A secreted luciferase for ex vivo monitoring of in vivo processes

Luciferases are widely used to monitor biological processes. Here we describe the naturally secreted Gaussia princeps luciferase (Gluc) as a highly sensitive reporter for quantitative assessment of cells in vivo by measuring its concentration in blood. The Gluc blood assay complements in vivo bioluminescence imaging, which has the ability to localize the signal and provides a multifaceted assessment of cell viability, proliferation and location in experimental disease and therapy models.     

Imaging radiotherapy-induced apoptosis

Prediction of response to therapy has been identified as an important tool to obtain a more customized treatment. It allows the selection of those patients who will benefit most from a particular therapy and prevents the exposure of patients to toxic, noneffective regimens. Recent technical advances and the introduction of novel markers in anatomical and functional imaging have created exciting opportunities for in vivo visualization and quantification of cell death. This review will focus on in vivo apoptosis imaging as a predictive marker for tumor response after radiation.     

Focal adhesion disassembly is an essential early event in hepatic stellate cell chemotaxis

Chemotaxis (i.e., directed migration) of hepatic stellate cells to areas of inflammation is a requisite event in the liver's response to injury. Previous studies of signaling pathways that regulate stellate cell migration suggest a key role for focal adhesions, but the exact function of these protein complexes in motility remains unclear. Focal adhesions attach a cell to its substrate and therefore must be regulated in a highly coordinated manner during migration. To test the hypothesis that focal adhesion turnover is an essential early event for chemotaxis in stellate cells, we employed a live-cell imaging technique in which chemotaxis was induced by locally stimulating the tips of rat stellate cell protrusions with platelet-derived growth factor-BB (PDGF). Focal adhesions were visualized with an antibody directed against vinculin, a structural component of the focal adhesion complex. PDGF triggered rapid disassembly of adhesions within 6.25 min, subsequent reassembly by 12.5 min, and continued adhesion assembly in concert with the spreading protrusion until the completion of chemotaxis. Blockade of adhesion disassembly by growing cells on fibronectin or treatment with nocodazole prevented a chemotactic response to PDGF. Augmentation of adhesion disassembly with ML-7 enhanced the chemotactic response to PDGF. These data suggest that focal adhesion disassembly is an essential early event in stellate cell chemotaxis in response to PDGF.     

Constitutive diffuse activation of phosphoinositide 3-kinase at the plasma membrane by v-Src suppresses the chemotactic response to PDGF by abrogating the polarity of PDGF receptor signalling

Cancer cells depend on chemotaxis for invasion and frequently overexpress and/or activate Src. We previously reported that v-Src accelerates motility by promoting phosphoinositide 3-kinase (PI3-K) signalling but abrogates chemotaxis. We here addressed the mechanism of the loss of chemotactic response to platelet-derived growth factor (PDGF) gradients in fibroblasts harbouring a thermosensitive v-Src kinase. At non-permissive temperature, PDGF receptor (PDGFR) signalling, assessed by phosphoY(751)-specific antibodies (a docking site for PI3-K), was not detected without PDGF and showed a concentration-dependent PDGF response. Both immunolabeling of PI3-K (p110) and live cell imaging of its product (phosphatidylinositol 3,4,5 tris-phosphate) showed PI3-K recruitment and activation at lamellipodia polarized towards a PDGF gradient. Centrosomes and PDGFR- and Src-bearing endosomes were also oriented towards this gradient. Upon v-Src thermoactivation, (i) Y(751) phosphorylation was moderately induced without PDGF and synergistically increased with PDGF; (ii) PI3-K was recruited and activated all along the plasma membrane without PDGF and did not polarize in response to a PDGF gradient; and (iii) polarization of centrosomes and of PDGFR-bearing endosomes were also abrogated. Thus, PDGF can further increase PDGFR auto-phosphorylation despite strong Src kinase activity, but diffuse downstream activation of PI3-K by Src abrogates cell polarization and chemotaxis: "signalling requires silence".     

