Implementing liquid biopsies into clinical decision making for cancer immunotherapy.

During the last decade, novel immunotherapeutic strategies, in particular antibodies directed against immune checkpoint inhibitors, have revolutionized the treatment of different malignancies leading to an improved survival of patients. Identification of immune-related biomarkers for diagnosis, prognosis, monitoring of immune responses and selection of patients for specific cancer immunotherapies is urgently required and therefore areas of intensive research. Easily accessible samples in particular liquid biopsies (body fluids), such as blood, saliva or urine, are preferred for serial tumor biopsies.Although monitoring of immune and tumor responses prior, during and post immunotherapy has led to significant advances of patients' outcome, valid and stable prognostic biomarkers are still missing. This might be due to the limited capacity of the technologies employed, reproducibility of results as well as assay stability and validation of results. Therefore solid approaches to assess immune regulation and modulation as well as to follow up the nature of the tumor in liquid biopsies are urgently required to discover valuable and relevant biomarkers including sample preparation, timing of the collection and the type of liquid samples. This article summarizes our knowledge of the well-known liquid material in a new context as liquid biopsy and focuses on collection and assay requirements for the analysis and the technical developments that allow the implementation of different high-throughput assays to detect alterations at the genetic and immunologic level, which could be used for monitoring treatment efficiency, acquired therapy resistance mechanisms and the prognostic value of the liquid biopsies.

Pronephric tubule morphogenesis in zebrafish depends on Mnx mediated repression of irx1b within the intermediate mesoderm.

Mutations in the homeobox transcription factor MNX1 are the major cause of dominantly inherited sacral agenesis. Studies in model organisms revealed conserved mnx gene requirements in neuronal and pancreatic development while Mnx activities that could explain the caudal mesoderm specific agenesis phenotype remain elusive. Here we use the zebrafish pronephros as a simple yet genetically conserved model for kidney formation to uncover a novel role of Mnx factors in nephron morphogenesis. Pronephros formation can formally be divided in four stages, the specification of nephric mesoderm from the intermediate mesoderm (IM), growth and epithelialisation, segmentation and formation of the glomerular capillary tuft. Two of the three mnx genes in zebrafish are dynamically transcribed in caudal IM in a time window that proceeds segmentation. We show that expression of one mnx gene, mnx2b, is restricted to the pronephric lineage and that mnx2b knock-down causes proximal pronephric tubule dilation and impaired pronephric excretion. Using expression profiling of embryos transgenic for conditional activation and repression of Mnx regulated genes, we further identified irx1b as a direct target of Mnx factors. Consistent with a repression of irx1b by Mnx factors, the transcripts of irx1b and mnx genes are found in mutual exclusive regions in the IM, and blocking of Mnx functions results in a caudal expansion of the IM-specific irx1b expression. Finally, we find that knock-down of irx1b is sufficient to rescue proximal pronephric tubule dilation and impaired nephron function in mnx-morpholino injected embryos. Our data revealed a first caudal mesoderm specific requirement of Mnx factors in a non-human system and they demonstrate that Mnx-dependent restriction of IM-specific irx1b activation is required for the morphogenesis and function of the zebrafish pronephros.

Pou5f1/Oct4 promotes cell survival via direct activation of mych expression during zebrafish gastrulation.

Myc proteins control cell proliferation, cell cycle progression, and apoptosis, and play important roles in cancer as well in establishment of pluripotency. Here we investigated the control of myc gene expression by the Pou5f1/Oct4 pluripotency factor in the early zebrafish embryo. We analyzed the expression of all known zebrafish Myc family members, myca, mycb, mych, mycl1a, mycl1b, and mycn, by whole mount in situ hybridization during blastula and gastrula stages in wildtype and maternal plus zygotic pou5f1 mutant (MZspg) embryos, as well as by quantitative PCR and in time series microarray data. We found that the broad blastula and gastrula stage mych expression, as well as late gastrula stage mycl1b expression, both depend on Pou5f1 activity. We analyzed ChIP-Seq data and found that both Pou5f1 and Sox2 bind to mych and mycl1b control regions. The regulation of mych by Pou5f1 appears to be direct transcriptional activation, as overexpression of a Pou5f1 activator fusion protein in MZspg embryos induced strong mych expression even when translation of zygotically expressed mRNAs was suppressed. We further showed that MZspg embryos develop enhanced apoptosis already during early gastrula stages, when apoptosis was not be detected in wildtype embryos. However, Mych knockdown alone did not induce early apoptosis, suggesting potentially redundant action of several early expressed myc genes, or combination of several pathways affected in MZspg. Experimental mych overexpression in MZspg embryos did significantly, but not completely suppress the apoptosis phenotype. Similarly, p53 knockdown only partially suppressed apoptosis in MZspg gastrula embryos. However, combined knockdown of p53 and overexpression of Mych completely rescued the MZspg apoptosis phenotype. These results reveal that Mych has anti-apoptotic activity in the early zebrafish embryo, and that p53-dependent and Myc pathways are likely to act in parallel to control apoptosis at these stages.

