Publications

Notice of republication An incorrect version of S1 Fig was published in error. This article was republished on July 27, 2020 to correct for this error. Please download this article again to view the correct version.

Adult tissue stem cells can form self-organizing 3D organoids in vitro. Organoids resemble small units of their organ of origin and have great potential for tissue engineering, as well as models of disease. However, current culture technology limits the size, architecture and complexity of organoids. Here, we review the establishment of intestinal and hepatic organoids and discuss how the convergence of organoids and biofabrication technologies can help overcome current limitations, and thereby further advance the translational application of organoids in tissue engineering and regenerative medicine.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 °C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

<p>Patient and sample characteristics</p>

ABSTRACT Vγ9Vδ2T cells have the unique ability to recognize a broad range of malignant transformed cells. The tumor targeting event involving BTN2A1 and BTN3A1 dimers on the tumor cell surface is critical, leading to full activation of the TCR. Although the molecular mechanisms governing TCR engagement and T cell activation are well-characterized, the role of Vγ9Vδ2 T cells in cancer immune surveillance remains to be fully elucidated, particularly the mechanisms that enable these cells to discriminate between healthy and malignant cells at an early stage of malignant transformation. We employed two independent, genetically engineered step-wise mutagenesis models of human colorectal and breast cancer that mimic the transformation steps leading to tumor formation. We demonstrate that various single oncogenic mutations introduced into healthy organoids or cells, are sufficient to upregulate surface expressed BTN2A1 and enable Vγ9Vδ2 TCR binding to tumor cells. However, full activation of T cells through a Vγ9Vδ2TCR required additional subsequent phosphorylation of juxtamembrane (JTM) amino acids of BTN3A1, leading to the activating heterodimerization of BTN2A1 and 3A1. Using a protein interactome mapping pipeline, we identified PHLDB2, SYNJ2 and CARMIL1 as key players in controlling these delicate dual surface dynamics of BTN2A1 and 3A1 during early transformation. This mode of action allowed Vγ9Vδ2TCR T cells to control tumors in vitro and in vivo, emphasizing the crucial role of these molecules from early mutagenesis, to advanced cancer stages, and highlighting the therapeutic potential of a Vγ9Vδ2TCR.

<title>Abstract</title> Clinical outcome for patients suffering from head and neck squamous cell carcinoma (HNSCC) remains poor. This is mostly due to highly invasive tumors that cause loco-regional relapses after initial therapeutic intervention. The molecular pathways governing the detrimental invasive growth modes in HNSCC remain however understudied. Here, we have established HNSCC patient derived organoid (PDO) models that recapitulate 3-dimensional invasion <italic>in vitro</italic>. Single cell mRNA sequencing was applied to study the differences between non-invasive and invasive conditions, and in a collective versus single cell invading PDO model. Differential expression analysis under invasive conditions in Collagen gels reveals an overall upregulation of a YAP-centered transcriptional program, irrespective of the invasion mode. However, we find that collectively invading HNSCC PDO cells show elevated levels of YAP transcription targets when compared to single cell invasion. Also, collectively invading cells are characterized by increased nuclear translocation of YAP within the invasive strands, which coincides with Collagen-I matrix alignment at the invasive front. Using gene set enrichment analysis, we identify immune cell-like migratory pathways in the single cell invading HNSCC PDO, while collective invasion is characterized by overt upregulation of adhesion and migratory pathways. Lastly, based on a clinical head and neck cancer cohort, we demonstrate that the identified collective invasion signature provides a candidate prognostic platform for survival in HNSCC. By uncoupling collective and single cell invasive programs, we have established invasion signatures that may guide new therapeutic options.

Research on the correspondence between retirement intentions and subsequent behaviour is scarce. We aimed to explore possible associations between retirement intentions and behaviour, using five-year high-quality quantitative panel data on Norwegian senior workers. Retirement intentions operate at different levels of firmness: (a) considerations; (b) preferences; and (c) decisions. Compared to work continuation considerations, a targeted age for retirement improved predictive power whether the target was preferred or decided, and particularly so if the target ( i.e. the preferred or decided age of retirement), corresponded with a normative retirement age. Because more workers are able to state a preferred age of retiring than a decision about when to retire, preferences may be better proxies for retirement behaviour than decisions, when the issue is planning for policies. The correspondence between intentions and behaviour varies primarily by health, education and type of work. Older workers with poor health, and workers with low education, often retire earlier than they prefer. Blue-collar workers often retire earlier than they had decided. These findings illustrate the possible effect of labour market resources, not only for senior workers' labour market participation, but also for their opportunities to work up to the age they prefer or had decided. Even for white-collar workers and those in good health, constraints seem to apply when they wish to retire late.

Additional file 10. Table S9