Activatable Zymography Probes Enable In Situ 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 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.

Protease activity sensors enable real-time treatment response monitoring in lymphangioleiomyomatosis

“…We first injected 1×10 6 105K cells s.c. into the flanks of female nude mice and excised tumours when they reached volumes of ∼500 mm 3 . We incubated homogenates of these tumours, as well as lungs from healthy nude mice, with a panel of 16 quenched fluorescent protease substrates expected to be broadly cleaved by metallo, serine and cysteine proteases, and monitored fluorescence over time ( supplementary figure S4a ) [ 22 , 24 ]. We found that multiple substrates were preferentially cleaved by Tsc2 -deficient tumour homogenates relative to healthy lung ( supplementary figure S4b ).…”

Synthetic Circuit-Driven Expression of Heterologous Enzymes for Disease Detection

“…By exploiting the unique properties that emerge at the nanoscale, multifunctional nanoparticles could be engineered to target and penetrate disease sites, either to detect the presence or activity of circuit-generated biomarkers or to sense orthogonal signals to multiplex the inputs to the synthetic circuit. Nanosystems have been extensively used to produce both in vivo imaging signals , and ex vivo readouts in accessible biofluids or tissues . Tuning similar nanoparticle sensors toward circuit-produced outputs could provide multiple levels of disease specificity and sensitivity that could ultimately yield highly accurate engineered detection systems.…”

“…Nanosystems have been extensively used to produce both in vivo imaging signals 22,41 and ex vivo readouts in accessible biofluids or tissues. 14 Tuning similar nanoparticle sensors toward circuit-produced outputs could provide multiple levels of disease specificity and sensitivity that could ultimately yield highly accurate engineered detection systems.…”

“…In synthetic biology, the forward engineering of biological systems toward this goal relies on the use of biological parts to create novel sense-and-respond functionalities inside living cells, enabling circuit-inspired control at the genetic, transcriptional, or protein levels. − In nanoparticle engineering, material and chemical systems are treated as the programmable entity and can be engineered as multifunctional, cooperative systems that can detect and respond to molecular events at the nanoscale. , In recent years, both these fields have seen the emergence of engineered, exogenous agents that can interrogate biological states in vivo to generate amplified readouts of disease . Specifically, several technologies, both in synthetic biology and nanotechnology, have leveraged enzymatic activity to measure or produce biomarkers of disease. − Because they harness the unique substrate specificity and catalytic signal amplification properties of enzymes, these engineered activity-based diagnostics have the potential to overcome some of the sensitivity and specificity limitations of standard diagnostics. Within this sphere, the integration of synthetic biology with nanoparticle sensing tools could yield hybrid sensors that interface directly with the body to produce highly specific, amplified disease readouts.…”

Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies

“…Conversely, capillary electrophoresis gained considerable interest in the field of study of monoclonal antibodies due to its high resolution and efficiency of separation of monoclonal antibodies and their derivatives [ 47 , 48 ], and it offers rapid, reproducible, and objective assessment that does not require antigen binding for signal detection. Other in situ zymography approaches have been introduced utilizing a quenched protease substrate conjugated to an integrin-targeted nanoparticle [ 17 ] or contained within a complexed poly-arginine probe for cell surface staining upon activation [ 49 ]. In contrast to QZ, these approaches are also limited by the requirement for target engagement or cell surface labeling for fluorescence-based staining detection of protease activity.…”