Publications by authors named "Bostjan Markelc"

39 Publications

Mutational burden, MHC-I expression and immune infiltration as limiting factors for in situ vaccination by TNFα and IL-12 gene electrotransfer.

Bioelectrochemistry 2021 Aug 28;140:107831. Epub 2021 Apr 28.

Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska cesta 2, SI-1000 Ljubljana, Slovenia; Faculty of Health Sciences, University of Ljubljana, Zdravstvena pot 5, SI-1000 Ljubljana, Slovenia.

In situ vaccination is a promising immunotherapeutic approach, where various local ablative therapies are used to induce an immune response against tumor antigens that are released from the therapy-killed tumor cells. We recently proposed using intratumoral gene electrotransfer for concomitant transfection of a cytotoxic cytokine tumor necrosis factor-α (TNFα) to induce in situ vaccination, and an immunostimulatory cytokine interleukin 12 (IL-12) to boost the primed immune response. Here, our aim was to test the local and systemic effectiveness of the approach in tree syngeneic mouse tumor models and associate it with tumor immune profiles, characterized by tumor mutational burden, immune infiltration and expression of PD-L1 and MHC-I on tumor cells. While none of the tested characteristic proved predictive for local effectiveness, high tumor mutational burden, immune infiltration and MHC-I expression were associated with higher abscopal effectiveness. Hence, we have confirmed that both the abundance and presentation of tumor antigens as well as the absence of immunosuppressive mechanisms are important for effective in situ vaccination. These findings provide important indications for future development of in situ vaccination based treatments, and for the selection of tumor types that will most likely benefit from it.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bioelechem.2021.107831DOI Listing
August 2021

PARP inhibitor olaparib has a potential to increase the effectiveness of electrochemotherapy in BRCA1 mutated breast cancer in mice.

Bioelectrochemistry 2021 Aug 1;140:107832. Epub 2021 May 1.

Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia; University of Ljubljana, Faculty of Health Sciences, Zdravstvena pot 5, SI - 1000 Ljubljana, Slovenia. Electronic address:

Electrochemotherapy (ECT), a local therapy, has different effectiveness among tumor types. In breast cancer, its effectiveness is low; therefore, combined therapies are needed. The aim of our study was to combine ECT with PARP inhibitor olaparib, which could inhibit the repair of bleomycin or cisplatin induced DNA damage and potentiate the effectiveness of ECT. The effects of combined therapy were studied in BRCA1 mutated (HCC1937) and non-mutated (HCC1143) triple negative breast cancer cell lines. Therapeutic effectiveness was studied in 2D and 3D cell cultures and in vivo on subcutaneous HCC1937 tumor model in mice. The underlying mechanism of combined therapy was determined with the evaluation of γH2AX foci. Combined therapy of ECT with bleomycin and olaparib potentiated the effectiveness of ECT in BRCA1 mutated HCC1937, but not in non-mutated HCC1143 cells. The combined therapy had a synergistic effect, which was due to the increased number of DNA double strand breaks. Addition of olaparib to ECT with bleomycin in vivo in HCC1937 tumor model had only minimal effect, indicating repetitive olaparib treatment would be needed. This study demonstrates that DNA repair inhibiting drugs, like olaparib, have the potential to increase the effectiveness of ECT with bleomycin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bioelechem.2021.107832DOI Listing
August 2021

Potentiation of electrochemotherapy effectiveness by immunostimulation with IL-12 gene electrotransfer in mice is dependent on tumor immune status.

J Control Release 2021 Apr 8;332:623-635. Epub 2021 Mar 8.

Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska cesta 2, SI-1000 Ljubljana, Slovenia; Faculty of Health Sciences, University of Ljubljana, Zdravstvena pot 5, SI-1000 Ljubljana, Slovenia. Electronic address:

Electrochemotherapy (ECT) exhibits high therapeutic effectiveness in the clinic, achieving up to 80% local tumor control but without a systemic (abscopal) effect. Therefore, we designed a combination therapy consisting of ECT via intratumoral application of bleomycin, oxaliplatin or cisplatin with peritumoral gene electrotransfer of a plasmid encoding interleukin-12 (p. t. IL-12 GET). Our hypothesis was that p. t. IL-12 GET potentiates the effect of ECT on local and systemic levels and that the potentiation varies depending on tumor immune status. Therefore, the combination therapy was tested in three immunologically different murine tumor models. In poorly immunogenic B16F10 melanoma, IL-12 potentiated the antitumor effect of ECT with biologically equivalent low doses of cisplatin, oxaliplatin or bleomycin. The most pronounced potentiation was observed after ECT using cisplatin, resulting in a complete response rate of 38% and an abscopal effect. Compared to B16F10 melanoma, better responsiveness to ECT was observed in more immunogenic 4 T1 mammary carcinoma and CT26 colorectal carcinoma. In both models, p. t. IL-12 GET did not significantly improve the therapeutic outcome of ECT using any of the chemotherapeutic drugs. Collectively, the effectiveness of the combination therapy depends on tumor immune status. ECT was more effective in more immunogenic tumors, but GET exhibited greater contribution in less immunogenic tumors. Thus, the selection of the therapy, namely, either ECT alone or combination therapy with p. t. IL-12, should be predominantly based on tumor immune status.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jconrel.2021.03.009DOI Listing
April 2021

PD1 blockade potentiates the therapeutic efficacy of photothermally-activated and MRI-guided low temperature-sensitive magnetoliposomes.

J Control Release 2021 Apr 4;332:419-433. Epub 2021 Mar 4.

School of Pharmacy, Queen's University Belfast, Belfast, United Kingdom. Electronic address:

This study investigates the effect of PD1 blockade on the therapeutic efficacy of novel doxorubicin-loaded temperature-sensitive liposomes. Herein, we report photothermally-activated, low temperature-sensitive magnetoliposomes (mLTSL) for efficient drug delivery and magnetic resonance imaging (MRI). The mLTSL were prepared by embedding small nitrodopamine palmitate (NDPM)-coated iron oxide nanoparticles (IO NPs) in the lipid bilayer of low temperature-sensitive liposomes (LTSL), using lipid film hydration and extrusion. Doxorubicin (DOX)-loaded mLTSL were characterized using dynamic light scattering, differential scanning calorimetry, electron microscopy, spectrofluorimetry, and atomic absorption spectroscopy. Photothermal experiments using 808 nm laser irradiation were conducted. In vitro photothermal DOX release studies and cytotoxicity was assessed using flow cytometry and resazurin viability assay, respectively. In vivo DOX release and tumor accumulation of mLTSL(DOX) were assessed using fluorescence and MR imaging, respectively. Finally, the therapeutic efficacy of PD1 blockade in combination with photothermally-activated mLTSL(DOX) in CT26-tumor model was evaluated by monitoring tumor growth, cytokine release and immune cell infiltration in the tumor tissue. Interestingly, efficient photothermal heating was obtained by varying the IO NPs content and the laser power, where on-demand burst DOX release was achievable in vitro and in vivo. Moreover, our mLTSL exhibited promising MR imaging properties with high transverse r relaxivity (333 mM s), resulting in superior MR imaging in vivo. Furthermore, mLTSL(DOX) therapeutic efficacy was potentiated in combination with anti-PD1 mAb, resulting in a significant reduction in CT26 tumor growth via immune cell activation. Our study highlights the potential of combining PD1 blockade with mLTSL(DOX), where the latter could facilitate chemo/photothermal therapy and MRI-guided drug delivery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jconrel.2021.03.002DOI Listing
April 2021

Reciprocal interactions between tumour cell populations enhance growth and reduce radiation sensitivity in prostate cancer.

Commun Biol 2021 01 4;4(1). Epub 2021 Jan 4.

CRUK and MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK.

