GFP Imaging Research Articles

GFP and FAP imaging of neuropeptide release in Drosophila.

Genetics in Drosophila have revealed the role of neuropeptides in development and behavior. However, determining when and where neuropeptides are released has been challenging. Furthermore, the cell biology underlying neuropeptide release has largely been unexplored. Thus, it has not been possible to determine whether changes in neuropeptide immunofluorescence reflect traffic and/or release, and in neurons where such changes are not detectable, conclusions about neuropeptide release have been formulated based on the assumption that electrical and Ca2+ recordings are accurate and quantitative predictors of release. Recently, the advent of optical detection of neuropeptides tagged with fluorescent proteins and fluorogen-activating proteins (FAPs) has made it feasible to directly image vesicle traffic and exocytosis that mediates neuropeptide release in peripheral synapses and in the brain. In fact, these approaches have led to the discovery of unexpected insights concerning neuropeptide release. Here procedures are presented for optimizing fluorescence imaging of neuropeptides tagged with green fluorescent protein or a FAP.

Comparative exploration of mammalian deafness gene homologues in the Drosophila auditory organ shows genetic correlation between insect and vertebrate hearing.

Johnston's organ, the Drosophila auditory organ, is anatomically very different from the mammalian organ of Corti. However, recent evidence indicates significant cellular and molecular similarities exist between vertebrate and invertebrate hearing, suggesting that Drosophila may be a useful platform to determine the function of the many mammalian deafness genes whose underlying biological mechanisms are poorly characterized. Our goal was a comprehensive screen of all known orthologues of mammalian deafness genes in the fruit fly to better understand conservation of hearing mechanisms between the insect and the fly and ultimately gain insight into human hereditary deafness. We used bioinformatic comparisons to screen previously reported human and mouse deafness genes and found that 156 of them have orthologues in Drosophila melanogaster. We used fluorescent imaging of T2A-GAL4 gene trap and GFP or YFP fluorescent protein trap lines for 54 of the Drosophila genes and found 38 to be expressed in different cell types in Johnston's organ. We phenotypically characterized the function of strong loss-of-function mutants in three genes expressed in Johnston's organ (Cad99C, Msp-300, and Koi) using a courtship assay and electrophysiological recordings of sound-evoked potentials. Cad99C and Koi were found to have significant courtship defects. However, when we tested these genes for electrophysiological defects in hearing response, we did not see a significant difference suggesting the courtship defects were not caused by hearing deficiencies. Furthermore, we used a UAS/RNAi approach to test the function of seven genes and found two additional genes, CG5921 and Myo10a, that gave a statistically significant delay in courtship but not in sound-evoked potentials. Our results suggest that many mammalian deafness genes have Drosophila homologues expressed in the Johnston's organ, but that their requirement for hearing may not necessarily be the same as in mammals.

Glioma Cell Invasion Is Dependent Upon the DNA Damage Response Kinase ATR

Abstract AIMS Gliomas have high levels of DNA replication stress and clinical trials of inhibitors of the key replication stress response protein ATR (ataxia telangiectasia and rad 3 related protein) as radiosensitisers are planned. We aimed to investigate the effect of ATR inhibition on the ability of glioma cells to infiltrate and invade the brain. METHOD Live cell imaging in a panel of primary glioma cultures following siRNA or pharmacological inhibition of ATR. Invasion following treatment of murine orthotopic gliomas was determined by immunohistochemistry. Intravital imaging of GFP expressing murine orthotopic xenografts via an intracranial window model of glioma was undertaken. RESULTS Invading margins of human glioma samples demonstrated increased pATR expression relative to core. Live cell imaging demonstrated reduced cell velocity following ATR inhibition (Berzosertib/BAY1895344) or siRNA. Cytoplasmic vacuolation occurred following ATRi or siRNA which were single walled structures which engulf high molecular weight dextran, compatible with blockade of macropinosome processing. Live cell imaging with GFP-integrin α5 and integrin recycling assays showed sequestration of integrins within macropinosomes and reduced integrin cycling. Intravital in vivo imaging of murine xenograft tumours confirmed vacuolation and dextran uptake following ATRi, whilst a further in vivo study demonstrated a reduction in invading tumour cells. CONCLUSION We demonstrate a novel role for ATR in facilitating macropinocytic vesicle trafficking and integrin recycling in GBM cells which results in a profound motility defect in vitro and in vivo. ATR inhibitors are entering early phase trials as radiation sensitisers and we propose that therapeutic benefit will extend beyond DNA damage potentiation.

Cell-Permeable Nanobodies Allow Dual-Color Super-Resolution Microscopy in Untransfected Living Cells.

