Hemodynamic molecular imaging of tumor-associated enzyme activity in the living brain
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Curated by eLife
Evaluation Summary:
This manuscript will be of interest to readers in the field of magnetic resonance imaging and responsive imaging probes. In this work, a new imaging probe is designed and applied in proof-of-principle animal models, with future promise for relevance in models that have higher relevance to human disease.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Molecular imaging could have great utility for detecting, classifying, and guiding treatment of brain disorders, but existing probes offer limited capability for assessing relevant physiological parameters. Here, we describe a potent approach for noninvasive mapping of cancer-associated enzyme activity using a molecular sensor that acts on the vasculature, providing a diagnostic readout via local changes in hemodynamic image contrast. The sensor is targeted at the fibroblast activation protein (FAP), an extracellular dipeptidase and clinically relevant biomarker of brain tumor biology. Optimal FAP sensor variants were identified by screening a series of prototypes for responsiveness in a cell-based bioassay. The best variant was then applied for quantitative neuroimaging of FAP activity in rats, where it reveals nanomolar-scale FAP expression by xenografted cells. The activated probe also induces robust hemodynamic contrast in nonhuman primate brain. This work thus demonstrates a potentially translatable strategy for ultrasensitive functional imaging of molecular targets in neuromedicine.
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Author Response:
Reviewer #1 (Public Review):
Molecular probes that respond to disease-specific activities to produce a diagnostic readout have had a major impact in the clinical management of cancer. The current study extends the teams previous work on the development of a molecular sensor for the cancer-associated, fibroblast activation protein (FAP). The molecular sensor is based on CGRP (Calcitonin gene-related peptide), a potent vasodilator of human arteries which mediates relaxation of arteries via activation of the CGRP(1)-type receptor. The sensor is fused to biotin with a linker sequence that contains FAP cleavage sites. In its intact form, the sensor fails to activate the CGRP-receptor, however, in the presence of FAP, proteolytic release of CGRP from inhibition, leads to CGRP-receptor engagement, which is then detected by …
Author Response:
Reviewer #1 (Public Review):
Molecular probes that respond to disease-specific activities to produce a diagnostic readout have had a major impact in the clinical management of cancer. The current study extends the teams previous work on the development of a molecular sensor for the cancer-associated, fibroblast activation protein (FAP). The molecular sensor is based on CGRP (Calcitonin gene-related peptide), a potent vasodilator of human arteries which mediates relaxation of arteries via activation of the CGRP(1)-type receptor. The sensor is fused to biotin with a linker sequence that contains FAP cleavage sites. In its intact form, the sensor fails to activate the CGRP-receptor, however, in the presence of FAP, proteolytic release of CGRP from inhibition, leads to CGRP-receptor engagement, which is then detected by changes in MRI contrast. Receptor activation on the vasculature, provides a diagnostic readout via local changes in hemodynamic image contrast for MRI. This is a technical report that provides a proof of principle evidence that a sensor for FAP proteolytic activity can be used in rodent models, with a robust signal to noise. However, the discussion and abstract overstate the clinical impact of the findings.
We are grateful for the Reviewer’s overall assessment and have made a number of additions and edits to the manuscript to characterize and clarify the clinical potential.
Reviewer #2 (Public Review):
In this manuscript, the authors create an imaging probe for magnetic resonance imaging (MRI) that is based on triggering vasodilation in a protease-dependent manner. The imaging probe is a steric blocking domain fused to the N-terminus of a vasoactive peptide connected by a linker that is sensitive to proteolytic cleavage by fibroblast activation protein (FAP). The linker design was optimized for FAP-mediated cleavage and led to a 34-fold increase in activity when there was FAP present. The imaging probe detected cells overexpressing FAP implanted into the rat striatum when infused directly at the transplantation site or into the cerebrospinal fluid. The authors also create a kinetic model to determine FAP catalysis rate, k, from temporal MRI signal. Lastly, the authors demonstrate in a proof-of-concept experiment that the vasoactive peptide is able to create imaging contrast in a nonhuman primate brain.
This group has previously described using peptide-mediated vasodilation as a method for image contrast in MRI. In this work, they advance this concept to make the peptide activity triggered by protease cleavage, thus creating an activity-based molecular imaging probe. The design presented in this work could likely be adapted to a wide range of proteases through substitution of the substrate that links the steric blocking domain and the vasoactive peptide allowing for the study of a wide range of protease activity in diseases that affect the brain. The creation of activity-based imaging probes is an important area of study for advancing precision medicine because the imaging signal may more accurately represent disease prognosis and stratification over conventional imaging probes.
A claim made in the abstract that is provided with limited support is the whether the probe allows for quantitative analysis of FAP activity. A useful measure for a diagnostic would be whether the imaging signal can quantitate the amount of enzyme activity. In this study, all in vivo experiments were conducted with the injection of a single concentration of cells overexpressing FAP transgene.