Intratumoural heterogeneity (ITH) contributes to local recurrence following radiotherapy in prostate cancer. Recent studies also show that ecological interactions between heterogeneous tumour cell populations can lead to resistance in chemotherapy. Here, we evaluated whether interactions between heterogenous populations could impact growth and response to radiotherapy in prostate cancer. Using mixed 3D cultures of parental and radioresistant populations from two prostate cancer cell lines and a predator-prey mathematical model to investigate various types of ecological interactions, we show that reciprocal interactions between heterogeneous populations enhance overall growth and reduce radiation sensitivity. The type of interaction influences the time of regrowth after radiation, and, at the population level, alters the survival and cell cycle of each population without eliminating either one. These interactions can arise from oxygen constraints and from cellular cross-talk that alter the tumour microenvironment. These findings suggest that ecological-type interactions are important in radiation response and could be targeted to reduce local recurrence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42003-020-01529-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782740PMC
January 2021

Radiation Induced Upregulation of DNA Sensing Pathways is Cell-Type Dependent and Can Mediate the Off-Target Effects.

Cancers (Basel) 2020 Nov 13;12(11). Epub 2020 Nov 13.

Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska cesta 2, 1000 Ljubljana, Slovenia.

Irradiation of tumors generates danger signals and inflammatory cytokines that promote the off-target bystander and abscopal effects, evident especially when radiotherapy is administered in combination with the immune checkpoint inhibitors (ICI). The underlying mechanisms are not fully understood; however, cGAS-STING pathway was recognized as the main mediator. In our study, we demonstrate by immunofluorescent staining that tumor cells as well as macrophages, cell types abundant in the tumor microenvironmeent (TME) accumulate DNA in their cytosol soon after irradiation. This accumulation activated several distinct DNA sensing pathways, most prominently activated DNA sensors being DDX60, DAI, and p204 in tumor cells and DDX60, DAI, p204, and RIG-I in macrophages as determined by PCR and immunofluorescence imaging studies. This was accompanied by increased expression of cytokines evaluated by flow cytometry, TNFα, and IFNβ in tumor cells and IL1β and IFNβ in macrophages, which can alter the TME and mediate off-target effects (bystander or abscopal effects). These results give insight into the mechanisms involved in the stimulation of antitumor immunity by radiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12113365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696780PMC
November 2020

Abnormal morphology biases hematocrit distribution in tumor vasculature and contributes to heterogeneity in tissue oxygenation.

Proc Natl Acad Sci U S A 2020 11 27;117(45):27811-27819. Epub 2020 Oct 27.

Mathematical Institute, University of Oxford, Oxford OX2 6GG, United Kingdom;

Oxygen heterogeneity in solid tumors is recognized as a limiting factor for therapeutic efficacy. This heterogeneity arises from the abnormal vascular structure of the tumor, but the precise mechanisms linking abnormal structure and compromised oxygen transport are only partially understood. In this paper, we investigate the role that red blood cell (RBC) transport plays in establishing oxygen heterogeneity in tumor tissue. We focus on heterogeneity driven by network effects, which are challenging to observe experimentally due to the reduced fields of view typically considered. Motivated by our findings of abnormal vascular patterns linked to deviations from current RBC transport theory, we calculated average vessel lengths [Formula: see text] and diameters [Formula: see text] from tumor allografts of three cancer cell lines and observed a substantial reduction in the ratio [Formula: see text] compared to physiological conditions. Mathematical modeling reveals that small values of the ratio λ (i.e., [Formula: see text]) can bias hematocrit distribution in tumor vascular networks and drive heterogeneous oxygenation of tumor tissue. Finally, we show an increase in the value of λ in tumor vascular networks following treatment with the antiangiogenic cancer agent DC101. Based on our findings, we propose λ as an effective way of monitoring the efficacy of antiangiogenic agents and as a proxy measure of perfusion and oxygenation in tumor tissue undergoing antiangiogenic treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2007770117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668105PMC
November 2020

FGF2 alters macrophage polarization, tumour immunity and growth and can be targeted during radiotherapy.

Nat Commun 2020 08 13;11(1):4064. Epub 2020 Aug 13.

Oxford Institute for Radiation Oncology, University of Oxford, Oxford, OX3 7DQ, UK.