Super‐resolution microscopy in living cells can be restricted by the availability of small molecule probes, which only exist against few targets and genetically encoded tags. Here, we expand the applicability of live‐cell STED by engineering cell‐permeable and highly fluorescent nanobodies as intracellular targeting agents. To ensure bright fluorescent signals at low concentrations we used the concept of intramolecular photostabilization by ligating a fluorophore along with the photostabilizer trolox to the nanobody using expressed protein ligation (EPL). Furthermore, these semi‐synthetic nanobodies are equipped with a cleavable cell‐penetrating peptide for efficient cellular entry, which enables super‐resolution imaging of GFP and mCherry, as well as two endogenous targets, nuclear lamins and the DNA replication and repair protein PCNA. We monitored cell division and DNA replication via confocal and STED microscopy thus demonstrating the utility of these new intracellular tools for functional analysis.

Nose-to-brain delivery of disulfiram nanoemulsion in situ gel formulation for glioblastoma targeting therapy

Glioblastoma (GBM) is a difficult-to-treat cancer, likely attributed to the blood brain barrier and drug resistance. Nose-to-brain drug delivery is a direct and non-invasive pathway for brain targeting with low systemic toxicity. Disulfiram (DSF) has shown its effectiveness against GBM, especially with copper ion (Cu). In this work, we designed a DSF loaded ion-sensitive nanoemulsion in situ gel (DSF-INEG) that was delivered intranasally along with Cu to the rat brains for the GBM treatment. The developed DSF-INEG nanomedicine showed a suitable particle size of 63.4 ± 1.1 nm and zeta potential of –23.5 ± 0.2 mV with a favorable gelling ability and prolonged DSF release. The results in vitro indicate DSF-INEG/Cu effectively inhibited the proliferation of both C6 and U87 cells. Besides, the excellent brain-targeting efficacy via nose-to-brain delivery was proved by the highest fluorescence signal of Cy5.5-INEG in the rat brains. Moreover, GFP imaging showed enhanced tumor growth inhibition of the rats by the DSF-INEG/Cu treatment, and their median survival time was 1.6 and 1.2 folds than those of the rats in the control and DSF/Cu treated groups, respectively, with no obvious histopathological damage to normal tissues. Overall, DSF-INEG/Cu could be a promising intranasal nanomedicine for effective GBM treatment.

Mutational analysis of Arabidopsis thaliana ABCE2 identifies important motifs for its RNA silencing suppressor function.

ATP‐binding cassette sub‐family E member 1 (ABCE1) is recognized as a strongly conserved ribosome recycling factor, indispensable for translation in archaea and eukaryotes, however, its role in plants remains largely unidentified. Arabidopsis thaliana encodes two paralogous ABCE proteins (AtABCE1 and AtABCE2), sharing 81% identity. We previously reported that AtABCE2 functions as a suppressor of RNA silencing and that its gene is ubiquitously expressed. Here we describe the structural requirements of AtABCE2 for its suppressor function.Using agroinfiltration assays, we transiently overexpressed mutated versions of AtABCE2 together with GFP, to induce silencing in GFP transgenic Nicotiana benthamiana leaves. The influence of mutations was analysed at both local and systemic levels by in vivo imaging of GFP, Northern blot analysis of GFP siRNAs and observation of plants under UV light.Mutants of AtABCE2 with impaired ATP binding in either active site I or II failed to suppress GFP RNA silencing. Mutations disrupting ATP hydrolysis influenced the suppression of silencing differently at active site I or II. We also found that the N‐terminal iron–sulphur cluster domain of AtABCE2 is crucial for its suppressor function.Meaningfully, the observed structural requirements of AtABCE2 for RNA silencing suppression were found to be similar to those of archaeal ABCE1 needed for ribosome recycling. AtABCE2 might therefore suppress RNA silencing via supporting the competing RNA degradation mechanisms associated with ribosome recycling.

Abstract 5026: Efficacy of anti-PD-1, IDO inhibitor, chemotherapy and bone-targeting agent on tumor growth in a syngeneic bone metastasis model of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive cancer with treatment options limited to standard-of-care chemotherapy. TNBC patients develop metastases in high incidence, and almost all patients have bone metastases at end-stage disease. Bone metastases are mainly osteolytic, causing severe bone loss that can be prevented by bone-targeting agents. Currently, there is no efficient cure for bone metastases. Activation of the patient’s own immune system by immunotherapies is widely studied in various cancers, but their efficacy on bone metastases is not well established. In this study, we aimed to evaluate the efficacy of immunotherapies in comparison to standard-of-care compounds in a preclinical TNBC bone metastasis model. Mouse 4T1-GFP TNBC cells were inoculated intracardially to immunocompetent female Balb/c mice to model bone metastasis. Treatment of single agents of chemotherapy (cyclophosphamide, 100 mg/kg), bone-targeting agent (zoledronic acid, 0.1 mg/kg), and immunotherapies programmed cell death 1 antibody (mouse anti-PD-1, 10 mg/kg) and indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitor (epacadostat, 100 mg/kg), and the combination of anti-PD-1 and epacadostat were started on the day following the cancer cell inoculations. The study was terminated about 2 weeks after the inoculations. Tumor burden was evaluated at sacrifice by GFP imaging in skeleton and soft tissues, and tumor-induced bone loss by X-ray imaging. Tumor infiltrating lymphocytes (TILs) were evaluated by immunohistochemical stainings of CD3+, CD4+ and CD8+ T cells. Cyclophosphamide decreased skeletal tumor burden and the area of tumor-induced osteolytic bone lesions. It also decreased the number of soft tissue metastases in ovaries and adrenal glands. Zoledronic acid decreased osteolytic lesion area but had no effect on tumor burden. Anti-PD-1, epacadostat and their combination had no effect on tumor burden, on the development of osteolytic lesions, or on the number of soft tissue metastases. Low number or no TILs were observed in the tumors growing in bone obtained from vehicle-treated mice. Immunotherapies did not prevent tumor growth in bone or tumor-induced bone changes in the syngeneic TNBC model. This is probably a consequence of the low number of TILs in the tumors growing in bone. Further studies are needed to evaluate the effects of treatment combinations and to increase the responsiveness to immunotherapies especially in bone metastases. Citation Format: Tiina Emilia Kähkönen, Mari I. Suominen, Jenni H. Mäki-Jouppila, Jussi M. Halleen, Jenni Bernoulli. Efficacy of anti-PD-1, IDO inhibitor, chemotherapy and bone-targeting agent on tumor growth in a syngeneic bone metastasis model of triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5026.

