Combined ion beam irradiation platform and 3D fluorescence microscope for cellular cancer research.

To improve particle radiotherapy, we need a better understanding of the biology of radiation effects, particularly in heavy ion radiation therapy, where global responses are observed despite energy deposition in only a subset of cells. Here, we integrated a high-speed swept confocally-aligned planar excitation (SCAPE) microscope into a focused ion beam irradiation platform to allow real-time 3D structural and functional imaging of living biological samples during and after irradiation. We demonstrate dynamic imaging of the acute effects of irradiation on 3D cultures of U87 human glioblastoma cells, revealing characteristic changes in cellular movement and intracellular calcium signaling following ionizing irradiation.

Inhibition of autophagic flux differently modulates cannabidiol-induced death in 2D and 3D glioblastoma cell cultures.

Radiotherapy combined with chemotherapy is the major treatment modality for human glioblastoma multiforme (GBM). GBMs eventually relapse after treatment and the average survival of GBM patients is less than two years. There is some evidence that cannabidiol (CBD) can induce cell death and increases the radiosensitivity of GBM by enhancing apoptosis. Beside initiation of death, CBD has been demonstrated as an inducer of autophagy. In the present study, we address the question whether CBD simultaneously induces a protective effect in GBM by upregulating autophagy. Addition of chloroquine that suppressed autophagic flux to 2D GBM cultures increased CBD-induced cell death, presenting proof for the protective autophagy. Blockage of autophagy upregulated radiation-induced cytotoxicity but only modestly affected the levels of cell death in CBD- or CBD/γ-irradiated 3D GBM cultures. Furthermore, CBD enhanced the pro-apoptotic activities of JNK1/2 and MAPK p38 signaling cascades while partially downregulated the pro-survival PI3K-AKT cascade, thereby changing a balance between cell death and survival. Suppression of JNK activation partially reduced CBD-induced cell death in 3D GBM cultures. In contrast, co-treatment of CBD-targeted cells with inhibitors of PI3K-AKT-NF-κB, IKK-NF-κB or JAK2-STAT3 pathways killed surviving GBM cells in both 2D and 3D cultures, potentially improving the therapeutic ratio of GBM.

High-Dose Radiation Increases Notch1 in Tumor Vasculature.

PURPOSE: The aim of this study is to characterize the effects of high-dose radiation therapy (HDRT) on Notch signaling components of the tumor vasculature. METHODS AND MATERIALS: Human umbilical vein endothelial cells monolayers were exposed to different single fraction doses of irradiation; ribonucleic acid RNA was isolated and polymerase chain reaction was performed for Notch signaling components. The vascular response to radiation therapy was examined in a xenograft model of neuroblastoma. Tumors were treated with 0 Gy, 2 Gy, and 12 Gy single fraction doses and analyzed by double immunofluorescence staining for Notch1, Notch ligands Jagged1 and Dll4, and the endothelial cell (EC) marker endomucin. To assess the role of Notch in vivo, NGP xenograft tumors expressing Fc or Notch1-1-24-decoy (a novel Notch inhibitor) were treated with 0 Gy and 12 Gy. Immunofluorescence staining for endomucin and endomucin/αSMA was performed to analyze the effect of combination treatment on tumor EC and endothelial-to-mesenchymal-transition (EndMT), respectively. RESULTS: In human umbilical vein endothelial cells monolayers doses ≥8 Gy increased expression of NOTCH1, JAG1, and Notch target genes HEY1 and HEY2 as early as 6 hours after irradiation. In vivo, 12 Gy significantly increased Notch1 and Jagged1 in tumor ECs compared with 0 Gy or 2 Gy after 72 hours. Combining HDRT with Notch inhibition using the Notch1-1-24-decoy resulted in a greater loss of EC coverage of tumor vessels than HDRT alone at 6 hours and 72 hours post treatment. Notch inhibition reduced EndMT induced by HDRT, as indicated by diminished αSMA staining in ECs. CONCLUSIONS: HDRT induced Notch1 expression and increased Notch1 signaling in the endothelial component of tumor vasculature, which was not observed with lower doses. This increase in Notch1 activation might protect tumor vessels from HDRT induced damage and regulate EndMT process.

Reprogramming mediated radio-resistance of 3D-grown cancer cells.

In vitro 3D growth of tumors is a new cell culture model that more closely mimics the features of the in vivo environment and is being used increasingly in the field of biological and medical research. It has been demonstrated that cancer cells cultured in 3D matrices are more radio-resistant compared with cells in monolayers. However, the mechanisms causing this difference remain unclear. Here we show that cancer cells cultured in a 3D microenvironment demonstrated an increase in cells with stem cell properties. This was confirmed by the finding that cells in 3D cultures upregulated the gene and protein expression of the stem cell reprogramming factors such as OCT4, SOX2, NANOG, LIN28 and miR-302a, compared with cells in monolayers. Moreover, the expression of ß-catenin, a regulating molecule of reprogramming factors, also increased in 3D-grown cancer cells. These findings suggest that cancer cells were reprogrammed to become stem cell-like cancer cells in a 3D growth culture microenvironment. Since cancer stem cell-like cells demonstrate an increased radio-resistance and chemo-resistance, our results offer a new perspective as to why. Our findings shed new light on understanding the features of the 3D growth cell model and its application in basic research into clinical radiotherapy and medicine.

Cytoplasmic dynein and LIS1 are required for microtubule advance during growth cone remodeling and fast axonal outgrowth.

