Fluorescent Epidermal Growth Factor Research Articles

Abstract 4301: Detection and characterization of regions of hypoxia within orthotopic pancreatic tumors using multispectral optoacoustic tomography

Abstract Optical imaging is a vital tool in cancer research for visualization of tumors in preclinical models. However, resolution for bioluminescence and fluorescence modalities is restricted to a depth of five millimeters, as light is attenuated at increased depth and autofluorescence can create background noise. Light propagation becomes diffuse at only a few millimeters, resulting in light scattering and absorption. To improve imaging resolution and acquisition in real-time, multispectral optoacoustic tomography (MSOT) was utilized to visualize molecular phenomena such as the tissue oxygenation of characteristically hypoxic aggressive pancreatic tumors. SCID mice were orthotopically implanted with S2VP10L and MIA PaCa-2 pancreatic cancer cells that highly express the epidermal growth factor receptor (EGFR), which exhibits extracellular domain binding of the epidermal growth factor (EGF) ligand. Cohorts were then intravenously injected with fluorescent EGF ligand probes and imaged semiweekly to follow tumor growth and morphology. Medical Grade Air levels were set to 0.9 L and oxygen at 0.1 L to assess the correlation of increased hypoxia with tumor growth and progression. Pancreatic tumor cells were imaged concurrently with evaluating levels of oxygenated and deoxygenated hemoglobin, providing a distinct picture of the tumor and its vascularization. Use of MSOT to detect regions of hypoxia within tumor was validated using pimonidazole immunohistochemical staining of tumor tissue. For the first time, our data demonstrate regions of tumor hypoxia in living subjects, at depth, and noninvasively, via optoacoustic tomography. Dynamic assessment of tumor microenvironment, especially regions of tumor hypoxia, is essential to effectively target and treat tumors that are otherwise resistant to treatment. Citation Format: Shanice V. Hudson, Charles Kimbrough, Michael Egger, Anil Khanal, Michelle E. Smith, William E. Grizzle, Lacey R. McNally. Detection and characterization of regions of hypoxia within orthotopic pancreatic tumors using multispectral optoacoustic tomography. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4301. doi:10.1158/1538-7445.AM2014-4301

Prospective isolation of adult neural stem cells from the mouse subependymal zone

Neural stem cells (NSCs) have the remarkable capacity to self-renew and the lifelong ability to generate neurons in the adult mammalian brain. However, the molecular and cellular mechanisms contributing to these behaviors are still not understood. Now that prospective isolation of the NSCs has become feasible, these mechanisms can be studied. Here we describe a protocol for the efficient isolation of adult NSCs, by the application of a dual-labeling strategy on the basis of their glial identity and ciliated nature. The cells are isolated from the lateral ventricular subependymal zone (SEZ) of adult hGFAP-eGFP (human glial fibrillary acidic protein-enhanced green fluorescent protein) transgenic mice by fluorescence-activated cell sorting. Staining against prominin1 (CD133) allows the isolation of the NSCs (hGFAP-eGFP(+)/prominin1(+)), which can be further subdivided by labeling with the fluorescent epidermal growth factor. This protocol, which can be completed in 7 h, allows the assessment of quantitative changes in SEZ NSCs and the examination of their molecular and functional characteristics.

MRI-coupled Fluorescence Tomography Quantifies EGFR Activity in Brain Tumors

This report demonstrates the diagnostic potential of magnetic resonance imaging (MRI)-coupled fluorescence molecular tomography (FMT) to determine epidermal growth factor receptor (EGFR) status in brain cancer. Two orthotopic glioma xenograft models were used in this study: one represented high EGFR expression and the other low expression. Nude mice were inoculated with cells from either one of the tumor lines or were used in a sham surgery control group. Animals were imaged using a unique MRI-FMT scanner 48 hours after intravenous injection of a near-infrared fluorophore bound to epidermal growth factor (EGF) ligand. Coronal images of fluorescence activity of the injected dye in the mouse brain were recovered using the MRI images as anatomical templates. In vivo images of fluorescence activity showed significant differences between animal populations, an observation confirmed by receiver operating characteristic analysis that revealed 100% sensitivity and specificity between animal groups implanted with EGFR((+)) and EGFR((-)) tumor lines. Similar performance was observed between EGFR((+)) and sham surgery control animals. This preclinical study suggests that MRI-FMT with fluorescent EGF provides excellent discrimination between tumors based on EGFR status. Reliable quantification of receptor status using minimally invasive techniques would be an important innovation for investigating new and existing cancer treatments that target these cellular mechanisms in research animals, and may be applied to identify receptor amplification in human brain cancer patients. This study represents the first systematic multianimal validation of receptor-specific imaging using MRI-guided fluorescence tomography.