Abstract 2690: Qualitatively distinct evolutionary games played by EGFR driven NSCLC

Abstract Background: Assuming cell-autonomous fitness within a population is often insufficient for understanding phenotypic shifts and resulting equilibria that can occur within ecosystems. Non-cell-autonomous interactions, whether competitive or cooperative in nature, influence the evolutionary trajectory of the population as a whole. We represented the interactions between gefitinib resistant and sensitive lung adenocarcinoma cells as evolutionary games to better understand population shifts that occur within heterogeneous tumors across a range of microenvironmental contexts. Methods: Co-cultures of fluorescently labeled PC-9 parental and evolved resistant lineages were plated across a range of relative initial proportions in 384 and 96 well formats. Bright field, GFP, and Texas Red images of each well were obtained in 3-4 hour intervals using an automated cell culture system. Least squares regressions were performed on the growth rate against the initial proportion of subpopulations in each well. Evolutionary games were quantified by converting the linear equations into payoff matrices correspond to each of the different conditions. Results: Interactions between the parental and resistant types within 11 different environmental drug conditions (DMSO, gefitinib, etoposide, erlotinib, doxorubicin, paclitaxel, gemcitabine, olaparib, osimertinib, lapatinib, and neratinib) were characterized and the corresponding payoff matrices derived. These games were plotted in a game space that lets us easily distinguish qualitatively distinct games. The distribution of each condition within the space was derived from the uncertainty of the preceding analysis. For example, while the interaction profile within gefitinib completely localizes to the “Deadlock” region and olaparib to the “Battle of the Sexes” region, the range of games played within paclitaxel crosses the boundary between the “Hawk-Dove” and “Concord” regions. Discussion: Our measurement of growth trajectories of the two different types across the different environmental contexts provided insight into the range of intercellular interactions that can occur in the presence of the various selective pressures. The qualitatively distinct regions of the constructed game space were predictive of the resulting phenotypic equilibrium of the heterogeneous co-cultures. For example, games played within the “Deadlock” region invariably lead to extinction of the parental lineage; however, interactions within the “Battle of the Sexes” region allowed for quantitatively predictable coexistence of both subpopulations. As such, the relative positions of the different eco-evolutionary dynamics correspond to different drugs within this space can be utilized to develop therapeutic strategies that steer the interactions (i.e. ‘treat the game') into regions that are associated with more therapeutically favorable equilibrium. Citation Format: Nathan Farrokhian, Artem Kaznatcheev, Patrick Ellsworth, Masahiro Hitomi, Emily Dolson, Jacob G. Scott. Qualitatively distinct evolutionary games played by EGFR driven NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2690.

HemoSYS: An Image‐based Toolkit for Quantifying Hemodynamics in the Microcirculation

