging probe incorporating S16 perfectly classified diseased and healthy prostates, supporting the relevance of <i>ex vivo</i> activity assays to <i>in vivo</i> 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. SIGNIFICANCE: Visualization of protease activity within the native tissue context using AZPs provides new biological insights into protease dysregulation in cancer and guides the design of conditional diagnostics and therapeutics.