Chemotherapy-induced metastasis: mechanisms and translational opportunities.

Tumors often overcome the cytotoxic effects of chemotherapy through either acquired or environment-mediated drug resistance. In addition, signals from the microenvironment obfuscate the beneficial effects of chemotherapy and may facilitate progression and metastatic dissemination. Seminal mediators in chemotherapy-induced metastasis appear to be a wide range of hematopoietic, mesenchymal and immune progenitor cells, originating from the bone marrow.

Macrophages orchestrate breast cancer early dissemination and metastasis.

Cancer cell dissemination during very early stages of breast cancer proceeds through poorly understood mechanisms. Here we show, in a mouse model of HER2 breast cancer, that a previously described sub-population of early-evolved cancer cells requires macrophages for early dissemination. Depletion of macrophages specifically during pre-malignant stages reduces early dissemination and also results in reduced metastatic burden at end stages of cancer progression.

A permanent window for the murine lung enables high-resolution imaging of cancer metastasis.

Stable, high-resolution intravital imaging of the lung has become possible through the utilization of vacuum-stabilized imaging windows. However, this technique is extremely invasive and limited to only hours in duration. Here we describe a minimally invasive, permanently implantable window for high-resolution intravital imaging of the murine lung that allows the mouse to survive surgery, recover from anesthesia, and breathe independently.

Pyk2 and FAK differentially regulate invadopodia formation and function in breast cancer cells.

The nonreceptor tyrosine kinase Pyk2 is highly expressed in invasive breast cancer, but the mechanism by which it potentiates tumor cell invasiveness is unclear at present. Using high-throughput protein array screening and bioinformatic analysis, we identified cortactin as a novel substrate and interactor of proline-rich tyrosine kinase 2 (Pyk2). Pyk2 colocalizes with cortactin to invadopodia of invasive breast cancer cells, where it mediates epidermal growth factor-induced cortactin tyrosine phosphorylation both directly and indirectly via Src-mediated Abl-related gene (Arg) activation, leading to actin polymerization in invadopodia, extracellular matrix degradation, and tumor cell invasion.

An improved MS2 system for accurate reporting of the mRNA life cycle.

The MS2-MCP system enables researchers to image multiple steps of the mRNA life cycle with high temporal and spatial resolution. However, for short-lived mRNAs, the tight binding of the MS2 coat protein (MCP) to the MS2 binding sites (MBS) protects the RNA from being efficiently degraded, and this confounds the study of mRNA regulation. Here, we describe a reporter system (MBSV6) with reduced affinity for the MCP, which allows mRNA degradation while preserving single-molecule detection determined by single-molecule FISH (smFISH) or live imaging.

Asymmetric localization of DLC1 defines avian trunk neural crest polarity for directional delamination and migration.

Following epithelial-mesenchymal transition, acquisition of avian trunk neural crest cell (NCC) polarity is prerequisite for directional delamination and migration, which in turn is essential for peripheral nervous system development. However, how this cell polarization is established and regulated remains unknown. Here we demonstrate that, using the RHOA biosensor in vivo and in vitro, the initiation of NCC polarization is accompanied by highly activated RHOA in the cytoplasm at the cell rear and its fluctuating activity at the front edge.

Rac3 regulates breast cancer invasion and metastasis by controlling adhesion and matrix degradation.

The initial step of metastasis is the local invasion of tumor cells into the surrounding tissue. Invadopodia are actin-based protrusions that mediate the matrix degradation necessary for invasion and metastasis of tumor cells. We demonstrate that Rac3 GTPase is critical for integrating the adhesion of invadopodia to the extracellular matrix (ECM) with their ability to degrade the ECM in breast tumor cells.

Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes in Neuroimaging.

Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial phytochromes but were not systematically compared in neurons. To fluoresce, NIR FPs utilize an enzymatic derivative of heme, the linear tetrapyrrole biliverdin, as a chromophore whose level in neurons is poorly studied. Here, we evaluated NIR FPs of the iRFP protein family, which were reported to be the brightest in non-neuronal mammalian cells, in primary neuronal culture, in brain slices of mouse and monkey, and in mouse brain in vivo.

Combining Structure-Function and Single-Molecule Studies on Cytoplasmic Dynein.