Characterization and regulation of the hb9/mnx1 beta-cell progenitor specific enhancer in zebrafish.

Differentiation of insulin producing beta-cells is a genetically well defined process that involves functions of various conserved transcription factors. Still, the transcriptional mechanisms underlying specification and determination of beta-cell fate are poorly defined. Here we provide the description of a beta-cell progenitor specific enhancer as a model to study initial steps of beta-cell differentiation. We show that evolutionary non-conserved upstream sequences of the zebrafish hb9 gene are required and sufficient for regulating expression in beta-cells prior to the onset of insulin expression. This enhancer contains binding sites for paired-box transcription factors and two E-boxes that in EMSA studies show interaction with Pax6b and NeuroD, respectively. We show that Pax6b is a potent activator of endodermal hb9 expression and that this activation depends on the beta-cell enhancer. Using genetic approaches we show that pax6b is crucial for maintenance but not induction of pancreatic hb9 transcription. As loss of Pax6b or Hb9 independently results in the loss of insulin expression, the data reveal a novel cross-talk between the two essential regulators of early beta-cell differentiation. While we find that the known pancreatic E-box binding proteins NeuroD and Ngn3 are not required for hb9 expression we also show that removal of both E-boxes selectively eliminates pancreatic specific reporter expression. The data provide evidence for an Ngn3 independent pathway of beta-cell specification that requires function of currently not specified E-box binding factors.

Pou5f1 contributes to dorsoventral patterning by positive regulation of vox and modulation of fgf8a expression.

Pou5f1/Oct-4 in mice is required for maintenance of embryonic pluripotent cell populations. Zebrafish pou5f1 maternal-zygotic mutant embryos (spiel ohne grenzen; MZspg) lack endoderm and have gastrulation and dorsoventral patterning defects. A contribution of Pou5f1 to the control of bmp2b, bmp4 and vox expression has been suggested, however the mechanisms remained unclear and are investigated in detail here. Low-level overexpression of a Pou5f1-VP16 activator fusion protein can rescue dorsalization in MZspg mutants, indicating that Pou5f1 acts as a transcriptional activator during dorsoventral patterning. Overexpression of larger quantities of Pou5f1-VP16 can ventralize wild-type embryos, while overexpression of a Pou5f1-En repressor fusion protein can dorsalize embryos. Lack of Pou5f1 causes a transient upregulation of fgf8a expression after mid-blastula transition, providing a mechanism for delayed activation of bmp2b in MZspg embryos. Overexpression of the Pou5f1-En repressor induces fgf8, suggesting an indirect mechanism of Pou5f1 control of fgf8a expression. Transcription of vox is strongly activated by Pou5f1-VP16 even when translation of zygotically expressed transcripts is experimentally inhibited by cycloheximide. In contrast, bmp2b and bmp4 are not activated under these conditions. We show that Pou5f1 binds to phylogenetically conserved Oct/Pou5f1 sites in the vox promoter, both in vivo (ChIP) and in vitro. Our data reveals a set of direct and indirect interactions of Pou5f1 with the BMP dorsoventral patterning network that serve to fine-tune dorsoventral patterning mechanisms and coordinate patterning with developmental timing.

The Rac1 regulator ELMO1 controls vascular morphogenesis in zebrafish.