Recent evidence has implicated dynein and its regulatory factors dynactin and LIS1 in neuronal and non-neuronal cell migration. In the current study we sought to test whether effects on neuronal cell motility might reflect, in part, a role for these proteins in the growth cone. In chick sensory neurons subjected to acute laminin treatment dynein, dynactin, and LIS1 were mobilized strikingly and rapidly to the leading edge of the growth cone, where they were seen to be associated with microtubules converging into the laminin-induced axonal outgrowths. To interfere acutely with LIS1 and dynein function and to minimize secondary phenotypic effects, we injected antibodies to these proteins just before axon initiation. Antibody to both proteins produced an almost complete block of laminin-induced growth cone remodeling and the underlying reorganization of microtubules. Penetration of microtubules into the peripheral zone of differentiating axonal growth cones was decreased dramatically by antibody injection, as judged by live analysis of enhanced green fluorescent protein-tubulin and the microtubule tip-associated EB3 (end-binding protein 3). Dynein and LIS1 inhibition had no detectable effect on microtubule assembly but reduced the ability of microtubules to resist retrograde actin flow. In hippocampal neurons dynein, dynactin, and LIS1 were enriched in axonal growth cones at stage 3, and both growth cone organization and axon elongation were altered by LIS1 RNA interference. Together, our data indicate that dynein and LIS1 play a surprisingly prominent role in microtubule advance during growth cone remodeling associated with axonogenesis. These data may explain, in part, the role of these proteins in brain developmental disease and support an important role in diverse aspects of neuronal differentiation and nervous system development.

Initiating morphological changes associated with long-term facilitation in Aplysia is independent of transcription or translation in the cell body.

In Aplysia, the growth of axonal arbor and the formation of new presynaptic varicosities are thought to contribute to long-term facilitation (LTF) produced by serotonin (5-HT). While it is known that there is a requirement for both transcription and translation in LTF and in the accompanying morphological changes, the mechanisms mediating the initiation and maintenance of these changes are poorly understood. We used long-term labeling of the presynaptic sensory neuron to carry out repeated imaging of axonal morphology, coupled with electrophysiology, to further elucidate the macromolecular requirements of this process. Robust synaptic facilitation, axonal growth, and the formation of axonal varicosities were elicited by 5-HT even when transcription was blocked with actinomycin. Increases in synaptic efficacy and varicosity number were detected 12 h after exposure to 5-HT but did not persist to 24 h. Even when sensory neuron cell bodies were removed, eliminating the contributions of both somal transcription and translation, 5-HT elicited these transient morphological and electrophysiological responses. New sensory varicosities contacting the postsynaptic neuron were filled with the neuropeptide sensorin. Under all conditions, global inhibition of protein synthesis completely blocked the formation of new axonal branches and varicosities. These results demonstrate that neither transcription nor somal translation is required to initiate the axonal growth that often accompanies long-term synaptic plasticity-protein synthesis in the axon is sufficient. Macromolecular synthesis in the cell body is, however, required to maintain the enlarged arbor.

Microtubule and Rac 1-dependent F-actin in growth cones.

Extracellular cues control the rate and direction of growth of neuronal processes in large part by regulating the cytoskeleton of the growth cone. The actin filament network of the peripheral region is thought to be the primary target for these cues, with consequences for the advance and organization of microtubules. Binding of laminin to integrin receptors is a cue that accelerates the growth of processes from many types of neurons. It was applied acutely to sympathetic neurons in culture to study its effects on the cytoskeleton of the growth cone. Microtubules advance to the edge of the growth cone and bundle in response to laminin, and it was found that small veils of membrane appear near the ends of some of those microtubules. To examine more clearly the relationship between the microtubules and the appearance of actin-rich structures at the periphery, a low dose of cytochalasin D was used to deplete the peripheral region of the growth cone of pre-existing F-actin. The subsequent addition of laminin resulted in the bundling of ends of dynamic (tyrosinated) microtubules at the distal edge of the growth cone, most of which were associated with foci of F-actin. Observations of labeled actin within living growth cones confirmed that these foci formed in response to laminin. Suppression of microtubule dynamics with drugs eliminated the actin foci; washout of drug restored them. Rac 1 did not co-concentrate with F-actin in the peripheral region of the growth cone in the absence of laminin, but did co-concentrate with the foci of F-actin that formed in response to laminin. Inhibition of Rac 1 functioning prevented the formation of the foci and also inhibited laminin-induced neurite growth with or without cytochalasin. These results indicate that extracellular cues can affect actin in the growth cone via microtubules, as well as affect microtubules via actin. They also point to the mediation of microtubule-dependent accumulation of F-actin at the front of the growth cone as a role of Rac 1 in neurite growth.

Caveolae-associated proteins in cardiomyocytes: caveolin-2 expression and interactions with caveolin-3.

Caveolin-3 the muscle-specific caveolin isoform, acts like the more ubiquitously expressed caveolin-1 to sculpt caveolae, specialized membrane microdomains that serve as platforms to organize signal transduction pathways. Caveolin-2 is a structurally related isoform that alone does not drive caveolae biogenesis; rather, caveolin-2 cooperates with caveolin-1 to form caveolae in nonmuscle cells. Although caveolin-2 might be expected to interact in an fashion analogous to that of caveolin-3, it generally has not been detected in cardiomyocytes. This study shows that caveolin-2 and caveolin-3 are detected at low levels in ventricular myocardium and increase dramatically with age or when neonatal cardiomyocytes are placed in culture. In contrast, flotillins (caveolin functional homologs) are expressed at relatively constant levels in these preparations. In neonatal cardiac cultures, caveolin-2 and -3 expression is not influenced by thyroid hormone (a postnatal regulator of other cardiac gene products). The further evidence that caveolin-2 coimmunoprecipitates with caveolin-3 and floats with caveolin-3 by isopycnic centrifugation in cardiomyocyte cultures suggests that caveolin-2 may play a role in caveolae biogenesis and influence cardiac muscle physiology.