Regulation of the programming of tumour-associated macrophages (TAMs) controls tumour growth and anti-tumour immunity. We examined the role of FGF2 in that regulation. Tumours in mice genetically deficient in low-molecular weight FGF2 (FGF2) regress dependent on T cells. Yet, TAMS not T cells express FGF receptors. Bone marrow derived-macrophages from Fgf2 mice co-injected with cancer cells reduce tumour growth and express more inflammatory cytokines. FGF2 is induced in the tumour microenvironment following fractionated radiation in murine tumours consistent with clinical reports. Combination treatment of in vivo tumours with fractionated radiation and a blocking antibody to FGF2 prolongs tumour growth delay, increases long-term survival and leads to a higher iNOS/CD206 TAM ratio compared to irradiation alone. These studies show for the first time that FGF2 affects macrophage programming and is a critical regulator of immunity in the tumour microenvironment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-17914-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426415PMC
August 2020

A lineage-tracing tool to map the fate of hypoxic tumour cells.

Dis Model Mech 2020 07 30;13(7). Epub 2020 Jul 30.

Department of Radiation Oncology (Maastro), GROW School for Oncology and Developmental Biology, Maastricht University Medical Centre, 6202 AZ Maastricht, The Netherlands

Intratumoural hypoxia is a common characteristic of malignant treatment-resistant cancers. However, hypoxia-modification strategies for the clinic remain elusive. To date, little is known on the behaviour of individual hypoxic tumour cells in their microenvironment. To explore this issue in a spatial and temporally controlled manner, we developed a genetically encoded sensor by fusing the O-labile hypoxia-inducible factor 1α (HIF-1α) protein to eGFP and a tamoxifen-regulated Cre recombinase. Under normoxic conditions, HIF-1α is degraded but, under hypoxia, the HIF-1α-GFP-Cre-ER fusion protein is stabilised and in the presence of tamoxifen activates a tdTomato reporter gene that is constitutively expressed in hypoxic progeny. We visualise the random distribution of hypoxic tumour cells from hypoxic or necrotic regions and vascularised areas using immunofluorescence and intravital microscopy. Once tdTomato expression is induced, it is stable for at least 4 weeks. Using this system, we could show that the post-hypoxic cells were more proliferative than non-labelled cells. Our results demonstrate that single-cell lineage tracing of hypoxic tumour cells can allow visualisation of their behaviour in living tumours using intravital microscopy. This tool should prove valuable for the study of dissemination and treatment response of post-hypoxic tumour cells at single-cell resolution.This article has an associated First Person interview with the joint first authors of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dmm.044768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406318PMC
July 2020

Intraoperative electrochemotherapy of colorectal liver metastases: A prospective phase II study.

Eur J Surg Oncol 2020 09 25;46(9):1628-1633. Epub 2020 Apr 25.

Institute of Oncology Ljubljana, Zaloska 2, Ljubljana, SI-1000, Slovenia; University of Ljubljana, Faculty of Health Sciences, Zdravstvena Pot 5, Ljubljana, SI - 1000, Slovenia. Electronic address:

Background And Objectives: A previous pilot study proved the feasibility, safety and efficacy of electrochemotherapy in the treatment of colorectal liver metastases. The aim of this study was to evaluate long-term effectiveness and safety of electrochemotherapy in the treatment of unresectable colorectal liver metastases.

Patients And Methods: In this prospective phase II study, patients with metachronous colorectal liver metastases were included. In all patients, at least one metastasis was unresectable due to its central location or a too-small future remnant liver volume. Patients were treated by electrochemotherapy using intravenously administered bleomycin during open surgery. Treated were 84 metastases in 39 patients. Local tumor control, progression-free survival and overall survival were evaluated.

Results: The objective response was 75% (63% CR, 12% PR). The median duration of the response was 20.8 months for metastases in CR and 9.8 months for metastases in PR. The therapy was significantly more effective for metastases smaller than 3 cm in diameter than for larger ones. There was no difference in response according to the metastatic location, i.e., metastases in central vs. peripheral locations. Progression-free survival was better in patients who responded well to electrochemotherapy compared to those metastases that had a partial response or progressive disease. However, there was no difference in overall survival, with a median of 29.0 months.