Cytoplasmic dynein is the largest and most intricate cytoskeletal motor protein. It is responsible for a vast array of biological functions, ranging from the transport of organelles and mRNAs to the movement of nuclei during neuronal migration and the formation and positioning of the mitotic spindle during cell division. Despite its megadalton size and its complex design, recent success with the recombinant expression of the dynein heavy chain has advanced our understanding of dynein's molecular mechanism through the combination of structure-function and single-molecule studies.

Designing brighter near-infrared fluorescent proteins: insights from structural and biochemical studies.

Brighter near-infrared (NIR) fluorescent proteins (FPs) are required for multicolor microscopy and deep-tissue imaging. Here, we present structural and biochemical analyses of three monomeric, spectrally distinct phytochrome-based NIR FPs, termed miRFPs. The miRFPs are closely related and differ by only a few amino acids, which define their molecular brightness, brightness in mammalian cells, and spectral properties.

Time-lapsed, large-volume, high-resolution intravital imaging for tissue-wide analysis of single cell dynamics.

Pathologists rely on microscopy to diagnose disease states in tissues and organs. They utilize both high-resolution, high-magnification images to interpret the staining and morphology of individual cells, as well as low-magnification overviews to give context and location to these cells. Intravital imaging is a powerful technique for studying cells and tissues in their native, live environment and can yield sub-cellular resolution images similar to those used by pathologists.

The Selective Tie2 Inhibitor Rebastinib Blocks Recruitment and Function of Tie2 Macrophages in Breast Cancer and Pancreatic Neuroendocrine Tumors.

Tumor-infiltrating myeloid cells promote tumor progression by mediating angiogenesis, tumor cell intravasation, and metastasis, which can offset the effects of chemotherapy, radiation, and antiangiogenic therapy. Here, we show that the kinase switch control inhibitor rebastinib inhibits Tie2, a tyrosine kinase receptor expressed on endothelial cells and protumoral Tie2-expressing macrophages in mouse models of metastatic cancer. Rebastinib reduces tumor growth and metastasis in an orthotopic mouse model of metastatic mammary carcinoma through reduction of Tie2 myeloid cell infiltration, antiangiogenic effects, and blockade of tumor cell intravasation mediated by perivascular Tie2/Vegf-A macrophages in the tumor microenvironment of metastasis (TMEM).

The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis.

Macrophage interactions with other cells, either locally or at distances, are imperative in both normal and pathological conditions. While soluble means of communication can transmit signals between different cells, it does not account for all long distance macrophage interactions. Recently described tunneling nanotubes (TNTs) are membranous channels that connect cells together and allow for transfer of signals, vesicles, and organelles.

A new era of studying p53-mediated transcription activation.

To prevent tumorigenesis, p53 stimulates transcription by facilitating the recruitment of the transcription machinery on target gene promoters. Cryo-Electron Microscopy studies on p53-bound RNA Polymerase II (Pol II) reveal that p53 structurally regulates Pol II to affect its DNA binding and elongation, providing new insights into p53-mediated transcriptional regulation.

Bacterial Phytochromes, Cyanobacteriochromes and Allophycocyanins as a Source of Near-Infrared Fluorescent Probes.

Bacterial photoreceptors absorb light energy and transform it into intracellular signals that regulate metabolism. Bacterial phytochrome photoreceptors (BphPs), some cyanobacteriochromes (CBCRs) and allophycocyanins (APCs) possess the near-infrared (NIR) absorbance spectra that make them promising molecular templates to design NIR fluorescent proteins (FPs) and biosensors for studies in mammalian cells and whole animals. Here, we review structures, photochemical properties and molecular functions of several families of bacterial photoreceptors.

Glioblastoma cellular cross-talk converges on NF-κB to attenuate EGFR inhibitor sensitivity.

In glioblastoma (GBM), heterogeneous expression of amplified and mutated epidermal growth factor receptor (EGFR) presents a substantial challenge for the effective use of EGFR-directed therapeutics. Here we demonstrate that heterogeneous expression of the wild-type receptor and its constitutively active mutant form, EGFRvIII, limits sensitivity to these therapies through an interclonal communication mechanism mediated by interleukin-6 (IL-6) cytokine secreted from EGFRvIII-positive tumor cells. IL-6 activates a NF-κB signaling axis in a paracrine and autocrine manner, leading to bromodomain protein 4 (BRD4)-dependent expression of the prosurvival protein survivin (BIRC5) and attenuation of sensitivity to EGFR tyrosine kinase inhibitors (TKIs).

Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism.

Breast cancer cells disseminate through TIE2/MENA/MENA-dependent cancer cell intravasation sites, called tumor microenvironment of metastasis (TMEM), which are clinically validated as prognostic markers of metastasis in breast cancer patients. Using fixed tissue and intravital imaging of a PyMT murine model and patient-derived xenografts, we show that chemotherapy increases the density and activity of TMEM sites and Mena expression and promotes distant metastasis. Moreover, in the residual breast cancers of patients treated with neoadjuvant paclitaxel after doxorubicin plus cyclophosphamide, TMEM score and its mechanistically connected MENA isoform expression pattern were both increased, suggesting that chemotherapy, despite decreasing tumor size, increases the risk of metastatic dissemination.

How to Increase Brightness of Near-Infrared Fluorescent Proteins in Mammalian Cells.

Numerous near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial photoreceptors but lack of their systematic comparison makes researcher's choice rather difficult. Here we evaluated side-by-side several modern NIR FPs, such as blue-shifted smURFP and miRFP670, and red-shifted mIFP and miRFP703. We found that among all NIR FPs, miRFP670 had the highest fluorescence intensity in various mammalian cells.

Quantitative mRNA imaging throughout the entire Drosophila brain.

We describe a fluorescence in situ hybridization method that permits detection of the localization and abundance of single mRNAs (smFISH) in cleared whole-mount adult Drosophila brains. The approach is rapid and multiplexable and does not require molecular amplification; it allows facile quantification of mRNA expression with subcellular resolution on a standard confocal microscope. We further demonstrate single-mRNA detection across the entire brain using a custom Bessel beam structured illumination microscope (BB-SIM).

p53 Dynamically Directs TFIID Assembly on Target Gene Promoters.

p53 is a central regulator that turns on vast gene networks to maintain cellular integrity in the presence of various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive.

Tumor Cell Invadopodia: Invasive Protrusions that Orchestrate Metastasis.

Invadopodia are a subset of invadosomes that are implicated in the integration of signals from the tumor microenvironment to support tumor cell invasion and dissemination. Recent progress has begun to define how tumor cells regulate the plasticity necessary for invadopodia to assemble and function efficiently in the different microenvironments encountered during dissemination in vivo. Exquisite mapping by many laboratories of the pathways involved in integrating diverse invadopodium initiation signals, from growth factors, to extracellular matrix (ECM) and cell-cell contact in the tumor microenvironment, has led to insight into the molecular basis of this plasticity.

Near-Infrared Fluorescent Proteins, Biosensors, and Optogenetic Tools Engineered from Phytochromes.

Phytochrome photoreceptors absorb far-red and near-infrared (NIR) light and regulate light responses in plants, fungi, and bacteria. Their multidomain structure and autocatalytic incorporation of linear tetrapyrrole chromophores make phytochromes attractive molecular templates for the development of light-sensing probes. A subclass of bacterial phytochromes (BphPs) utilizes heme-derived biliverdin tetrapyrrole, which is ubiquitous in mammalian tissues, as a chromophore.

Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells.

Breast carcinoma cells use specialized, actin-rich protrusions called invadopodia to degrade and invade through the extracellular matrix. Phosphorylation of the actin nucleation-promoting factor and actin-stabilizing protein cortactin downstream of the epidermal growth factor receptor-Src-Arg kinase cascade is known to be a critical trigger for invadopodium maturation and subsequent cell invasion in breast cancer cells. The functions of cortactin phosphorylation in this process, however, are not completely understood.

Near-infrared optogenetic pair for protein regulation and spectral multiplexing.

Multifunctional optogenetic systems are in high demand for use in basic and biomedical research. Near-infrared-light-inducible binding of bacterial phytochrome BphP1 to its natural PpsR2 partner is beneficial for simultaneous use with blue-light-activatable tools. However, applications of the BphP1-PpsR2 pair are limited by the large size, multidomain structure and oligomeric behavior of PpsR2.