Bioluminescence technology for imaging cell proliferation

As continuous cell proliferation caused by genetic alterations leads to cancer, monitoring abnormal cell proliferation in sporadic tumor models is important in the context of tumor generation, development and response to therapy. Bioluminescence imaging technology, which visualizes the conversion of chemical energy into visible light by luciferase enzymes, is an established method to measure cell numbers in grafted tumors in vivo, but has not been used to monitor cell proliferation per se. To measure cell proliferation noninvasively, transgenic mice have been developed that express the luciferase gene under the control of the E2F1 promoter. When these reporter mice are crossed with genetically defined mouse models of human cancer, the proliferative activity of the tumor cells can be monitored with proportional light production. These technologies support more detailed preclinical trials and could enable other biological pathways to be monitored in living cells.     

Platelet-derived growth factor is constitutively secreted from neuronal cell bodies but not from axons

Neurons synthesise and secrete many growth and survival factors but it is not usually clear whether they are released locally at the cell body or further afield from axons or axon terminals. Without this information, we cannot predict the site(s) of action or the biological functions of many neuron-derived factors. For example, can neuronal platelet-derived growth factor (PDGF) be secreted from axons and reach glial cells in nerve-fibre (white-matter) tracts? To address this question, we expressed PDGF-A in retinal ganglion neurons in transgenic mice and tested for release of PDGF from cell bodies in the retina and from axons in the optic nerve. In both the retina and optic nerve, there are glial cells that express PDGF receptor alpha (PDGFR alpha) [1] and divide in response to PDGF [2-5], so we could detect functional PDGF indirectly through the mitogenic response of glia at both locations. Expressing PDGF-A in neurons under the control of the neuron-specific enolase promoter (NSE-PDGF-A) resulted in a striking hyperplasia of retinal astrocytes, demonstrating that PDGF is secreted from the cell bodies of neurons in the retina [4]. In contrast, glial proliferation in the optic nerve was unaffected, indicating that PDGF is not released from axons. When PDGF was expressed directly in the optic nerve under the control of an astrocyte-specific promoter (GFAP-PDGF-A), oligodendrocyte progenitors hyperproliferated, resulting in a hypertrophic optic nerve. We conclude that PDGF is constitutively secreted from neuronal cell bodies in vivo, but not from axons in white-matter tracts.     

Calcitonin gene-related peptide promotes Schwann cell proliferation

Schwann cells in culture divide in response to defined mitogens such as PDGF and glial growth factor (GGF), but proliferation is greatly enhanced if agents such as forskolin, which increases Schwann cell intracellular cAMP, are added at the same time as PDGF or GGF (Davis, J. B., and P. Stroobant. 1990. J. Cell Biol. 110:1353-1360). The effect of forskolin is probably due to an increase in numbers of PDGF receptors (Weinmaster, G., and G. Lemke. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:915-920. Neuropeptides and beta-adrenergic agonists have been reported to have no effect on potentiating the mitogenic response of either PDGF or GGF. We show that the neuropeptide calcitonin gene- related peptide (CGRP) increases Schwann cell cAMP levels, but the cells rapidly desensitize. We therefore stimulated the cells in pulsatile fashion to partly overcome the effects of desensitization and show that CGRP can synergize with PDGF to stimulate Schwann cell proliferation, and that CGRP is as effective as forskolin in the pulsatile regime. CGRP is a good substrate for the neutral endopeptidase 24.11. Schwann cells in vivo have this protease on their surface, so the action of CGRP could be terminated by this enzyme and desensitization prevented. We therefore suggest that CGRP may play an important role in stimulating Schwann cell proliferation by regulating the response of mitogenic factors such as PDGF.     

Magnetic resonance tracking of dendritic cells in melanoma patients for monitoring of cellular therapy

The success of cellular therapies will depend in part on accurate delivery of cells to target organs. In dendritic cell therapy, in particular, delivery and subsequent migration of cells to regional lymph nodes is essential for effective stimulation of the immune system. We show here that in vivo magnetic resonance tracking of magnetically labeled cells is feasible in humans for detecting very low numbers of dendritic cells in conjunction with detailed anatomical information. Autologous dendritic cells were labeled with a clinical superparamagnetic iron oxide formulation or (111)In-oxine and were co-injected intranodally in melanoma patients under ultrasound guidance. In contrast to scintigraphic imaging, magnetic resonance imaging (MRI) allowed assessment of the accuracy of dendritic cell delivery and of inter- and intra-nodal cell migration patterns. MRI cell tracking using iron oxides appears clinically safe and well suited to monitor cellular therapy in humans.     

