Real-time imaging of polymersome nanoparticles in zebrafish embryos engrafted with melanoma cancer cells: Localization, toxicity and treatment analysis.

BACKGROUND: The developing zebrafish is an emerging tool in nanomedicine, allowing non-invasive live imaging of the whole animal at higher resolution than is possible in the more commonly used mouse models. In addition, several transgenic fish lines are available endowed with selected cell types expressing fluorescent proteins; this allows nanoparticles to be visualized together with host cells. METHODS: Here, we introduce the zebrafish neural tube as a robust injection site for cancer cells, excellently suited for high resolution imaging. We use light and electron microscopy to evaluate cancer growth and to follow the fate of intravenously injected nanoparticles. FINDINGS: Fluorescently labelled mouse melanoma B16 cells, when injected into this structure proliferated rapidly and stimulated angiogenesis of new vessels. In addition, macrophages, but not neutrophils, selectively accumulated in the tumour region. When injected intravenously, nanoparticles made of Cy5-labelled poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate) (PEG-PDPA) selectively accumulated in the neural tube cancer region and were seen in individual cancer cells and tumour associated macrophages. Moreover, when doxorubicin was released from PEG-PDPA, in a pH dependant manner, these nanoparticles could strongly reduce toxicity and improve the treatment outcome compared to the free drug in zebrafish xenotransplanted with mouse melanoma B16 or human derived melanoma cells. INTERPRETATION: The zebrafish has the potential of becoming an important intermediate step, before the mouse model, for testing nanomedicines against patient-derived cancer cells. FUNDING: We received funding from the Norwegian research council and the Norwegian cancer society.

Ex vivo live imaging of single cell divisions in mouse neuroepithelium.

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line (1-3) and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP (4). In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Increased expression of Grainyhead-like-3 rescues spina bifida in a folate-resistant mouse model.

Neural tube defects (NTDs), such as spina bifida, are common and severe birth defects in humans but the underlying causes are poorly understood. The pathogenesis and etiology of spina bifida in the curly tail mouse closely resemble defects in humans, providing a well-characterized model of NTDs. Grainyhead-like-3 (Grhl3), which encodes a transcription factor, was recently identified as a candidate gene for curly tail based on chromosomal location and the occurrence of spina bifida in Grhl3 null mice. However, the causative curly tail mutation has not been established, while the relationship between Grhl3 gene expression and the known cellular defect leading to NTDs in curly tail is unknown. Spina bifida in curly tail results from a cell type-specific proliferation defect in the hindgut endoderm, and we find that Grhl3 is expressed specifically in this tissue during the final stages of spinal neural tube closure in wild type embryos. Moreover, Grhl3 expression is diminished in the spinal region of neurulation-stage curly tail embryos. Curly tail mice do not carry a coding region mutation in Grhl3, however, we found a putative regulatory mutation upstream of the Grhl3 gene, which may be responsible for the expression deficit. In order to test the hypothesis that spina bifida in curly tail mice results from insufficient expression of Grhl3, we generated Grhl3-expressing curly tail mice by bacterial artificial chromosome-mediated transgenesis and demonstrated complete rescue of spina bifida. This study provides evidence for a critical role of diminished Grhl3 expression in causing spinal NTDs in the curly tail mouse model.