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Invasive cancer cells develop small actin-based protrusions called invadopodia, which perform a primordial role in metastasis and extracellular matrix remodelling. Neural Wiskott-Aldrich syndrome protein (N-WASp) is a scaffold protein which can directly bind to actin monomers and Arp2/3 and is a crucial player in the formation of an invadopodium precursor. Expression modulation has pointed to an important role for N-WASp in invadopodium formation but the role of its C-terminal VCA domain in this process remains unknown. In this study, we generated alpaca nanobodies against the N-WASp VCA domain and investigated if these nanobodies affect invadopodium formation. By using this approach, we were able to study functions of a selected functional/structural N-WASp protein domain in living cells, without requiring overexpression, dominant negative mutants or siRNAs which target the gene, and hence the entire protein. When expressed as intrabodies, the VCA nanobodies significantly reduced invadopodium formation in both MDA-MB-231 breast cancer and HNSCC61 head and neck squamous cancer cells. Furthermore, expression of distinct VCA Nbs (VCA Nb7 and VCA Nb14) in PC-3 prostate cancer cells resulted in reduced overall matrix degradation without affecting MMP9 secretion/activation or MT1-MMP localisation at invadopodial membranes. From these results, we conclude that we have generated nanobodies targeting N-WASp which reduce invadopodium formation and functioning, most likely via regulation of N-WASp—Arp2/3 complex interaction, indicating that this region of N-WASp plays an important role in these processes. [ABSTRACT FROM AUTHOR]

Purpose: Gelsolin amyloidosis (AGel), also known as familial amyloidosis, Finnish type (FAF), is an autosomal, dominant, incurable disease caused by a point mutation (G654A/T) in the gelsolin (GSN) gene. The mutation results in loss of a Ca2+-binding site in the second gelsolin domain. Subsequent incorrect folding exposes a cryptic furin cleavage site, leading to the formation of a 68-kDa C-terminal cleavage product (C68) in the trans-Golgi network. This C68 fragment is cleaved by membrane type 1-matrix metalloproteinase (MT1-MMP) during secretion into the extracellular environment, releasing 8- and 5-kDa amyloidogenic peptides. These peptides aggregate and cause disease-associated symptoms. We set out to investigate whether AGel-specific nanobodies could be used to monitor amyloidogenic gelsolin buildup.Procedures: Three nanobodies (FAF Nb1-3) raised against the 8-kDa fragment were screened as AGel amyloid imaging agents in WT and AGel mice using 99mTc-based single-photon emission computed tomography (SPECT)/X-ray tomography (CT), biodistribution analysis, and immunofluorescence (IF). The quantitative characteristics were analyzed in a follow-up study with a Nb11-expressing mouse model.Results: All three nanobodies possess the characteristics desired for a 99mTc-based SPECT/CT imaging agent, high specificity and a low background signal. FAF Nb1 was identified as the most potent, based on its superior signal-to-noise ratio and signal specificity. As a proof of concept, we implemented 99mTc-FAF Nb1 in a follow-up study of the Nb11-expressing AGel mouse model. Using biodistribution analysis and immunofluorescence, we demonstrated the validity of the data acquired via 99mTc-FAF Nb1 SPECT/CT.Conclusion: These findings demonstrate the potential of this nanobody as a non-invasive tool to image amyloidogenic gelsolin deposition and assess the therapeutic capacity of AGel therapeutics currently under development. We propose that this approach can be extended to other amyloid diseases, thereby contributing to the development of specific therapies. [ABSTRACT FROM AUTHOR]