Engineering Mesoscopic 3D Tumor Models with a Self-Organizing Vascularized Matrix.

Advanced in vitro systems such as multicellular spheroids and lab-on-a-chip devices have been developed, but often fall short in reproducing the tissue scale and self-organization of human diseases. A bioprinted artificial tumor model is introduced with endothelial and stromal cells self-organizing into perfusable and functional vascular structures. This model uses 3D hydrogel matrices to embed multicellular tumor spheroids, allowing them to grow to mesoscopic scales and to interact with endothelial cells. It is shown that angiogenic multicellular tumor spheroids promote the growth of a vascular network, which in turn further enhances the growth of cocultivated tumor spheroids. The self-developed vascular structure infiltrates the tumor spheroids, forms functional connections with the bioprinted endothelium, and can be perfused by erythrocytes and polystyrene microspheres. Moreover, cancer cells migrate spontaneously from the tumor spheroid through the self-assembled vascular network into the fluid flow. Additionally, tumor type specific characteristics of desmoplasia, angiogenesis, and metastatic propensity are preserved between patient-derived samples and tumors derived from this same material growing in the bioreactors. Overall, this modular approach opens up new avenues for studying tumor pathophysiology and cellular interactions in vitro, providing a platform for advanced drug testing while reducing the need for in vivo experimentation.

Combining Engineered U1 snRNA and Antisense Oligonucleotides to Improve the Treatment of a BBS1 Splice Site Mutation.

Manipulation of pre-mRNA processing is a promising approach toward overcoming disease-causing mutations and treating human diseases. We show that a combined treatment applying two splice-manipulating technologies improves therapeutic efficacies to correct mutation-induced splice defects. Previously, we identified a family affected by retinitis pigmentosa caused by the homozygous BBS1 splice donor site mutation c.479G > A. The mutation leads to both exon 5 skipping and intron 5 retention. We developed a therapeutic approach applying lentivirus-mediated gene delivery of engineered U1 small nuclear RNA (U1), which resulted in increased levels of correctly spliced BBS1. Herein, we show that the therapeutic effect of the engineered U1 efficiently reverted exon skipping but failed to reduce the intron retention. To complement the engineered U1 treatment, we identified four different antisense oligonucleotides (AONs) that block intron 5 retention in BBS1 transcripts. A treatment using engineered U1 in combination with AONs showed the highest therapeutic efficacy and increased the amount of correctly spliced BBS1 transcripts. We did not detect elevated levels of apoptotic cell death in AON-treated cell lines. In conclusion, engineered U1 or AONs provide efficient therapies with complementary effects and can be combined to increase efficacy of therapeutic approaches to correct splice defects.

Comparative Analysis of Gene Regulatory Network Components in the Auditory Hindbrain of Mice and Chicken.

The neurons in the mammalian and avian auditory hindbrain nuclei share a number of significant morphological and physiological properties for fast, secure and precise neurotransmission, such as giant synapses, voltage-gated K+ channels and fast AMPA receptors. Based on the independent evolution of the middle ear in these two vertebrate lineages, on different embryonic origins of the nuclei and on marked differences on the circuit level, these similarities are assumed to reflect convergent evolution. Independent acquisition of similar phenotypes can be produced by divergent evolution of genetic mechanisms or by similar molecular mechanisms. The distinction between these two possibilities requires knowledge of the gene regulatory networks (GRNs) that orchestrate the development of auditory hindbrain structures. We therefore compared the expression pattern of GRN components, both transcription factors (TFs) and noncoding RNA, during terminal differentiation of the auditory hindbrain structures in mouse and chicken when neurons acquire their final morphological and electrophysiological properties. In general, we observed broad expression of these genes in the mouse auditory cochlear nucleus complex and the superior olivary complex at both postnatal day 4 (P4) and at P25, and for the chicken at the equivalent developmental stages, i.e. embryonic day 13 (E13) and at P14-P17. Our data are in agreement with a model based on similar molecular mechanisms underlying terminal differentiation and maintenance of neuronal cell identity in the auditory hindbrain of different vertebrate lineages. This conservation might reflect developmental constraints arising from the tagmatic organization of rhombomeres and the evolutionarily highly conserved GRNs operating in these structures.