Conclusions: Electrochemotherapy has proven to be safe and effective in the treatment of colorectal liver metastases, with a durable response. It provides local tumor control that enables patients with unresectable metastases to receive further treatments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejso.2020.04.037DOI Listing
September 2020

Multiple cytosolic DNA sensors bind plasmid DNA after transfection.

Nucleic Acids Res 2019 11;47(19):10235-10246

Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA.

Mammalian cells express a variety of nucleic acid sensors as one of the first lines of defense against infection. Despite extensive progress in the study of sensor signaling pathways during the last decade, the detailed mechanisms remain unclear. In our previous studies, we reported increased type I interferon expression and the upregulation of several proposed cytosolic DNA sensors after transfection of several tumor cell types with plasmid DNA (pDNA). In the present study, we sought to reveal the early events in the cytosolic sensing of this nucleic acid in a myoblast cell line. We demonstrated that DNA-dependent activator of interferon regulatory factors/Z-DNA binding protein 1 (DAI/ZBP1) bound plasmid DNA in the cytosol within 15 minutes of transfection and at consistent levels for 4 h. Interferon activated gene 204 protein (p204) and DEAH box helicase 9 (DHX9) also bound pDNA, peaking 15 and 30 min respectively. Plasmid DNA was not detectably bound by DEAD box helicase 60 (DDX60) protein, despite a similar level of mRNA upregulation to DAI/ZBP1, or by cyclic GMP-AMP synthase (cGAS), despite its presence in the cell cytosol. Taken together, these results indicate several DNA sensors may participate and cooperate in the complex process of cytosolic DNA sensing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821305PMC
November 2019

Type I IFN protects cancer cells from CD8+ T cell-mediated cytotoxicity after radiation.

J Clin Invest 2019 10;129(10):4224-4238

CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, United Kingdom.

Treatment of tumors with ionizing radiation stimulates an antitumor immune response partly dependent on induction of IFNs. These IFNs directly enhance dendritic cell and CD8+ T cell activity. Here we show that resistance to an effective antitumor immune response is also a result of IFN signaling in a different cellular compartment of the tumor, the cancer cells themselves. We abolished type I IFN signaling in cancer cells by genetic elimination of its receptor, IFNAR1. Pronounced immune responses were provoked after ionizing radiation of tumors from 4 mouse cancer cell lines with Ifnar1 knockout. This enhanced response depended on CD8+ T cells and was mediated by enhanced susceptibility to T cell-mediated killing. Induction of Serpinb9 proved to be the mechanism underlying control of susceptibility to T cell killing after radiation. Ifnar1-deficient tumors had an augmented response to anti-PD-L1 immunotherapy with or without radiation. We conclude that type I IFN can protect cancer cells from T cell-mediated cytotoxicity through regulation of Serpinb9. This result helps explain why radiation of tumors can stimulate antitumor immunity yet also result in resistance. It further suggests potential targets for intervention to improve therapy and to predict responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI127458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763250PMC
October 2019

Pre-clinical investigation of the synergy effect of interleukin-12 gene-electro-transfer during partially irreversible electropermeabilization against melanoma.

J Immunother Cancer 2019 06 26;7(1):161. Epub 2019 Jun 26.

Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, BP 64182, UMR 5089, 205 Route de Narbonne, F-31077, Toulouse Cedex, France.

Background: Melanoma is a very aggressive skin tumor that can be cured when diagnosed and treated in its early stages. However, at the time of identification, the tumor is frequently in a metastatic stage. Intensive research is currently ongoing to improve the efficacy of the immune system in eliminating cancer cells. One approach is to boost the activation of cytotoxic T cells by IL-12 cytokine that plays a central role in the activation of the immune system. In parallel, physical methods such as electropermeabilization-based treatments are currently under investigation and show promising results.

Methods: In this study, we set electrical parameters to induce a partial-irreversible electropermeabilization (pIRE) of melanoma to induce a sufficient cell death and potential release of tumor antigens able to activate immune cells. This protocol mimics the situation where irreversible electropermeabilization is not fully completed. Then, a peritumoral plasmid IL-12 electrotransfer was combined with pIRE treatment. Evaluation of the tumor growth and survival was performed in mouse strains having a different immunological background (C57Bl/6 (WT), nude and C57Bl6 (TLR9-/-)).

