Mounting in vitro, in vivo and clinical evidence suggest an important role for filopodia in driving cancer cell invasion. Using a high-throughput microscopic-based drug screen, we identify FDA-approved calcium channel blockers (CCBs) as potent inhibitors of filopodia formation in cancer cells. Unexpectedly, we discover that L-type calcium channels are functional and frequently expressed in cancer cells suggesting a previously unappreciated role for these channels during tumorigenesis. We further demonstrate that, at filopodia, L-type calcium channels are activated by integrin inside-out signalling, integrin activation and Src. Moreover, L-type calcium channels promote filopodia stability and maturation into talin-rich adhesions through the spatially restricted regulation of calcium entry and subsequent activation of the protease calpain-1. Altogether we uncover a novel and clinically relevant signalling pathway that regulates filopodia formation in cancer cells and propose that cycles of filopodia stabilization, followed by maturation into focal adhesions, directs cancer cell migration and invasion.

In vitro and in vivo behavior of nanoparticles (NPs) is often studied by tracing the NPs with fluorescent dyes. This requires stable incorporation of dyes within the NPs, as dye leakage may give a wrong interpretation of NP biodistribution, cellular uptake and intracellular distribution. Furthermore, NP labeling with trace amounts of dye should not alter NP properties such as interactions with cells or tissues.To allow for versatile NP studies with a variety of fluorescence-based assays, labeling of NPs with different dyes is desirable. Hence, when new dyes are introduced, simple and fast screening methods to assess labeling stability and NP-cell interactions are needed. For this purpose we have used a previously described generic flow cytometry assay; incubation of cells with NPs at 4°C and 37°C. Cell-NP interaction is confirmed by cellular fluorescence after 37°C incubation, and NP dye retention is confirmed when no cellular fluorescence is detected at 4°C.Three different NP-platforms labeled with six different dyes were screened, and a great variability in dye retention was observed. Surprisingly, incorporation of trace amounts of certain dyes was found to reduce, or even inhibit NP uptake. This work highlights the importance of thoroughly evaluating every dye-NP combination before pursuing fluorescent NP applications.3

Background Poly(alkyl cyanoacrylate) (PACA) nanoparticles have shown promise as drug carriers both to solid tumors and across the blood–brain barrier. Efficient drug delivery requires both high cellular uptake of the nanoparticles and release of the drug from the nanoparticles. Release of hydrophobic drugs from PACA nanoparticles is primarily governed by nanoparticle degradation, and this process has been poorly studied at the cellular level. Here we use the hydrophobic model drug Nile Red 668 (NR668) to investigate intracellular degradation of PACA nanoparticles by measuring changes in NR668 fluorescence emission and lifetime, as the spectral properties of NR668 depend on the hydrophobicity of the dye environment. We also assess the potential of poly(butyl cyanoacrylate) (PBCA) and poly(octyl cyanoacrylate) (POCA) nanoparticles for intracellular drug delivery in the prostate cancer cell line PC3 and rat brain endothelial cell line RBE4 and the role of endocytosis pathways in PACA nanoparticle uptake in those cell lines.ResultsFluorescence lifetime imaging, emission spectra analysis and Förster resonance energy transfer indicated that the intracellular degradation was in line with the degradation found by direct methods such as gas chromatography and scanning electron microscopy, showing that PBCA has a faster degradation rate compared to POCA. The combined P(BCA/OCA) nanoparticles had an intermediate degradation rate. The uptake of POCA and PBCA nanoparticles was much higher in RBE4 than in PC3 cells. Endocytosis inhibition studies showed that both clathrin- and caveolin-mediated endocytosis were involved in PACA nanoparticle uptake, and that the former played a predominant role, particularly in PC3 cells.ConclusionsIn the present study, we used three different optical techniques to show that within a 24-hour period PBCA nanoparticles degraded significantly inside cells, releasing their payload into the cytosol, while POCA nanoparticles remained intact. This indicates that it is possible to tune the intracellular drug release rate by choosing appropriate monomers from the PACA family or by using hybrid PACA nanoparticles containing different monomers. In addition, we showed that the uptake of PACA nanoparticles depends not only on the monomer material, but also on the cell type, and that different cell lines can use different internalization pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-015-0156-7) contains supplementary material, which is available to authorized users.