Development of a quantitative, cell-based, high-content screening assay for epidermal growth factor receptor modulators

To develop a robust, cell-based, high-content screening (HCS) assay based on receptor internalization for the identification of novel modulators of the epidermal growth factor receptor (EGFR). Agonist-induced receptor internalization is part of the signaling cascade of EGFR. Fluorescent-tagged epidermal growth factor (EGF) was used to visualize the internalized receptor-ligand complex. The fluorescent intracellular spots were detected and measured with an ArrayScan HCS reader. Compounds that can competitively bind to EGFR or interfere with EGFR internalization process would result in a reduced number and intensity of intracellular fluorescent spots. This assay was validated, optimized, and applied to a large-scale screening of a library containing 48,000 synthetic compounds. The competition between fluorescent EGF and unlabeled EGF reveals the IC(50) of unlabeled EGF is approximately 0.2 nmol/L, which is comparable with other published reports. Thirteen compounds with a relatively high degree of interference with EGFR internalization were identified. One of the compounds was proven to be agonist of the EGFR since it induced phosphorylation of the receptor and extracellular signal-regulated protein kinase (ERK). This automated, objective, and easy-to-use assay provided abundant information, quantitative results, and demonstrated the potential use of HCS methods in searching membrane receptor modulators.

A Quantitative Cell-Based High-Content Screening Assay for the Epidermal Growth Factor Receptor-Specific Activation of Mitogen-Activated Protein Kinase

The complexity of mitogen-activated protein kinase (MAPK) signaling pathways and their activation by different stimuli makes assaying the activation of particular MAPKs by specific receptors a challenging problem. The multiplexing capability of quantitative high-content screening (HCS) assays enables the simultaneous monitoring and correlation, in the same cell, of an MAPK's specific activation with a particular receptor's post-signaling behavior, such as its internalization. We demonstrate a cell-based HCS assay to quantify the epidermal growth factor (EGF) receptor-specific activation of the MAPK ERK. Activation was quantified by measuring immunofluorescently labeled phosphorylated extracellular signal-regulated protein kinases (ERK) in the nucleus. Specificity of ERK activation by the EGF receptor was simultaneously confirmed in the same cell by quantitatively monitoring fluorescent EGF's internalization and subsequent intracellular degradation. Quantitative analysis of the temporal behavior of these two activities showed that phosphorylated ERK's accumulation in the nucleus peaked at 5 min before falling to basal levels by 30 min. Cellular accumulation of fluorescent EGF was slower, peaking around 30 min, before being degraded. This assay strategy can serve as a paradigm to study other signaling pathways and their activation by specific receptors. The flexibility and multiplexing capability of HCS assays allow the use of additional targets to further qualify the specificity of response by including other MAPKs or receptors, to rule out cross-talk from competing signaling pathways, or to simultaneously monitor toxicity effects of compounds. This automated, non-subjective, easy-to-use assay procedure provides information rich, quantitative results, and demonstrates the potential of the HCS assay approach in deconvolving intracellular signaling pathways.

A Quantitative Cell-Based High-Content Screening Assay for the Epidermal Growth Factor Receptor-Specific Activation of Mitogen-Activated Protein Kinase

The complexity of mitogen-activated protein kinase (MAPK) signaling pathways and their activation by different stimuli makes assaying the activation of particular MAPKs by specific receptors a challenging problem. The multiplexing capability of quantitative high-content screening (HCS) assays enables the simultaneous monitoring and correlation, in the same cell, of an MAPK's specific activation with a particular receptor's post-signaling behavior, such as its internalization. We demonstrate a cell-based HCS assay to quantify the epidermal growth factor (EGF) receptor-specific activation of the MAPK ERK. Activation was quantified by measuring immunofluorescently labeled phosphorylated extracellular signal-regulated protein kinases (ERK) in the nucleus. Specificity of ERK activation by the EGF receptor was simultaneously confirmed in the same cell by quantitatively monitoring fluorescent EGF's internalization and subsequent intracellular degradation. Quantitative analysis of the temporal behavior of these two activities showed that phosphorylated ERK's accumulation in the nucleus peaked at 5 min before falling to basal levels by 30 min. Cellular accumulation of fluorescent EGF was slower, peaking around 30 min, before being degraded. This assay strategy can serve as a paradigm to study other signaling pathways and their activation by specific receptors. The flexibility and multiplexing capability of HCS assays allow the use of additional targets to further qualify the specificity of response by including other MAPKs or receptors, to rule out cross-talk from competing signaling pathways, or to simultaneously monitor toxicity effects of compounds. This automated, non-subjective, easy-to-use assay procedure provides information rich, quantitative results, and demonstrates the potential of the HCS assay approach in deconvolving intracellular signaling pathways.