Results: pIRE treatment induced apoptotic cell death and a temporary tumor growth delay in all mouse strains. In C57Bl/6 mice, we showed that peritumoral plasmid IL-12 electrotransfer combined with tumor pIRE treatment induced tumor regression correlating with a local secretion of IL-12 and IFN-γ. This combined treatment induced a growth delay of distant tumors and prevented the emergence of a second tumor in 50% of immunocompetent mice.

Conclusions: The combination of pIL-12 GET and pIRE not only enhanced survival but could bring a curative effect in wild type mice. This two-step treatment, named Immune-Gene Electro-Therapy (IGET), led to a systemic activation of the adaptive immune system and the development of an anti-tumor immune memory.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40425-019-0638-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6595571PMC
June 2019

Image-Based Artefact Removal in Laser Scanning Microscopy.

IEEE Trans Biomed Eng 2020 01 29;67(1):79-87. Epub 2019 Apr 29.

Recent developments in laser scanning microscopy have greatly extended its applicability in cancer imaging beyond the visualization of complex biology, and opened up the possibility of quantitative analysis of inherently dynamic biological processes. However, the physics of image acquisition intrinsically means that image quality is subject to a tradeoff between a number of imaging parameters, including resolution, signal-to-noise ratio, and acquisition speed. We address the problem of geometric distortion, in particular, jaggedness artefacts that are caused by the variable motion of the microscope laser, by using a combination of image processing techniques. Image restoration methods have already shown great potential for post-acquisition image analysis. The performance of our proposed image restoration technique was first quantitatively evaluated using phantom data with different textures, and then qualitatively assessed using in vivo biological imaging data. In both cases, the presented method, comprising a combination of image registration and filtering, is demonstrated to have substantial improvement over state-of-the-art microscopy acquisition methods.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1109/TBME.2019.2908345DOI Listing
January 2020

Aspirin blocks formation of metastatic intravascular niches by inhibiting platelet-derived COX-1/thromboxane A2.

J Clin Invest 2019 03 25;129(5):1845-1862. Epub 2019 Mar 25.

Cancer Research UK and MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, United Kingdom.

Because metastasis is associated with the majority of cancer-related deaths, its prevention is a clinical aspiration. Prostanoids are a large family of bioactive lipids derived from the activity of cyclooxygenase-1 (COX-1) and COX-2. Aspirin impairs the biosynthesis of all prostanoids through the irreversible inhibition of both COX isoforms. Long-term administration of aspirin leads to reduced distant metastases in murine models and clinical trials, but the COX isoform, downstream prostanoid, and cell compartment responsible for this effect are yet to be determined. Here, we have shown that aspirin dramatically reduced lung metastasis through inhibition of COX-1 while the cancer cells remained intravascular and that inhibition of platelet COX-1 alone was sufficient to impair metastasis. Thromboxane A2 (TXA2) was the prostanoid product of COX-1 responsible for this antimetastatic effect. Inhibition of the COX-1/TXA2 pathway in platelets decreased aggregation of platelets on tumor cells, endothelial activation, tumor cell adhesion to the endothelium, and recruitment of metastasis-promoting monocytes/macrophages, and diminished the formation of a premetastatic niche. Thus, platelet-derived TXA2 orchestrates the generation of a favorable intravascular metastatic niche that promotes tumor cell seeding and identifies COX-1/TXA2 signaling as a target for the prevention of metastasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI121985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486338PMC
March 2019

Electroporation-Induced Stress Response and Its Effect on Gene Electrotransfer Efficacy: In Vivo Imaging and Numerical Modeling.

IEEE Trans Biomed Eng 2019 09 23;66(9):2671-2683. Epub 2019 Jan 23.

Objective: Skin is an attractive target tissue for gene transfer due to its size, accessibility, and its immune competence. One of the promising delivery methods is gene delivery by means of electroporation (EP), i.e., gene electrotransfer (GET). To assess the importance of different effects of electroporation for successful GET we investigated: stress response and transfection efficacy upon different pulse protocols. Moreover, numerical modeling was used to explain experimental results and to test the agreement of experimental results with current knowledge about GET.