RATIONALE: Angiogenesis is regulated by the small GTPase Rac1. The ELMO1/DOCK180 complex forms a guanine nucleotide exchange factor for Rac1, regulating its activation during cell migration in different biological systems. OBJECTIVE: To investigate the function of ELMO1/DOCK180 in vascular development. METHODS AND RESULTS: In situ hybridization studies for elmo1 identified a vascular and neuronal expression in zebrafish. Morpholino-based expression silencing of elmo1 severely impaired the formation of the vasculature, including intersomitic vessels, the dorsal longitudinal anastomotic vessel, the parachordal vessel, and the development of the thoracic duct in tg(fli1:EGFP) embryos. Mechanistically, we identified Netrin-1 and its receptor Unc5B as upstream activators of the ELMO1/DOCK180 complex, regulating its functional interaction and leading to Rac1 activation in endothelial cells and vessel formation in zebrafish. CONCLUSIONS: Our data have identified a novel signaling cascade regulating vasculature formation in zebrafish.

Zebrafish Pou5f1-dependent transcriptional networks in temporal control of early development.

The transcription factor POU5f1/OCT4 controls pluripotency in mammalian ES cells, but little is known about its functions in the early embryo. We used time-resolved transcriptome analysis of zebrafish pou5f1 MZspg mutant embryos to identify genes regulated by Pou5f1. Comparison to mammalian systems defines evolutionary conserved Pou5f1 targets. Time-series data reveal many Pou5f1 targets with delayed or advanced onset of expression. We identify two Pou5f1-dependent mechanisms controlling developmental timing. First, several Pou5f1 targets are transcriptional repressors, mediating repression of differentiation genes in distinct embryonic compartments. We analyze her3 gene regulation as example for a repressor in the neural anlagen. Second, the dynamics of SoxB1 group gene expression and Pou5f1-dependent regulation of her3 and foxD3 uncovers differential requirements for SoxB1 activity to control temporal dynamics of activation, and spatial distribution of targets in the embryo. We establish a mathematical model of the early Pou5f1 and SoxB1 gene network to demonstrate regulatory characteristics important for developmental timing. The temporospatial structure of the zebrafish Pou5f1 target networks may explain aspects of the evolution of the mammalian stem cell networks.

PlexinA3 restricts spinal exit points and branching of trunk motor nerves in embryonic zebrafish.

The pioneering primary motor axons in the zebrafish trunk are guided by multiple cues along their pathways. Plexins are receptor components for semaphorins that influence motor axon growth and path finding. We cloned plexinA3 in zebrafish and localized plexinA3 mRNA in primary motor neurons during axon outgrowth. Antisense morpholino knock-down led to substantial errors in motor axon growth. Errors comprised aberrant branching of primary motor nerves as well as additional exit points of axons from the spinal cord. Excessively branched and supernumerary nerves were found in both ventral and dorsal pathways of motor axons. The trunk environment and several other types of axons, including trigeminal axons, were not detectably affected by plexinA3 knock-down. RNA overexpression rescued all morpholino effects. Synergistic effects of combined morpholino injections indicate interactions of plexinA3 with semaphorin3A homologs. Thus, plexinA3 is a crucial receptor for axon guidance cues in primary motor neurons.

Zebrafish mnx genes in endocrine and exocrine pancreas formation.

The pancreas consists of two components, which exert distinct homeostatic function, an endocrine part that secretes hormones including insulin and an exocrine part that produces digestive enzymes. In mouse, one of the factors essential for development of the pancreas is the Mnx-class homeobox transcription factor Hb9. Genetic studies showed that Hb9 is required for both initial morphogenesis of the pancreas as well as subsequent differentiation of insulin-producing beta-cells [Nat. Genet. 23 (1999) 71; Nat. Genet. 23 (1999) 67]. To get a better understanding of what role mnx genes play in pancreas development, we isolated and characterized mnx genes in the model organism zebrafish. We found one gene with homology to hb9 orthologs and two that display homology to the related chicken mnr2. Embryonic expression of the zebrafish mnx genes is very dynamic and is detected in derivatives of all three germ layers. Endodermal expression of hb9 takes place in the early gut endoderm and, later, in the endocrine pancreas and the swim bladder. In addition, one of the mnr2 genes, mnr2a, shows expression in an endodermal cell population that is initially intermingled with insulin-positive cells and that later becomes restricted to the exocrine pancreas. In knockdown studies using antisense morpholinos, we show that hb9 is essential for differentiation of the insulin-producing beta-cells but unlike mouse Hb9 is not needed for early morphogenesis of the pancreas. In contrast, mnr2a is required during late morphogenesis of the exocrine pancreas. In summary, our data suggest a tissue-specific mnx-expression code in the zebrafish pancreas and they reveal a novel role of an mnr2-related gene.