Efficient CPP-mediated Cre protein delivery to developing and adult CNS tissues.

BACKGROUND: Understanding and manipulating gene function in physiological conditions is a major objective for both fundamental and applied research. In contrast to other experimental settings, which use either purely genetic or gene delivery (viral or non-viral) strategies, we report here a strategy based on direct protein delivery to central nervous system (CNS) tissues. We fused Cre recombinase with cell-penetrating peptides and analyzed the intracellular biological activity of the resulting chimerical proteins when delivered into cells endowed with Cre-mediated reporter gene expression. RESULTS: We show that active Cre enzymatic conjugates are readily internalized and exert their enzymatic activity in the nucleus of adherent cultured cells. We then evaluated this strategy in organotypic cultures of neural tissue explants derived from reporter mice carrying reporter "floxed" alleles. The efficacy of two protocols was compared on explants, either by direct addition of an overlying drop of protein conjugate or by implantation of conjugate-coated beads. In both cases, delivery of Cre recombinase resulted in genomic recombination that, with the bead protocol, was restricted to discrete areas of embryonic and adult neural tissues. Furthermore, delivery to adult brain tissue resulted in the transduction of mature postmitotic populations of neurons. CONCLUSION: We provide tools for the spatially restricted genetic modification of cells in explant culture. This strategy allows to study lineage, migration, differentiation and death of neural cells. As a proof-of-concept applied to CNS tissue, direct delivery of Cre recombinase enabled the selective elimination of an interneuron subpopulation of the spinal cord, thereby providing a model to study early events of neurodegenerative processes. Thus our work opens new perspectives for both fundamental and applied cell targeting protocols using proteic cargoes which need to retain full bioactivity upon internalisation, as illustrated here with the oligomeric Cre recombinase.

Choroid-plexus-derived Otx2 homeoprotein constrains adult cortical plasticity.

Brain plasticity is often restricted to critical periods in early life. Here, we show that a key regulator of this process in the visual cortex, Otx2 homeoprotein, is synthesized and secreted globally from the choroid plexus. Consequently, Otx2 is maintained in selected GABA cells unexpectedly throughout the mature forebrain. Genetic disruption of choroid-expressed Otx2 impacts these distant circuits and in the primary visual cortex reopens binocular plasticity to restore vision in amblyopic mice. The potential to regulate adult cortical plasticity through the choroid plexus underscores the importance of this structure in brain physiology and offers therapeutic approaches to recovery from a broad range of neurodevelopmental disorders.

New blocking antibodies impede adhesion, migration and survival of ovarian cancer cells, highlighting MFGE8 as a potential therapeutic target of human ovarian carcinoma.

Milk Fat Globule--EGF--factor VIII (MFGE8), also called lactadherin, is a secreted protein, which binds extracellularly to phosphatidylserine and to αvß3 and αvß5 integrins. On human and mouse cells expressing these integrins, such as endothelial cells, phagocytes and some tumors, MFGE8/lactadherin has been shown to promote survival, epithelial to mesenchymal transition and phagocytosis. A protumoral function of MFGE8 has consequently been documented for a few types of human cancers, including melanoma, a subtype of breast cancers, and bladder carcinoma. Inhibiting the functions of MFGE8 could thus represent a new type of therapy for human cancers. Here, we show by immunohistochemistry on a collection of human ovarian cancers that MFGE8 is overexpressed in 45% of these tumors, and we confirm that it is specifically overexpressed in the triple-negative subtype of human breast cancers. We have established new in vitro assays to measure the effect of MFGE8 on survival, adhesion and migration of human ovarian and triple-negative breast cancer cell lines. Using these assays, we could identify new MFGE8-specific monoclonal antibodies, which efficiently blocked these three tumor-promoting effects of MFGE8. Our results suggest future use of MFGE8-blocking antibodies as new anti-cancer therapeutics in subgroups of ovarian carcinoma, and triple-negative breast carcinoma patients.

An essential role for decorin in bladder cancer invasiveness.

Muscle-invasive forms of urothelial carcinomas are responsible for most mortality in bladder cancer. Finding new treatments for invasive bladder tumours requires adequate animal models to decipher the mechanisms of progression, in particular the way tumours interact with their microenvironment. Herein, using the murine bladder tumour cell line MB49 and its more aggressive variant MB49-I, we demonstrate that the adaptive immune system efficiently limits progression of MB49, whereas MB49-I has lost tumour antigens and is insensitive to adaptive immune responses. Furthermore, we unravel a parallel mechanism developed by MB49-I to subvert its environment: de novo secretion of the proteoglycan decorin. We show that decorin overexpression in the MB49/MB49-I model is required for efficient progression, by promoting angiogenesis and tumour cell invasiveness. Finally, we show that these results are relevant to muscle-invasive human bladder carcinomas, which overexpress decorin together with angiogenesis- and adhesion/migration-related genes, and that decorin overexpression in the human bladder carcinoma cell line TCCSUP is required for efficient invasiveness in vitro. We thus propose decorin as a new therapeutic target for these aggressive tumours.