Methods: Double transgenic mice Hspa1b-LucF (+/+) Hspa1b-mPlum (+/+) were used to determine the level of stress sensed by the cell in the tissue in vivo that was exposed to EP. The effect of five different pulse protocols on stress levels sensed by the exposed cells and their efficacy for gene electrotransfer for two plasmids pEGFP-C1 (EGFP) and pCMV-tdTomato was tested.

Results: Quantification of the bioluminescence signal intensity shows that EP, regardless of the electric pulse parameters used, increased mean bioluminescence compared to the baseline bioluminescence signal of the non-exposed skin. The results of numerical modeling indicate that thermal stress alone is not sufficient to explain the measured bioluminescence signal. Of the tested pulse protocols, the highest expression of EGFP and tdTomato was achieved with HV-MV (high voltage - medium voltage) protocols, which agrees also with numerical model.

Significance: Although EP is widely used as a method for gene delivery, we show that the field could benefit from the use of mathematical modeling by introducing additional parameters such as EP induced stress and electrophoretic movement of plasmids.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1109/TBME.2019.2894659DOI Listing
September 2019

Safe and efficient novel approach for non-invasive gene electrotransfer to skin.

Sci Rep 2018 11 15;8(1):16833. Epub 2018 Nov 15.

Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, BP 64182, 205 Route de Narbonne, Toulouse, F-31077, France.

Gene transfer into cells or tissue by application of electric pulses (i.e. gene electrotransfer (GET)) is a non-viral gene delivery method that is becoming increasingly attractive for clinical applications. In order to make GET progress to wide clinical usage its efficacy needs to be improved and the safety of the method has to be confirmed. Therefore, the aim of our study was to increase GET efficacy in skin, by optimizing electric pulse parameters and the design of electrodes. We evaluated the safety of our novel approach by assaying the thermal stress effect of GET conditions and the biodistribution of a cytokine expressing plasmid. Transfection efficacy of different pulse parameters was determined using two reporter genes encoding for the green fluorescent protein (GFP) and the tdTomato fluorescent protein, respectively. GET was performed using non-invasive contact electrodes immediately after intradermal injection of plasmid DNA into mouse skin. Fluorescence imaging of transfected skin showed that a sophistication in the pulse parameters could be selected to get greater transfection efficacy in comparison to the standard ones. Delivery of electric pulses only mildly induced expression of the heat shock protein Hsp70 in a luminescent reporting transgenic mouse model, demonstrating that there were no drastic stress effects. The plasmid was not detected in other organs and was found only at the site of treatment for a limited period of time. In conclusion, we set up a novel approach for GET combining new electric field parameters with high voltage short pulses and medium voltage long pulses using contact electrodes, to obtain a high expression of both fluorescent reporter and therapeutic genes while showing full safety in living animals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-34968-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237991PMC
November 2018

STING-Dependent Interferon-λ1 Induction in HT29 Cells, a Human Colorectal Cancer Cell Line, After Gamma-Radiation.

Int J Radiat Oncol Biol Phys 2018 05 3;101(1):97-106. Epub 2018 Feb 3.

CRUK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, United Kingdom; Department of Oncology, University of Oxford, Oxford, United Kingdom. Electronic address:

Purpose: To investigate the induction of type III interferons (IFNs) in human cancer cells by gamma-rays.

Methods And Materials: Type III IFN expression in human cancer cell lines after gamma-ray irradiation in vitro was assessed by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Signaling pathways mediating type III IFN induction were examined by a variety of means, including immunoblotting, flow cytometry, confocal imaging, and reverse transcription-quantitative polymerase chain reaction. Key mediators in these pathways were further explored and validated using gene CRISPR knockout or short hairpin RNA knockdown.

Results: Exposure to gamma-rays directly induced type III IFNs (mainly IFNL1) in human cancer cell lines in dose- and time-dependent fashions. The induction of IFNL1 was primarily mediated by the cytosolic DNA sensors-STING-TBK1-IRF1 signaling axis, with a lesser contribution from the nuclear factor kappa b signaling in HT29 cells. In addition, type III IFN signaling through its receptors serves as a positive feedback loop, further enhancing IFN expression via up-regulation of the kinases in the STING-TBK1 signaling axis.

