Cancer cell invasion through tissue barriers is the intrinsic feature of metastasis, the most life-threatening aspect of cancer. Detailed observation and analysis of cancer cell behaviour in a 3D environment is essential for a full understanding of the mechanisms of cancer cell invasion. The inherent limits of optical microscopy resolution do not allow to for in-depth observation of intracellular structures, such as invadopodia of invading cancer cells. The required resolution can be achieved using electron microscopy techniques such as FIB-SEM. However, visualising cells in a 3D matrix using FIB-SEM is challenging due to difficulties with localisation of a specific cell deep within the resin block. We have developed a new protocol based on the near-infrared branding (NIRB) procedure that extends the pattern from the surface grid deep inside the resin. This 3D burned pattern allows for precise trimming followed by targeted 3D FIB-SEM. Here we present detailed 3D CLEM results combining confocal and FIB-SEM imaging of cancer cell invadopodia that extend deep into the collagen meshwork.

• MeSH
• blízká infračervená spektroskopie metody   MeSH
• fibrosarkom patologie   MeSH
• invazivní růst nádoru MeSH
• lidé MeSH
• mikroskopie elektronová rastrovací metody   MeSH
• nádorové buňky kultivované MeSH
• nádory prsu patologie   MeSH
• počítačové zpracování obrazu MeSH
• podozomy patologie   MeSH
• zobrazování trojrozměrné metody   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Avian schistosomes, the causative agents of human cercarial dermatitis (or swimmer's itch), die in mammals but the mechanisms responsible for parasite elimination are unknown. Here we examined the role of reactive nitrogen species, nitric oxide (NO) and peroxynitrite, in the immune response of mice experimentally infected with Trichobilharzia regenti, a model species of avian schistosomes remarkable for its neuropathogenicity. METHODS: Inducible NO synthase (iNOS) was localized by immunohistochemistry in the skin and the spinal cord of mice infected by T. regenti. The impact of iNOS inhibition by aminoguanidine on parasite burden and growth was then evaluated in vivo. The vulnerability of T. regenti schistosomula to NO and peroxynitrite was assessed in vitro by viability assays and electron microscopy. Additionally, the effect of NO on the activity of T. regenti peptidases was tested using a fluorogenic substrate. RESULTS: iNOS was detected around the parasites in the epidermis 8 h post-infection and also in the spinal cord 3 days post-infection (dpi). Inhibition of iNOS resulted in slower parasite growth 3 dpi, but the opposite effect was observed 7 dpi. At the latter time point, moderately increased parasite burden was also noticed in the spinal cord. In vitro, NO did not impair the parasites, but inhibited the activity of T. regenti cathepsins B1.1 and B2, the peptidases essential for parasite migration and digestion. Peroxynitrite severely damaged the surface tegument of the parasites and decreased their viability in vitro, but rather did not participate in parasite clearance in vivo. CONCLUSIONS: Reactive nitrogen species, specifically NO, do not directly kill T. regenti in mice. NO promotes the parasite growth soon after penetration (3 dpi), but prevents it later (7 dpi) when also suspends the parasite migration in the CNS. NO-related disruption of the parasite proteolytic machinery is partly responsible for this effect.

• MeSH
• centrální nervový systém parazitologie   MeSH
• guanidiny farmakologie   MeSH
• infekce červy třídy Trematoda farmakoterapie   MeSH
• kůže parazitologie   MeSH
• kyselina peroxydusitá farmakologie   MeSH
• lidé MeSH
• mícha parazitologie   MeSH
• myši MeSH
• oxid dusnatý farmakologie   MeSH
• proteasy účinky léků   metabolismus   MeSH
• proteiny červů účinky léků   metabolismus   MeSH
• ptáci parazitologie   MeSH
• Schistosoma účinky léků   růst a vývoj   patogenita   MeSH
• Schistosomatidae účinky léků   růst a vývoj   patogenita   MeSH
• schistosomóza farmakoterapie   MeSH
• synthasa oxidu dusnatého účinky léků   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH