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Droplet microfluidics is a powerful tool for a variety of biological applications including single-cell genetics, antibody discovery and directed evolution. All these applications make use of genetic libraries, illustrating the difficulty of generating chemically distinct droplets for screening applications. This protocol describes our Braille Display valving platform for on-demand generation of droplets with different chemical contents (16 different reagents and combinations thereof), as well as sorting droplets with different chemical properties, on the basis of fluorescence signals. The Braille Display platform is compact, versatile and cost efficient (only ~US$1,000 on top of a standard droplet microfluidics setup). The procedure includes manufacturing of microfluidic chips, assembly of custom hardware, co-encapsulation of cells and drugs into droplets, fluorescence detection of readout signals and data analysis using shared, freely available LabVIEW and Python packages. As a first application, we demonstrate the complete workflow for screening cancer cell drug sensitivities toward 74 conditions. Furthermore, we describe here an assay enabling the normalization of the observed drug sensitivity to the number of cancer cells per droplet, which additionally increases the robustness of the system. As a second application, we also demonstrate the sorting of droplets according to enzymatic activity. The drug screening application can be completed within 2 d; droplet sorting takes ~1 d; and all preparatory steps for manufacturing molds, chips and setting up the Braille controller can be accomplished within 1 week.

Canonical Wnt/β-catenin signaling is an established regulator of cellular state and its critical contributions to tumor initiation, malignant tumor progression and metastasis formation have been demonstrated in various cancer types. Here, we investigated how the binding of β-catenin to the transcriptional coactivators B-cell CLL/lymphoma 9 (Bcl9) and Bcl9-Like (Bcl9L) affected mammary gland carcinogenesis in the MMTV-PyMT transgenic mouse model of metastatic breast cancer. Conditional knockout of both Bcl9and Bcl9Lresulted into tumor cell death. In contrast, disrupting the interaction of Bcl9/Bcl9L with β-catenin, either by deletion of their HD2 domains or by a point mutation in the N-terminal domain of β-catenin (D164A), diminished primary tumor growth and tumor cell proliferation and reduced tumor cell invasion and lung metastasis. In comparison, the disruption of HD1 domain-mediated binding of Bcl9/Bcl9L to Pygopus had only moderate effects. Interestingly, interfering with the β-catenin-Bcl9/Bcl9L-Pygo chain of adapters only partially impaired the transcriptional response of mammary tumor cells to Wnt3a and TGFβ treatments. Together, the results indicate that Bcl9/Bcl9L modulate but are not critically required for canonical Wnt signaling in its contribution to breast cancer growth and malignant progression, a notion consistent with the “just-right” hypothesis of Wnt-driven tumor progression.

The emergence of low molecular weight kinase inhibitors as “targeted” drugs has led to remarkable advances in the treatment of cancer patients. The clinical benefits of these tumor therapies, however, vary widely in patient populations and with duration of treatment. Intrinsic and acquired resistance against such drugs limits their efficacy. In addition to the well studied mechanisms of resistance based upon drug transport and metabolism, genetic alterations in drug target structures and the activation of compensatory cell signaling have received recent attention. Adaptive responses can be triggered which counteract the initial dependence of tumor cells upon a particular signaling molecule and allow only a transient inhibition of tumor cell growth. These compensating signaling mechanisms are often based upon the relief of repression of regulatory feedback loops. They might involve cell autonomous, intracellular events or they can be mediated via the secretion of growth factor receptor ligands into the tumor microenvironment and signal induction in an auto- or paracrine fashion. The transcription factors Stat3 and Stat5 mediate the biological functions of cytokines, interleukins and growth factors and can be considered as endpoints of multiple signaling pathways. In normal cells this activation is transient and the Stat molecules return to their non-phosphorylated state within a short time period. In tumor cells the balance between activating and de-activating signals is disturbed resulting in the persistent activation of Stat3 or Stat5. The constant activation of Stat3 induces the expression of target genes, which cause the proliferation and survival of cancer cells, as well as their migration and invasive behavior. Activating components of the Jak-Stat pathway have been recognized as potentially valuable drug targets and important principles of compensatory signaling circuit induction during targeted drug treatment have been discovered in the context of kinase inhibition studies in HNSCC cells [1]. The treatment of HNSCC with a specific inhibitor of c-Src, initially resulted in reduced Stat3 and Stat5 activation and subsequently an arrest of cell proliferation and increased apoptosis. However, the inhibition of c-Src only caused a persistent inhibition of Stat5, whereas the inhibition of Stat3 was only transient. The activation of Stat3 was restored within a short time period in the presence of the c-Src inhibitor. This process is mediated through the suppression of PStat5 activity and the decrease in the expression of the Stat5 dependent target gene SOCS2, a negative regulator of Jak2. Jak2 activity is enhanced upon SOCS2 downregulation and causes the reactivation of Stat3. A similar observation has been made upon inhibition of Bmx, bone marrow kinase x-linked, activated in the murine glioma cell lines Tu-2449 and Tu-9648. Its inhibition resulted in a transient decrease of P-Stat3 and the induction of a compensatory Stat3 activation mechanism, possibly through the relief of negative feedback inhibition and Jak2 activation. These observations indicate that the inhibition of a single tyrosine kinase might not be sufficient to induce lasting therapeutic effects in cancer patients. Compensatory kinases and pathways might become activated and maintain the growth and survival of tumor cells. The definition of these escape pathways and their preemptive inhibition will suggest effective new combination therapies for cancer.