ObjectiveTo develop an easy‐to‐use image‐based toolkit for quantifying spatiotemporal hemodynamic changes in the healthy and pathologic microcirculation.MethodsWe combined the capabilities of two well‐known image processing platforms: Matlab® (MathWorks, MA) and ImageJ into a single framework, and created an interactive graphical user interface (GUI) to manage all aspects of user interactions. The resulting toolkit for quantifying hemodynamic changes, HemoSYS, can be utilized without any prior programming knowledge, and consists of five analysis modules: propagation, clustering, coupling, perturbation, and Fourier. We demonstrate the utility of HemoSYS by using it to quantify hemodynamic changes within the tumor microenvironment (TME) of a murine breast cancer model. To acquire multi‐variable microcirculatory dynamics of the TME, we used a multi‐contrast optical imaging system and obtained wide field (i.e. 5 × 7 mm2) images of tumor extent via fluorescence (FL) imaging of GFP tagged tumor cells, and concurrent image‐sequences of tumor microvasculature or blood volume (BV) via hemoglobin‐based intrinsic optical signal (IOS) imaging, intravascular oxygen saturation (HbSat) via IOS imaging, and blood flow (BF) via laser speckle contrast (LSC) imaging every 30 seconds, for 1 hour.ResultsFig. 1 shows a schematic of HemoSYS illustrating its input data and capabilities. Using HemoSYS on a cohort of 5 animals, we identified the tumor extent and interrogated: (i) the propagation characteristics of an acute hypoxic episode; (ii) the formation and cessation of clusters with distinct microcirculatory dynamics; (iii) the dysregulation of blood flow; (iv) the multi‐variable hemodynamic response to carbogen gas inhalation;and (v) the heterogeneity of microcirculatory dynamics in the frequency domain.ConclusionHemoSYS (freely available upon request) enables characterization of hemodynamic changes in microvascular beds using a systems biology approach. It can be used by anyone interested in quantifying hemodynamic changes from imaging data because it does not require any programming experience. While we demonstrated its utility using wide‐field optical imaging data, HemoSYS could be readily modified to accept input data from additional imaging modalities such as MR, PET, and Photoacoustic imaging. We believe that HemoSYS will become a tool‐of‐choice for image‐based characterization of the microcirculation in any preclinical disease model or tissue bed.Support or Funding InformationThis work was supported by NCI 1R01CA196701, 5R01CA138264 and a Kavli Neuroscience Distinguished Fellowship (JS).Schematic of HemoSYS illustrating its inputs and capabilities.HemoSYS accepts a broad range of inputs, from static images indicating an area of interest (e.g. the tumor extent) to multiple image‐sequences showing concurrent spatiotemporal changes in multiple hemodynamic variables (e.g. BV, HbSat and BF). One can then interactively use HemoSYS’s five modules (i.e. propagation, clustering, coupling, perturbation and Fourier) to quantify and characterize the underlying microcirculation.Figure 1

Abstract B144: Lack of immunotherapy efficacy in a syngeneic bone metastasis model of triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive cancer with treatment options limited to standard-of-care chemotherapy. TNBC patients develop metastases in high incidence, and almost all patients have bone metastases at end-stage disease. Bone metastases are mainly osteolytic, causing severe bone loss that can be prevented by bone-targeted agents. Currently, there is no efficient cure for bone metastases. Activation of the patient’s own immune system by immunotherapies is widely studied in various cancers, but their efficacy on bone metastases is not well established. In this study, we aimed to evaluate the efficacy of immunotherapies in comparison to standard-of-care compounds in a preclinical TNBC bone metastasis model. Materials and methods: Mouse 4T1-GFP TNBC cells were inoculated intracardially to immunocompetent female Balb/c mice to model bone metastasis. Treatment of single agents of chemotherapy (cyclophosphamide, 100 mg/kg), bone-targeting agent (zoledronic acid, 0.1 mg/kg), and immunotherapies programmed cell death 1 antibody (mouse anti-PD-1, 10 mg/kg) and indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitor (epacadostat, 100 mg/kg), and the combination of anti-PD-1 and epacadostat were started on the day following the cancer cell inoculations. The study was terminated 12-13 days after the inoculations. Tumor burden was evaluated by GFP imaging and tumor-induced bone loss by X-ray imaging at sacrifice. Tumor infiltrating lymphocytes (TILs) were evaluated by immunohistochemical stainings of CD3+, CD4+ and CD8+ T cells. Results: Cyclophosphamide decreased skeletal tumor burden and the area of tumor-induced osteolytic bone lesions. Zoledronic acid decreased the area of osteolytic lesions but had no effect on skeletal tumor burden. Anti-PD-1, epacadostat and their combination had no effect on skeletal tumor burden or in the development of osteolytic lesions. Low number or no TILs were observed in the tumors growing in bone obtained from vehicle-treated mice. Conclusions: Immunotherapies did not prevent tumor growth in bone or tumor-induced bone changes in the syngeneic TNBC model. This is probably caused by the low number of TILs in the tumors growing in bone. Further studies are needed to evaluate the effects of treatment combinations and to increase the responsiveness to immunotherapies especially in bone metastases. Citation Format: Tiina E Kähkönen, Mari I Suominen, Jenni HE Mäki-Jouppila, Jussi M Halleen, Jenni Bernoulli. Lack of immunotherapy efficacy in a syngeneic bone metastasis model of triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B144. doi:10.1158/1535-7163.TARG-19-B144

Abstract 2814: Systemic and local bone metastasis models for immuno-oncology drug development

