Non-Invasive Three-Dimensional Cell Analysis in Bioinks by Raman Imaging.

3D bioprinting is an emerging biofabrication strategy using bioinks, comprising cells and biocompatible materials, to produce functional tissue models. Despite progress in building increasingly complex objects, biological analyses in printed constructs remain challenging. Especially, methods that allow non-invasive and non-destructive evaluation of embedded cells are largely missing. Here, we implemented Raman imaging for molecular-sensitive investigations on bioprinted objects. Different aspects such as culture formats (2D, 3D-cast, and 3D-printed), cell types (endothelial cells and fibroblasts), and the selection of the biopolymer (alginate, alginate/nanofibrillated cellulose, alginate/gelatin) were considered and evaluated. Raman imaging allowed for marker-independent identification and localization of subcellular components against the surrounding biomaterial background. Furthermore, single-cell analysis of spectral signatures, performed by multivariate analysis, demonstrated discrimination between endothelial cells and fibroblasts and identified cellular features influenced by the bioprinting process. In summary, Raman imaging was successfully established to analyze cells in 3D culture in situ and evaluate them with regard to the localization of different cell types and their molecular phenotype as a valuable tool for quality control of bioprinted objects.

A self-enforcing CD44s/ZEB1 feedback loop maintains EMT and stemness properties in cancer cells.

Invasion and metastasis of carcinomas are often activated by induction of aberrant epithelial-mesenchymal transition (EMT). This is mainly driven by the transcription factor ZEB1, promoting tumor-initiating capacity correlated with increased expression of the putative stem cell marker CD44. However, the direct link between ZEB1, CD44 and tumourigenesis is still enigmatic. Remarkably, EMT-induced repression of ESRP1 controls alternative splicing of CD44, causing a shift in the expression from the variant CD44v to the standard CD44s isoform. We analyzed whether CD44 and ZEB1 regulate each other and show that ZEB1 controls CD44s splicing by repression of ESRP1 in breast and pancreatic cancer. Intriguingly, CD44s itself activates the expression of ZEB1, resulting in a self-sustaining ZEB1 and CD44s expression. Activation of this novel CD44s-ZEB1 regulatory loop has functional impact on tumor cells, as evident by increased tumor-sphere initiation capacity, drug-resistance and tumor recurrence. In summary, we identified a self-enforcing feedback loop that employs CD44s to activate ZEB1 expression. This renders tumor cell stemness independent of external stimuli, as ZEB1 downregulates ESRP1, further promoting CD44s isoform synthesis.

Linkage of autosomal-dominant common variable immunodeficiency to chromosome 4q.

The phenotype of common variable immunodeficiency (CVID) is characterized by recurrent infections owing to hypogammaglobulinemia, with deficiency in immunoglobulin (Ig)G and at least one of IgA or IgM. Family studies have shown a genetic association between CVID and selective IgA deficiency (IgAD), the latter being a milder disorder compatible with normal health. Approximately 20-25% of CVID cases are familial, if one includes families with at least one case of CVID and one of IgAD. Nijenhuis et al described a five-generation family with six cases of CVID, five cases of IgAD, and three cases of dysgammaglobulinemia. We conducted a genome-wide scan on this family seeking genetic linkage. One interval on chromosome 4q gives a peak multipoint LOD score of 2.70 using a strict model that treats only the CVID patients and one obligate carrier with dysgammaglobulinemia as affected. Extending the definition of likely affected to include IgAD boosts the peak multipoint LOD score to 3.38. The linkage interval spans at least from D4S2361 to D4S1572. We extended our study to a collection of 32 families with at least one CVID case and a second case of either CVID or IgAD. We used the same dominant penetrance model and genotyped and analyzed nine markers on 4q. The 32 families have a peak multipoint LOD score under heterogeneity of 0.96 between markers D4S423 and D4S1572 within the suggested linkage interval of the first family, and an estimated proportion of linked families (alpha) of 0.32, supporting the existence of a disease-causing gene for autosomal-dominant CVID/IgAD on chromosome 4q.

Analysis of families with common variable immunodeficiency (CVID) and IgA deficiency suggests linkage of CVID to chromosome 16q.

Common variable immunodeficiency (CVID) is an antibody deficiency syndrome that often co-occurs in families with selective IgA deficiency (IgAD). Vorechovský et al. (Am J Hum Genet 64:1096-1109, 1999; J Immunol 164:4408-4416, 2000) ascertained and genotyped 101 multiplex IgAD families and used them to identify and fine map the IGAD1 locus on chromosome 6p. We analyzed the original genotype data in a subset of families with at least one case of CVID and present evidence of a CVID locus on chromosome 16q with autosomal dominant inheritance. The peak (model-based) LOD score for the best marker D16S518 is 2.83 at theta=0.07, and a 4-marker LOD score under heterogeneity peaks at 3.00 with alpha=0.68. The (model-free) NPL score using the same markers peaks at the same location with a value of 3.38 (P=0.0001).