TNF-alpha suppresses the PDGF beta-receptor kinase

PDGF and TNF-alpha are both known to play important roles in inflammation, albeit frequently by opposing actions. Typically, TNF-alpha can attenuate PDGF beta-receptor signaling. Pretreatment of mouse 3T3 L1 fibroblasts with TNF-alpha greatly diminished their proliferative response to PDGF. However, TNF-alpha affected neither the binding of PDGF-BB to cell surface receptors nor the total amount of PDGF beta-receptor in the cells, but decreased the PDGF-induced in vitro kinase activity of the receptor. The phosphatase inhibitor ortho-vanadate did not prevent this effect. Ortho-phosphate labeling of cells prior to TNF-alpha treatment and PDGF-BB stimulation confirmed a decrease of in vivo phosphorylation of the PDGF beta-receptor. Two-dimensional mapping after tryptic cleavage as well as phosphoamino acid analysis demonstrated a general decrease in phosphorylation of all known tyrosine residues in the PDGF beta-receptor. The exact mechanism for this suppression remains to be clarified.     

Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex.

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.     

In chemotaxing fibroblasts, both high-fidelity and weakly biased cell movements track the localization of PI3K signaling

Cell movement biased by a chemical gradient, or chemotaxis, coordinates the recruitment of cells and collective migration of cell populations. During wound healing, chemotaxis of fibroblasts is stimulated by platelet-derived growth factor (PDGF) and certain other chemoattractants. Whereas the immediate PDGF gradient sensing response has been characterized previously at the level of phosphoinositide 3-kinase (PI3K) signaling, the sensitivity of the response at the level of cell migration bias has not yet been studied quantitatively. In this work, we used live-cell total internal reflection fluorescence microscopy to monitor PI3K signaling dynamics and cell movements for extended periods. We show that persistent and properly aligned (i.e., high-fidelity) fibroblast migration does indeed correlate with polarized PI3K signaling; accordingly, this behavior is seen only under conditions of high gradient steepness (>10% across a typical cell length of 50 μm) and a certain range of PDGF concentrations. Under suboptimal conditions, cells execute a random or biased random walk, but nonetheless move in a predictable fashion according to the changing pattern of PI3K signaling. Inhibition of PI3K during chemotaxis is accompanied by loss of both cell-substratum contact and morphological polarity, but after a recovery period, PI3K-inhibited fibroblasts often regain the ability to orient toward the PDGF gradient.     

Proliferation versus migration in platelet-derived growth factor signaling: the key role of endocytosis

It is common knowledge that platelet-derived growth factor (PDGF) is a critical regulator of mesenchymal cell migration and proliferation. Nevertheless, these two cellular responses are mutually exclusive. To solve this apparent contradiction, we studied the behavior of NIH3T3 fibroblasts in response to increasing concentrations of PDGF. We found that there is strong cell proliferation induction only with PDGF concentrations >5 ng/ml, whereas the cell migration response arises starting from 1 ng/ml and is negligible at higher PDGF concentrations. According to these phenotypic evidences, our data indicate that cells display a differential activation of the main signaling pathways in response to PDGF as a function of the stimulation dose. At low PDGF concentrations, there is maximal activation of signaling pathways linked to cytoskeleton rearrangement needed for cell motility, whereas high PDGF concentrations activate pathways linked to mitogenesis induction. Our results suggest a mechanism by which cells switch from a migrating to a proliferating phenotype sensing the increasing gradient of PDGF. In addition, we propose that the cell decision to proliferate or migrate relies on different endocytotic routes of the PDGF receptor in response to different PDGF concentrations.     