Abstract Bone metastases are common and count 30-70% of metastases in breast, lung, and bladder cancer, and 80% of multiple myeloma patients have bone disease. Despite recent progress in cancer treatment, bone metastases remain incurable. Novel immunotherapies have the potential to cure also bone metastatic disease. Here we have established and validated syngeneic models with a focus on bone metastasis that could be used in preclinical efficacy studies. Syngeneic models were established for breast (4T1-GFP), multiple myeloma (5TGM1), bladder (MBT-2) and lung cancer (KLN-205). The cells were inoculated into systemic circulation (4T1 intracardially and 5TGM1 into tail vein) or bone marrow (MBT-2 and KLN-205). In the 4T1 model, tumor burden was assessed by ex vivo GFP imaging and in the 5TGM1 model by measuring serum IgG2b paraprotein levels during the study. Tumor-induced bone changes were followed by X-ray imaging in all models. Hind limbs were analyzed by histology and immunohistochemistry for tumor-infiltrating lymphocytes (TILs). The effects of standard-of-care compounds were assessed in the 4T1 (cyclophosphamide, 100 mg/kg or zoledronic acid, 0.1 mg/kg) and 5TGM1 (bortezomib, 1 mg/kg) models. The effect of anti-PD-1 treatment (200 µg/dose) was evaluated in the MBT-2 model. In the 4T1 model, osteolytic bone lesions formed within 13 days. In addition to bone metastases, about 50% of the mice had metastases in lungs, ovaries, kidneys and adrenal glands based on GFP imaging. Cyclophosphamide decreased the tumor burden and the area of osteolytic bone lesions. Zoledronic acid decreased the osteolytic lesion area but had no effect on tumor burden. Low number or no TILs were observed. In the 5TGM1 model, osteolytic lesions were observed and the study was ended at day 35. Metastases in ovaries, kidneys and adrenal glands were observed in about 30% of the mice. Bortezomib decreased serum paraprotein compared to vehicle treated mice. In both models, cachexia and paraplegia were occasionally observed. Moderate number of CD3+ TILs were observed in the tumors. In the intratibial MBT-2 and KLN-205 models, large osteolytic lesions were observed within 25 days, and in the KLN-205 model also lung metastases were observed. Anti-PD-1 treatment decreased osteolytic tumor area in the MBT-2 model. Moderate number of CD3+ and low number of CD4+ and CD8+ TILs were observed in the tumors growing in bone and also in the lung metastases. A high incidence of bone metastases was observed in all models. The use of systemic models allows studying the effects of test compounds in prevention or treatment of metastases. Intratibial models can be used when the primary interest is in tumor growth in bone microenvironment. Mimicking the clinical situation, none of the SOC compounds could prevent tumor growth completely, and therefore combination therapies are warranted for better overall efficacy. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jenni H. Mäki-Jouppila, Jussi M. Halleen, Arne Scholz, Jenni Bernoulli. Systemic and local bone metastasis models for immuno-oncology drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2814.

SLIM microscopy allows for visualization of DNA-containing liposomes designed for sperm-mediated gene transfer in cattle.

Naked DNA has been shown to bind naturally to the sperm, a method called sperm-mediated gene transfer (SMGT). Based on these observations, we examined the efficiency of exogenous DNA binding to sperm using liposomes. In this experiment, we analyzed methods to select frozen-thawed bovine sperm, and evaluated the binding of exogenous DNA to those sperm. To determine the optimal selection method, we used Computer-Assisted Sperm Analysis (CASA). Percoll or Swim-Up were used to select sperm, followed by incubation up to 3h with the liposome-DNA complexes. The samples were collected after 1h and after 3h. We used enhanced green fluorescent protein (eGFP) in combination with the liposomes as a marker for exogenous DNA binding. Five treatments per selection method were analyzed: (1) no incubation, no liposomes and no DNA, (2) incubation with no liposomes and no DNA, (3) incubation with liposomes and no DNA, (4) incubation with liposomes and 1µg of DNA and (5) incubation with liposomes and 10µg of DNA. The CASA results for total motility and rapid motility were statistically significant (P < 0.01) between the control and the other treatments in the Percoll group as opposed to Swim-Up. Swim-Up was therefore chosen as the optimal selection method. In order to determine if the liposome-DNA complex had bound to sperm, real time PCR was used to detect GFP DNA and images of the sperm were analyzed using the Spatial Light Interference Microscopy (SLIM). SLIM confirmed the presence of liposomes on the sperm head and tail.

Identification of an RNA silencing suppressor encoded by citrus vein enation virus

In order to know if any protein encoded by citrus vein enation virus (CVEV) might act as an RNA silencing suppressor (RSS), five open reading frames (ORF) of CVEV, including the coat protein (CP) gene, were individually cloned into the binary expression vector pBI121. The silencing suppression ability of the five ORFs was tested by ‘viral minireplicon infectivity assays’ based on a beet yellows virus (BYV) minireplicon carrying a reporter gene (BYVminiR-GFP). GFP imaging and microscopic analyses were used to observe whether RNA silencing suppression occurred or not. In addition, real-time quantitative RT-PCR was used to quantify the mRNA expression of GFP in the injected region. The fluorescence intensity of leaves with CP expression was stronger than that of the empty vector negative control, but it was weaker than that of the positive control HC-Pro of tobacco etch virus (TEV), indicating that the CP of CVEV partly inhibited the GFP gene silencing. Co-expression of each of the other four ORFs with BYVminiR-GFP showed an almost complete quenching of GFP fluorescence in infiltrated areas, similar to that of the negative control. The amount of GFP mRNA in the leaves co-expressed with CP was nearly twice as much as that of either the negative control or other ORFs. These results suggest that CP of CVEV is a weak gene silencing suppressor.