Introduction:Foetal cells present in amniotic fluid (AF) have been used for many years to perform prenatal genetic screening. Recent reports suggested that these cells might have additional benefits. AF contains, in addition to committed and differentiated cells, a subpopulation with stem cell characteristics. AF-derived stem cells (AFS) have functions found in mesenchymal stem cells, but in addition, exhibit a potent expansion capacity and plasticity. AFS are able to undergo multi-lineage differentiation and produce progeny indicative of all three germ layers.Areas covered:The experimental approaches available to isolate AFS and their potential for tissue engineering, the repair of organs through cell replacement and tissue regeneration.Expert opinion:The deployment of AFS for tissue regeneration offers advantages over the use of embryonic or adult stem cells: i) AF represents a convenient and non-contested source for obtaining stem cells; ii) their derivation is relatively simple and rapid; iii) no feeder layers are required for their cultivation; iv) they display no spontaneous differentiation in culture; and v) their stem cell phenotype is not affected by long-term storage. The application of AFS for tissue replacement therapies in vivois at a very early stage, but existing studies indicate great potential for clinical use.

Signal transducers and activators of transcription (STATs) comprise a unique family of transcription factors, which transmit the interactions of cytokines, hormones and growth factors with their cell surface receptors into transcriptional programs. The mechanism of STAT activation has been well-established and comprises tyrosine phosphorylation, dimerization, nuclear translocation, binding to specific DNA response elements, recruitment of co-activators or co-repressors and transcriptional induction or repression of target genes. Gene deletion, microarrays, proteomics and chromatin immunoprecipitation experiments have revealed target genes with a broad range of functions regulated by STAT3 and STAT5. In the mammary gland, STAT5-induced genes contribute mainly to the prolactin dependent lobulo-alveolar development, whereas STAT3 induced genes control apoptosis during involution. Crucial effects have also been observed in other tissues. The germ line deletion of STAT3 or STAT5 causes early embryonal or perinatal lethality in mice. STAT5 is also required for proliferation of T- and B-cells and hematopoietic stem cell self-renewal. Deregulated STAT activity is often found associated with tumorigenesis and activated STATs seem to be limiting components in tumor cells. This review summarizes the functions of STAT3 and STAT5 in different cell types and the strategies that are used to counteract their action in tumor cells.Signal transducers and activators of transcription (STATs) comprise a unique family of transcription factors, which transmit the interactions of cytokines, hormones and growth factors with their cell surface receptors into transcriptional programs. The mechanism of STAT activation has been well-established and comprises tyrosine phosphorylation, dimerization, nuclear translocation, binding to specific DNA response elements, recruitment of co-activators or co-repressors and transcriptional induction or repression of target genes. Gene deletion, microarrays, proteomics and chromatin immunoprecipitation experiments have revealed target genes with a broad range of functions regulated by STAT3 and STAT5. In the mammary gland, STAT5-induced genes contribute mainly to the prolactin dependent lobulo-alveolar development, whereas STAT3 induced genes control apoptosis during involution. Crucial effects have also been observed in other tissues. The germ line deletion of STAT3 or STAT5 causes early embryonal or perinatal lethality in mice. STAT5 is also required for proliferation of T- and B-cells and hematopoietic stem cell self-renewal. Deregulated STAT activity is often found associated with tumorigenesis and activated STATs seem to be limiting components in tumor cells. This review summarizes the functions of STAT3 and STAT5 in different cell types and the strategies that are used to counteract their action in tumor cells.

Ohno Y, et al. Proc Natl Acad Sci USA 2010; In press.

An epigenetic modification prevents the production of a tumor-suppressing and anti-inflammatory microRNA in receptor-negative breast cancers.