Monitoring of in vivo function of superparamagnetic iron oxide labelled murine dendritic cells during anti-tumour vaccination

Dendritic cells (DCs) generated in vitro to present tumour antigens have been injected in cancer patients to boost in vivo anti-tumour immune responses. This approach to cancer immunotherapy has had limited success. For anti-tumour therapy, delivery and subsequent migration of DCs to lymph nodes leading to effective stimulation of effector T cells is thought to be essential. The ability to non-invasively monitor the fate of adoptively transferred DCs in vivo using magnetic resonance imaging (MRI) is an important clinical tool to correlate their in vivo behavior with response to treatment. Previous reports of superparamagnetic iron oxides (SPIOs) labelling of different cell types, including DCs, have indicated varying detrimental effects on cell viability, migration, differentiation and immune function. Here we describe an optimised labelling procedure using a short incubation time and low concentration of clinically used SPIO Endorem to successfully track murine DC migration in vivo using MRI in a mouse tumour model. First, intracellular labelling of bone marrow derived DCs was monitored in vitro using electron microscopy and MRI relaxometry. Second, the in vitro characterisation of SPIO labelled DCs demonstrated that viability, phenotype and functions were comparable to unlabelled DCs. Third, ex vivo SPIO labelled DCs, when injected subcutaneously, allowed for the longitudinal monitoring by MR imaging of their migration in vivo. Fourth, the SPIO DCs induced the proliferation of adoptively transferred CD4(+) T cells but, most importantly, they primed cytotoxic CD8(+) T cell responses to protect against a B16-Ova tumour challenge. Finally, using anatomical information from the MR images, the immigration of DCs was confirmed by the increase in lymph node size post-DC injection. These results demonstrate that the SPIO labelling protocol developed in this study is not detrimental for DC function in vitro and in vivo has potential clinical application in monitoring therapeutic DCs in patients with cancer.     

活体动物体内光学成像技术的研究进展及其应用

活体动物体内光学成像是利用基因改构进行内源性成像试剂或外源性成像试剂标记细胞、蛋白或DNA，从而非侵入性地报告小动物体内的特定生物学事件的技术。活体成像可以直观灵敏地监测基因的表达模式、标记和示踪细胞、探讨蛋白间的相互作用，因而这一技术被广泛地用于分析基因的表达模式、评价基因治疗效果、评估肿瘤的发生和转移、监测移植器官等。简要综述了现有活体动物体内光学成像技术的基本原理、技术进展和相关应用。     

Tumor blood vessel visualization

Significant advances have been made in understanding the role of tumor angiogenesis and its influence on tumor progression in cancer. Based on this knowledge, a series of inhibitors of angiogenesis have been developed and evaluated in preclinical and clinical trials. Since detailed information of tumor progression in response to therapy is important to assess the efficacy of anti-tumor treatment in vivo, noninvasive imaging techniques emerge more and more as important tools to monitor alterations in tumor growth and vessel recruitment, as well as metastatic spread over time. So far, remarkable efforts have been made to improve the technical capability of these imaging modalities based on better resolution, as well as to implement multimodal approaches combining molecular with anatomical information. Advanced imaging techniques not only allow the detection and monitoring of tumor development, but also facilitate a broad understanding of the cellular and molecular events that propagate tumor angiogenesis, as well as those occurring in response to therapy. This review provides an overview of different imaging techniques in preclinical settings of oncological research and discusses their potential impact on clinical translation. Imaging modalities will be presented that have been implemented to address key biological issues by exploring tumor angiogenic processes and evaluating antiangiogenic therapy.     

小动物体内可见光三维成像技术研究进展

活体动物体内可见光成像是采用生物发光和荧光为标记物,利用灵敏的仪器来监控活体动物体内的细胞活动、蛋白表达情况和基因行为。近年来,可见光成像在生物医学的各个方面得到了广泛的应用。随着成像技术和检测仪器的不断发展,现已从平面二维成像逐渐发展为立体三维成像。三维成像技术在靶点的空间定位、与器官的关系,及绝对定量方面都有了很大的进展。本文就三维成像技术的原理、应用和发展前景进行了简要的综述。     

Platelet-derived growth factor increases the in vivo activity of phospholipase C-gamma 1 and phospholipase C-gamma 2.