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge.

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and cardiovascular disease, have been shown to activate tissue and/or cell-specific miRNA expression associated with disease progression. The inhibition of miRNA expression offers the potential for a therapeutic intervention. However, traditional approaches to inhibit miRNAs, employing antagomir oligonucleotides, affect specific miRNA functions upon global delivery. Herein, we present a protocol for the in vivo cardio-specific inhibition of the miR-181 family in a rat model. A miRNA-sponge construct is designed to include 10 repeated anti-miR-181 binding sequences. The cardio-specific α-MHC promoter is cloned into the pEGFP backbone to drive the cardio-specific miR-181 miRNA-sponge expression. To create a stable cell line expressing the miR-181-sponge, myoblast H9c2 cells are transfected with the α-MHC-EGFP-miR-181-sponge construct and sorted by fluorescence-activated cell sorting (FACs) into GFP positive H9c2 cells which are cultured with neomycin (G418). Following stable growth in neomycin, monoclonal cell populations are established by additional FACs and single cell cloning. The resulting myoblast H9c2-miR-181-sponge-GFP cells exhibit a loss of function of miR-181 family members as assessed through the increased expression of miR-181 target proteins and compared to H9c2 cells expressing a scramble non-functional sponge. In addition, we develop a nanovector for the systemic delivery of the miR-181-sponge construct by complexing positively charged liposomal nanoparticles and negatively charged miR-181-sponge plasmids. In vivo imaging of GFP reveals that multiple tail vein injections of a nanovector over a three-week period are able to promote a significant expression of the miR-181-sponge in a cardio-specific manner. Importantly, a loss of miR-181 function is observed in the heart tissue but not in the kidney or the liver. The miRNA-sponge is a powerful method to inhibit tissue-specific miRNA expression. Driving the miRNA-sponge expression from a tissue-specific promoter provides specificity for the miRNA inhibition, which can be confined to a targeted organ or tissue. Furthermore, combining nanovector and miRNA-sponge technologies permits an effective delivery and tissue-specific miRNA inhibition in vivo.

Three dimensional two-photon brain imaging in freely moving mice using a miniature fiber coupled microscope with active axial-scanning

We present a miniature head mounted two-photon fiber-coupled microscope (2P-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. We show three-dimensional two-photon imaging of neuronal structure and record neuronal activity from GCaMP6s fluorescence from multiple focal planes in a freely-moving mouse. Two-color simultaneous imaging of GFP and tdTomato fluorescence is also demonstrated. Additionally, dynamic control of the axial scanning of the electrowetting lens allows tilting of the focal plane enabling neurons in multiple depths to be imaged in a single plane. Two-photon imaging allows increased penetration depth in tissue yielding a working distance of 450 μm with an additional 180 μm of active axial focusing. The objective NA is 0.45 with a lateral resolution of 1.8 μm, an axial resolution of 10 μm, and a field-of-view of 240 μm diameter. The 2P-FCM has a weight of only ~2.5 g and is capable of repeatable and stable head-attachment. The 2P-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening new opportunities in neuroscience research.

Abstract 1037: Progression from melanocytic nevi to melanoma is associated with increased genomic mutations in a UV-induced mouse model of human melanoma

Abstract Melanoma is the deadliest form of skin cancer with approximately 132,000 cases worldwide each year. Benign melanocytic nevi are nearly universal, and although progression of nevi to melanoma is very rare, 20-50% of melanoma appear to arise from a pre-existing nevus. UV exposure, particularly childhood sunburn, is believed to play an important role in the development of melanocytic nevi and melanoma, but the exact mechanism is unknown. Alterations in MAPK pathway genes, especially NRAS and BRAF, are common in both benign nevi and melanoma, but approximately 1/3 of melanomas do not have an identified driver mutation. Studying nevus initiation and progression prospectively in the human population is impractical due to the long latency to progression and repeated UV exposures Our laboratory has developed a hepatocyte growth factor (HGF) genetically engineered mouse model with “humanized” junctional distribution of melanocytes on an iDCT-GFP background with melanocyte-specific GFP expression, allowing melanocytic lesions to be tracked through percutaneous GFP imaging. Following a single relevant dose of UV modeling childhood sunburn, HGF iDCT-GFP develop discrete, small melanocytic lesions consistent with nevi. Most nevi remain stable over the lifetime of the mouse, but around 1 in 30 progress to melanoma usually starting at 6-12 months. The melanocytic lesions are histologically similar to human nevi and melanoma, label with melanocyte markers and tumors are transplantable into syngeneic mice. Melanomas that arise in the model are heterogeneous and include radial growth phase and vertical growth phase tumors and sometimes metastasize to liver and lung. Exome sequencing of 28 nevi and melanomas show that vertical growth phase melanomas have approximately 3x more mutations than radial growth phase melanomas or nevi. The increased number of mutations in vertical growth phase tumors is due to an increase in C&amp;gt;T transitions despite the lack of additional UV exposure. Interestingly, melanocytic nevi and melanomas with DNA repair pathway mutations average 3x more mutations than lesions without mutations in these pathways. Melanomas sometime contain mutations in hotspot locations from human melanomas, including GNAQ, but most do not have a previously identified dominant driver. Genes potentially involved in the initiation of melanocytic lesions or progression to aggressive melanomas and relevant to human melanoma have been identified and are being functionally tested using CRISPR to introduce point mutations or knock out genes and in vitro skin reconstitution assays. Identification of novel drivers and pathways involved in non-BRAF, non-NRAS melanoma has the potential to uncover biomarkers and new therapeutic targets to improve clinical outcomes for melanoma patients. Citation Format: Helen Michael, Chi-Ping Day, Howard Yang, Aleksandra Michalowski, Maxwell Lee, Glenn Merlino. Progression from melanocytic nevi to melanoma is associated with increased genomic mutations in a UV-induced mouse model of human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1037. doi:10.1158/1538-7445.AM2017-1037

