Transformations of extracellular matrix (ECM) accompany pathological tissue changes, yet how cell-ECM crosstalk drives these processes remains unknown as adequate tools to probe forces or mechanical strains in tissues are lacking. Here, we introduce a new nanoprobe to assess the mechanical strain of fibronectin (Fn) fibers in tissue, based on the bacterial Fn-binding peptide FnBPA5. FnBPA5 exhibits nM binding affinity to relaxed, but not stretched Fn fibers and is shown to exhibit strain-sensitive ECM binding in cell culture in a comparison with an established Fn-FRET probe. Staining of tumor tissue cryosections shows large regions of relaxed Fn fibers and injection of radiolabeled 111In-FnBPA5 in a prostate cancer mouse model reveals specific accumulation of 111In-FnBPA5 in tumor with prolonged retention compared to other organs. The herein presented approach enables to investigate how Fn fiber strain at the tissue level impacts cell signaling and pathological progression in different diseases.
Fibroblast growth factor 2 (FGF-2) is a small 18 kDa
protein with
clinical potential for ischemic heart disease, wound healing, and
spinal cord injury. However, the therapeutic potential of systemic
FGF-2 administration is challenged by its fast elimination. Therefore,
we deployed genetic codon expansion to integrate an azide functionality
to the FGF-2 N-terminus, which was site-directly decorated with poly(ethylene
glycol) (PEG) through bioorthogonal strain-promoted azide–alkyne
cycloaddition (SPAAC). PEGylated FGF-2 was as bioactive as wild-type
FGF-2 as demonstrated by cell proliferation and Erk phosphorylation
of fibroblasts. The PEGylated FGF-2 conjugate was radiolabeled with
[111In] Indium cation ([111In]In3+) to study its biodistribution through noninvasive imaging by single-photon
emission computed tomography (SPECT) and by quantitative activity
analysis of the respective organs in healthy mice. This study details
the biodistribution pattern of site-specific PEGylated FGF-2 in tissues
after intravenous (iv) administration compared to the unconjugated
protein. Low accumulation of the PEGylated FGF-2 variant in the kidney
and the liver was demonstrated, whereas specific uptake of PEGylated
FGF-2 into the retina was significantly diminished. In conclusion,
site-specific PEGylation of FGF-2 by SPAAC resulted in a superior
outcome for the synthesis yield and in conjugates with excellent biological
performances with a gain of half-life but reduced tissue access in
vivo.
The extracellular matrix (ECM) acts as reservoir for a plethora of growth factors and cytokines some of which are hypothesized to be regulated by ECM fiber tension. Yet, ECM fiber tension has never been mapped in healthy versus diseased organs. Using our recently developed tension nanoprobe derived from the bacterial adhesin FnBPA5, which preferentially binds to structurally relaxed fibronectin fibers, we discovered here that fibronectin fibers are kept under high tension in selected healthy mouse organs. In contrast, tumor tissues and virus-infected lymph nodes exhibited a significantly higher content of relaxed or proteolytically cleaved fibronectin fibers. This demonstrates for the first time that the tension of ECM fibers is significantly reduced upon pathological tissue transformations. This has wide implications, as the active stretching of fibronectin fibers adjusts critical cellular niche parameters and thereby tunes the reciprocal cell-ECM crosstalk. Mapping the tensional state of fibronectin fibers opens novel and unexpected diagnostic opportunities.
Fibroblast growth factor-2 (FGF-2) is a potent modulator of cell growth and regulation, with improper FGF-2 signaling being involved in impaired responses to injury or even cancer. Therefore, the exploitation of FGF-2 as a therapeutic drives the prerequisite for effective insight into drug disposition kinetics. In this article, we present an In-radiolabeled FGF-2 derivative for noninvasive imaging in small animals deploying single photon emission tomography (SPECT).In-FGF-2 is equally well suitable for in vitro and ex vivo investigations as I-FGF-2. Furthermore,In-FGF-2 permits the performance of in vivo imaging, for example for the analysis of FGF-2 containing pharmaceutical formulations in developmental or preclinical stages. In-FGF-2 had affinity for the low-molecular-weight heparin enoxaparin identical to that of unlabeled FGF-2 (K: 0.6 ± 0.07 μM and 0.33 ± 0.03 μM, respectively) as assessed by isothermal titration calorimetry. The binding of In-FGF-2 to heparan sulfate proteoglycans (HPSGs) and the biological activity were comparable to those of unlabeled FGF-2, with EC values of 12 ± 2 pM and 25 ± 6 pM, respectively. In vivo biodistribution in healthy nude mice indicated a predominant accumulation of In-FGF-2 in filtering organs and minor uptake in the retina and the salivary and pituitary glands, which was confirmed by SPECT imaging. Therefore,In-FGF-2 is a valid tracer for future noninvasive animal imaging of FGF-2 in pharmaceutical development.
To measure the discrimination of the nomogram, a receiver-operating characteristic curve was construed, and the area under the curve was calculated. However, the area under the curve was 0.72, a very high value considering that the limit of acceptability is 0.70-0.80. The calculation system developed by the Memorial Sloan-Kettering Cancer Center provides a predictive value on the histopathologic state of sentinel lymph nodes.
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