Upon binding to its cell surface receptor, platelet-derived growth factor (PDGF) causes the tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1) and stimulates the production of diacylglycerol and inositol 1,4,5-triphosphate. We showed that following stimulation by PDGF, rat-2 cells overexpressing PLC-gamma 1 display an increase in the levels of both tyrosine-phosphorylated PLC-gamma 1 and inositol phosphates compared with the parental rat-2 cells. This increased responsiveness to PDGF is a direct effect of PLC-gamma 1 overexpression, as a cell line expressing similar levels of an enzymatically inactive point mutant of PLC-gamma 1, PLC-gamma 1 335Q, did not show elevated inositol phosphate production in response to PDGF. Hematopoietic cells express PLC-gamma 2, a PLC isoform that is closely related to PLC-gamma 1. When rat-2 cells overexpressing PLC-gamma 2 were treated with PDGF, an increase in both the tyrosine phosphorylation and the in vivo activity of PLC-gamma 2 was observed. Aluminum fluoride (AIF4-), a universal activator of PLC linked to G-proteins, did not produce an increase in the levels of inositol phosphates in either of the overexpressing cell lines compared with parental rat-2 cells, demonstrating that PLC-gamma isoforms respond specifically to a receptor with tyrosine kinase activity.     

Control of progenitor cell number by mitogen supply and demand

BACKGROUND: Much is known about how cell proliferation is controlled at the single cell level, but much less about the control of cell numbers in developing populations. Cell number might be determined by an intracellular division limiter or, alternatively, by the availability of mitogens or other factors outside the cell. We investigated the relative importance of intracellular and extracellular controls for one well-defined population of neural precursor cells, namely the glial progenitors that give rise to oligodendrocytes in the mouse spinal cord. RESULTS: We found by cumulative BrdU labeling in vivo that the progenitor cell division cycle slows down markedly as their numbers increase during embryogenesis. When cultured in saturating PDGF, the main mitogen for these cells, their cell cycle accelerated and was independent of their prior rate of division in vivo. This shows that mitogens are limiting in vivo, and suggests that division normally slows down because the PDGF concentration declines. In PDGF-transgenic mice, cell number was proportional to the PDGF supply and apparently unsaturable; at ten times the normal rate of supply, cell number was still increasing but the animals were no longer viable. CONCLUSIONS: Progenitor cell proliferation in the embryo is limited by environmental factors, not a cell-intrinsic mechanism. The linear relationship between PDGF supply and final cell number strongly suggests that cells deplete the mitogenic activity in their environment at a rate proportional to the total number of cells. The cells might simply consume the available PDGF or they might secrete autocrine inhibitors, or both.     

Induction of platelet-derived growth factor receptor expression in smooth muscle cells and fibroblasts upon tissue culturing

The expression of platelet-derived growth factor (PDGF) receptors in porcine uterus and human skin in situ, was compared with that of cultured primary cells isolated from the same tissues. PDGF receptor expression was examined by monoclonal antibodies specific for the B type PDGF receptor and by RNA/RNA in situ hybridization with a probe constructed from a cDNA clone encoding the B type PDGF receptor. In porcine uterus tissue both mRNA and the protein product for the PDGF receptor were detected in the endometrium; the myometrium, in contrast, contained much lower amounts. Moreover, freshly isolated myometrial cells were devoid of PDGF receptors. However, after 1 d in culture receptors appeared, and after 2 wk of culturing essentially all of the myometrial cells stained positively with the anti-PDGF receptor antibodies and contained PDGF receptor mRNA. Similarly, B type PDGF receptors were not detected in normal human skin, but fibroblast-like cells from explant cultures of human skin possessed PDGF receptors. When determined by immunoblotting, porcine uterus myometrial membranes contained approximately 20% of the PDGF receptor antigen compared with the amount found in endometrial membranes. In addition, PDGF stimulated the phosphorylation of a 175-kD component, most likely representing autophosphorylation of the B type PDGF receptor in endometrial membranes, whereas only a marginal phosphorylation was seen in myometrial membranes. Taken together, these results demonstrate that PDGF receptor expression varies in normal tissues and that fibroblasts and smooth muscle cells do not uniformly express the receptor in situ. Furthermore, fibroblasts and smooth muscle cells that are released from tissues are induced to express PDGF receptors in response to cell culturing. The data suggest that, in addition to the availability of the ligand, PDGF-mediated cell growth in vivo is dependent on factors regulating expression of the receptor.