Lifetime imaging of GFP at CoxVIIIa reports respiratory supercomplex assembly in live cells

The assembly of respiratory complexes into macromolecular supercomplexes is currently a hot topic, especially in the context of newly available structural details. However, most work to date has been done with purified detergent-solubilized material and in situ confirmation is absent. We here set out to enable the recording of respiratory supercomplex formation in living cells. Fluorescent sensor proteins were placed at specific positions at cytochrome c oxidase suspected to either be at the surface of a CI1CIII2CIV1 supercomplex or buried within this supercomplex. In contrast to other loci, sensors at subunits CoxVIIIa and CoxVIIc reported a dense protein environment, as detected by significantly shortened fluorescence lifetimes. According to 3D modelling CoxVIIIa and CoxVIIc are buried in the CI1CIII2CIV1 supercomplex. Suppression of supercomplex scaffold proteins HIGD2A and CoxVIIa2l was accompanied by an increase in the lifetime of the CoxVIIIa-sensor in line with release of CIV from supercomplexes. Strikingly, our data provide strong evidence for defined stable supercomplex configuration in situ.

Mutant α-Synuclein Overexpression Induces Stressless Pacemaking in Vagal Motoneurons at Risk in Parkinson's Disease.

Overexpression of mutant α-synuclein causes Parkinson's disease, presumably by driving neurodegeneration in vulnerable neuronal target populations. However, the extent of α-synuclein pathology (e.g., Lewy bodies) is not directly related to the degree of neurodegeneration across various vulnerable neuronal populations. Here, we show that, in contrast to dopamine neurons in the substantia nigra, vagal motoneurons do not enhance their excitability and oxidative load in response to chronic mutant α-synuclein overexpression. Rather, by downregulating their voltage-activated calcium channels, vagal motoneurons acquire a stressless form of pacemaking that diminishes mitochondrial and cytosolic oxidative stress. Emulating this endogenous adaptive response to α-synuclein overexpression could lead to novel strategies to protect dopamine neurons and perhaps delay the onset of Parkinson's disease.

Abstract 835: Comparison of direct transplantation of colon cancer cells vs. tumor xenografts using orthotopic mouse model for colon cancer progression and metastasis

Abstract Orthotopic transplantation is the process of implantation of cancer cells or tumor xenograft tissues into the organ of origin of the cancer. This procedure enables cells to grow and proliferate in their natural microenvironment. The orthotopic transplantation model has been observed to be highly amenable to cancer cells and recapitulates the human disease with high fidelity. Orthotopic transplantation is very common in clinical cancer research using athymic nude mouse as a model system. A major advantage of the orthotopic transplantation model is its ability to study the development and progression of cancer metastasis. The identification and therapeutic validation of anti-metastatic agents is of urgent need since till date there are no effective anti-metastatic therapies available for colon cancer. The procedure of xenograft transplant is time consuming and costly because of the involvement of more number of mice. Popular method for orthotopic transplant is to grow a subcutaneous tumor injecting human cancer cells and then transplant a small piece of xenograft to the immune deficient mouse for clinical study. In this study, we directly injected the tumor cells below the sub-serosal layer of the caecum to develop colorectal cancer bypassing the formation of xenograft and its subsequent transplantation. We compared the effectiveness in tumor progression and metastasis between the cell transplant and xenograft implant to the caecum. GFP labelled HCT116 cells were used for this study to develop colorectal cancer. First, we injected 8 million cells subcutaneously to the athymic nude mice to generate xenograft for further transplant to the caecum. On another set of experimental animals, a total of 2 million cells were directly injected below the sub-serosal membrane to develop primary tumor on the colon. Whole body GFP imaging revealed that within a week of transplantation of cancer cells or xenografts, primary tumor starts to develop to the colon and metastasized within 4-5 weeks after implantation. In both the cases, we record a 100% of primary tumor formation with 50-60% liver and 75-80% lung metastasis. To confirm the metastasis in liver and lung we studied GAPDH expression on the tissues and conduct H&amp;E /immunofluorescence staining. Whole body or organ GFP imaging also reveals that pancreas, kidney and associated intestinal lymph glands are severely affected by both the orthotopic transplantation methods. In conclusion, direct sub-serosal injection of cells to develop colorectal cancer primary and metastatic deposits is cost effective and time saving and thus seems to potentially have an advantage over xenograft transplant method. Citation Format: Anathbandhu Chaudhuri, Liying Geng, Jing Wang. Comparison of direct transplantation of colon cancer cells vs. tumor xenografts using orthotopic mouse model for colon cancer progression and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 835.