We thank the Reviewer for this considered assessment. In this paper, we use kinetic modeling to quantify FAP activity over animals and over voxels in the experiments of Figures 2 and 3. Although these experiments followed implantation of a fixed number of FAP-expressing or control cells per animal, the three-dimensional distribution of FAP activity produced in the brain is heterogeneous, thus allowing for spatially resolved quantification of enzyme activity that addresses the Reviewer’s point. To this revision, we further add an analysis of enzymatic constants obtained on a per-voxel level from the data in Figure 4, indicating approximate linearity of the relationship between maximum signal change and the probe cleavage rate constant k. Values of k that range from 0 to 0.03 s^-1 are obtained from our analysis, corresponding to estimated localized FAP concentrations ranging from 0 to 20 nM in rat brains. These values are physically plausible and consistent with in vitro quantification of FAP activity over a range of transfected and patient-derived tumor cell numbers in data added to Figure 4 of the revised manuscript.
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Evaluation Summary:
This manuscript will be of interest to readers in the field of magnetic resonance imaging and responsive imaging probes. In this work, a new imaging probe is designed and applied in proof-of-principle animal models, with future promise for relevance in models that have higher relevance to human disease.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Molecular probes that respond to disease-specific activities to produce a diagnostic readout have had a major impact in the clinical management of cancer. The current study extends the teams previous work on the development of a molecular sensor for the cancer-associated, fibroblast activation protein (FAP). The molecular sensor is based on CGRP (Calcitonin gene-related peptide), a potent vasodilator of human arteries which mediates relaxation of arteries via activation of the CGRP(1)-type receptor. The sensor is fused to biotin with a linker sequence that contains FAP cleavage sites. In its intact form, the sensor fails to activate the CGRP-receptor, however, in the presence of FAP, proteolytic release of CGRP from inhibition, leads to CGRP-receptor engagement, which is then detected by changes in MRI …
Reviewer #1 (Public Review):
Molecular probes that respond to disease-specific activities to produce a diagnostic readout have had a major impact in the clinical management of cancer. The current study extends the teams previous work on the development of a molecular sensor for the cancer-associated, fibroblast activation protein (FAP). The molecular sensor is based on CGRP (Calcitonin gene-related peptide), a potent vasodilator of human arteries which mediates relaxation of arteries via activation of the CGRP(1)-type receptor. The sensor is fused to biotin with a linker sequence that contains FAP cleavage sites. In its intact form, the sensor fails to activate the CGRP-receptor, however, in the presence of FAP, proteolytic release of CGRP from inhibition, leads to CGRP-receptor engagement, which is then detected by changes in MRI contrast. Receptor activation on the vasculature, provides a diagnostic readout via local changes in hemodynamic image contrast for MRI. This is a technical report that provides a proof of principle evidence that a sensor for FAP proteolytic activity can be used in rodent models, with a robust signal to noise. However, the discussion and abstract overstate the clinical impact of the findings.
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Reviewer #2 (Public Review):
In this manuscript, the authors create an imaging probe for magnetic resonance imaging (MRI) that is based on triggering vasodilation in a protease-dependent manner. The imaging probe is a steric blocking domain fused to the N-terminus of a vasoactive peptide connected by a linker that is sensitive to proteolytic cleavage by fibroblast activation protein (FAP). The linker design was optimized for FAP-mediated cleavage and led to a 34-fold increase in activity when there was FAP present. The imaging probe detected cells overexpressing FAP implanted into the rat striatum when infused directly at the transplantation site or into the cerebrospinal fluid. The authors also create a kinetic model to determine FAP catalysis rate, k, from temporal MRI signal. Lastly, the authors demonstrate in a proof-of-concept …
Reviewer #2 (Public Review):
In this manuscript, the authors create an imaging probe for magnetic resonance imaging (MRI) that is based on triggering vasodilation in a protease-dependent manner. The imaging probe is a steric blocking domain fused to the N-terminus of a vasoactive peptide connected by a linker that is sensitive to proteolytic cleavage by fibroblast activation protein (FAP). The linker design was optimized for FAP-mediated cleavage and led to a 34-fold increase in activity when there was FAP present. The imaging probe detected cells overexpressing FAP implanted into the rat striatum when infused directly at the transplantation site or into the cerebrospinal fluid. The authors also create a kinetic model to determine FAP catalysis rate, k, from temporal MRI signal. Lastly, the authors demonstrate in a proof-of-concept experiment that the vasoactive peptide is able to create imaging contrast in a nonhuman primate brain.
This group has previously described using peptide-mediated vasodilation as a method for image contrast in MRI. In this work, they advance this concept to make the peptide activity triggered by protease cleavage, thus creating an activity-based molecular imaging probe. The design presented in this work could likely be adapted to a wide range of proteases through substitution of the substrate that links the steric blocking domain and the vasoactive peptide allowing for the study of a wide range of protease activity in diseases that affect the brain. The creation of activity-based imaging probes is an important area of study for advancing precision medicine because the imaging signal may more accurately represent disease prognosis and stratification over conventional imaging probes.
A claim made in the abstract that is provided with limited support is the whether the probe allows for quantitative analysis of FAP activity. A useful measure for a diagnostic would be whether the imaging signal can quantitate the amount of enzyme activity. In this study, all in vivo experiments were conducted with the injection of a single concentration of cells overexpressing FAP transgene.
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