Opioid control of MAP kinase cascade

Activation of G protein-coupled receptors (GPCRs) may result in phosphorylation of extracellular signal-regulated kinases 1/2 (ERK 1/2). The signaling pathway involves ectodomain shedding, generating epidermal growth factor (EGF)-like ligands, which in turn stimulate the mitogen-activated protein kinase (MAPK) via EGF receptors. The present study investigates into the control of MAPKs by opioidergic GPCRs in human embryonic kidney cells (HEK 293). Experiments were conducted with cells expressing opioid receptors, G protein-coupled receptor kinases, and ERKs. The outcome of our studies let us suggest that EGF-like ligands released by opioid receptor stimulation utilize different EGF receptors to phosphorylate ERKs, while EGF utilizes type 1 receptors. Differences between multiple opioid receptors are apparent with respect to the activation of ERKs. EGF rapidly triggers internalization of the fluorescent EGF receptor type 1, but we failed to observe any sequestration of this receptor type upon exposure of cells to an opioid, since opioids most likely trigger stimulation of a different EGF receptor type. In conclusion, G protein-coupled opioid receptors control the MAPK cascade in a similar fashion as described for non-opioid GPCRs, although distinct differences exist between μ-, δ- and κ-receptors. EGF-induced ERK activation is mediated by EGF receptor type 1 while opioid receptor activation seems to brings about stimulation via EGF receptor type.

Cytoplasmic transport of Stat3 by receptor-mediated endocytosis.

Signal transducer and activator of transcription (STAT) proteins are cytoplasmic transcription factors that translocate to the nucleus and regulate gene expression upon activation of cytokine or growth factor receptors. While this translocation event is essential for gene regulation by STATs, their mechanism of transport through the cytoplasm to the nucleus has remained elusive. We now report that cytoplasmic transport of Stat3 is an active process that requires receptor-mediated endocytosis. Stat3 co-localizes with endocytic vesicles in transit from the cell membrane to the perinuclear region in response to growth factor stimulation. Consistent with a role for receptor endocytosis in growth factor signaling, disruption of endocytosis with specific inhibitors blocks Stat3 nuclear translocation and Stat3-dependent gene regulation. These results indicate that receptor-mediated endocytosis may be a general mechanism of transport through the cytoplasm for a subset of cytoplasmic signaling proteins destined for the nucleus.

Epidermal growth factor receptor distribution during chemotactic responses.

To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast to other studies indicating that the EGFR might be localized to certain regions of the plasma membrane, we found that the chimera is homogeneously distributed on the plasma membrane and becomes most concentrated in vesicles after endocytosis. In spatial gradients of EGF, endocytosed receptor accumulates on the upgradient side of the cell. Visualization of the binding of fluorescent EGF to cells reveals that the affinity properties of the receptor, together with its expression level on cells, can provide an initial amplification step in spatial gradient sensing.

Epidermal growth factor induces electrically silent Na+ influx in human fibroblasts.

Addition of epidermal growth factor (EGF) to quiescent confluent cultures of human foreskin fibroblasts causes a rapid, nearly 2-fold stimulation of unidirectional Na+ influx and a doubling of the rate of the Na+,K+ pump, whereas K+ efflux remains unaltered. The diuretic amiloride, an inhibitor of Na+/H+ exchange, completely blocks EGF-induced Na+ influx, Na+,K+-pump activity, and DNA synthesis without affecting the cellular binding, visible clustering, and internalization of 125I-labeled and fluorescent EGF. In the absence of EGF, the induction of amiloride-sensitive Na+ influx and Na+,K+-pump activity can be mimicked by exposing the cells to weak acids. Neither the rapid stimulation of Na+ influx by EGF nor its inhibition by amiloride is accompanied by a detectable change in membrane potential (mean value of -66 mV), as evidenced by direct intracellular recording. In contrast, a rapid but transient membrane depolarization of about 50 mV, due to an unselective permeability increase, is observed in response to serum-growth factors. These results (i) indicate that EGF rapidly activates an electroneutral, previously inactive Na+ transport system in the plasma membrane of quiescent fibroblasts, and (ii) suggest that EGF-induced Na+ influx occurs in exchange for intracellular protons. The data further imply that early changes in membrane potential are not necessary for the initiation of a mitogenic response.