Lateral Gene Transfer Contributing to Leukemogenesis

The uncontrolled proliferation of genetically mutated cells is the commonly understood mechanism for cancer growth and invasion, with accumulation of new mutations in daughter cells leading to clonal diversity of cancer derived from a single founding event. The genetic alterations are passed to new generations by cell division and vertical gene transfer. Viral transmission of oncogenes represents a known mechanism of lateral gene transfer in cancer initiation. Some experimental systems have also suggested that circulating DNA or micro-vesicles may contribute to lateral oncogene transfer in tumorigenesis. We hypothesized that interactions between leukemic cells and adjacent normal hematopoietic stem or progenitor cells may provide an alternative mechanism for the accumulation of mutated genes and the multiplicity of distinct clones in leukemia.To test this hypothesis, we performed experiments to determine whether tumorigenic properties could be transferred from a tumor cell line to normal mouse bone marrow cells using both in vivo and in vitro and systems. B6-GFP+ mice were injected i.v. with 200,000 C1498-Luc cells (a B6-derived NKT-cell-like mouse tumor cell line expressing luciferase and DSRed). Bioluminescent imaging was used to monitor the progression of tumor cell growth in recipients. At 1 month after tumor-cell inoculation, marrow from these mice was harvested and FACS-sorted for GFP+ cells (to eliminate C1498 cells), and then cultured on irradiated stromal cell layers in 96-well plates in a limiting dilution analysis for Poisson analysis of GFP+ clonogenic precursor frequency on day 9. On day 10, cells were harvested from culture and GFP+ cells resorted onto fresh stromal layers for second and third determinations of GFP+ clonogenic precursor frequency on days 15 and 18. As shown in Figure 1, the frequency of clonogenic precursors increased with each successive determination for marrow from C1498-injected mice, while control cultures from non-injected mice showed no increase in precursor frequency, suggesting that exposure to C1498 cells conferred a growth advantage to the marrow cells in the tumor-cell injected mice. Similar results were obtained using an in vitro system of co-culture using C1498 cells and GFP+ bone marrow cells, followed by serial rounds of GFP+ sorting and Poisson analysis, showing increases in clonogenic frequency over 5 successive sorts and re-cultures over a 2-month period, while control cultures showed decreased clonogenic frequencies over the course of the experiment.To confirm these observations in vivo, B6-GFP mice were injected with C1498-Luc and marrow was harvested after a month and sorted for GFP+ cells. The sorted marrow was transplanted into 11Gy-irradiated (FVB x B6albino)F1 recipients (5 x 106 cells per recipient, n=5). Control recipients were irradiated and transplanted with GFP+ marrow from non-injected donors. All recipients developed full hematopoietic engraftment with GFP+ cells. At 6 months post-transplant, a tumor was observed near the left shoulder of one of the recipients of C1498-exposed GFP+ marrow. Figure 2 shows IVIS GFP imaging of this mouse with the GFP+ tumor along with control animals. The tumor was not positive for luciferase expression. The mouse was sacrificed and the tumor excised and a portion was dissociated for flow cytometric analysis and culturing (with other segments reserved for subsequent histological and genetic analysis). Both GFP+ and non-GFP cells were found in the dissociated tumor cell suspension. The GFP+ cells were hematopoietic in origin (CD45+) and exhibited a mixed phenotype containing markers expressed on C1498 (DX5+) and myeloid lineage cells (CD11b+) as well as Sca-1, a stem cell marker. Cultures of the GFP+ tumor yielded a population of GFP+ mononuclear cells.These data are consistent with a model in which growth-promoting or transforming genes from cancer cells become incorporated within a healthy hematopoietic stem or progenitor cell, which contributes to the genetic diversity of the cancer through the initiation a new transformed clone. Genetic analysis with deep sequencing will compare the DNA sequences between the parental C1498 cell line, sorted populations of clonogenic GFP+ cells obtained from the in vitro and in vivo experiments, and the GFP+ tumor cells to confirm the transformation of healthy bone marrow hematopoietic stem cells with genetic sequences derived from the C1498 cells. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.