Conclusions: Our results suggest that IFNL1 can be up-regulated in human cancer cell lines after gamma-ray treatment. In HT29 cells this induction occurs via the STING pathway, adding another layer of complexity to the understanding of radiation-induced antitumor immunity, and may provide novel insights into IFN-based cancer treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijrobp.2018.01.091DOI Listing
May 2018

Increased permeability of blood vessels after reversible electroporation is facilitated by alterations in endothelial cell-to-cell junctions.

J Control Release 2018 04 21;276:30-41. Epub 2018 Feb 21.

Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, BP 64182, 205 Route de Narbonne, F-31077, France. Electronic address:

Delivery of electric field pulses, i.e. electroporation (EP), to tissues has been shown to have a blood flow modifying effect. Indeed, the diameter of blood vessels exposed to EP is immediately reduced resulting in blood flow abrogation, followed by an increase in vascular permeability. The main cause of the increased permeability remains unknown. The aim of this study was to determine whether the in vivo effects of EP on permeability of blood vessels are linked to the permeabilization of endothelial cells' membrane (EC) and/or disruption of cell-to-cell junctions. We used a dorsal window chamber model in C57Bl/6 mice coupled with multiphoton microscopy and fluorescently labelled antibodies against PECAM-1 (CD31) to visualize endothelial cell-to-cell junctions. Clinically validated EP parameters were used and behavior of cell-to-cell junctions, in combination with leakage of 70 kDa fluorescein isothiocyanate labelled dextran (FD), was followed in time. After EP, a constriction of blood vessels was observed and correlated with the change in the shape of ECs. This was followed by an increase in permeability of blood vessels for 70 kDa FD and a decrease in the volume of labelled cell-to-cell junctions. Both parameters returned to pre-treatment values in 50% of mice. For the remaining 50%, we hypothesize that disruption of cell-to-cell junctions after EP may trigger the platelet activation cascade. Our findings show for the first time in vivo that alterations in cell-to-cell junctions play an important role in the response of blood vessels to EP and explain their efficient permeabilization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jconrel.2018.02.032DOI Listing
April 2018

Segmentation of Vasculature From Fluorescently Labeled Endothelial Cells in Multi-Photon Microscopy Images.

IEEE Trans Med Imaging 2019 01 9;38(1):1-10. Epub 2017 Aug 9.

Vasculature is known to be of key biological significance, especially in the study of tumors. As such, considerable effort has been focused on the automated segmentation of vasculature in medical and pre-clinical images. The majority of vascular segmentation methods focus on bloodpool labeling methods; however, particularly, in the study of tumors, it is of particular interest to be able to visualize both the perfused and the non-perfused vasculature. Imaging vasculature by highlighting the endothelium provides a way to separate the morphology of vasculature from the potentially confounding factor of perfusion. Here, we present a method for the segmentation of tumor vasculature in 3D fluorescence microscopic images using signals from the endothelial and surrounding cells. We show that our method can provide complete and semantically meaningful segmentations of complex vasculature using a supervoxel-Markov random field approach. We show that in terms of extracting meaningful segmentations of the vasculature, our method outperforms both state-of-the-art method, specific to these data, as well as more classical vasculature segmentation methods.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1109/TMI.2017.2725639DOI Listing
January 2019

Microvessel Chaste: An Open Library for Spatial Modeling of Vascularized Tissues.

Biophys J 2017 May;112(9):1767-1772

Department of Computer Science, University of Oxford, Oxford, United Kingdom.

Spatial models of vascularized tissues are widely used in computational physiology. We introduce a software library for composing multiscale, multiphysics models for applications including tumor growth, angiogenesis, osteogenesis, coronary perfusion, and oxygen delivery. Composition of such models is time consuming, with many researchers writing custom software. Recent advances in imaging have produced detailed three-dimensional (3D) datasets of vascularized tissues at the scale of individual cells. To fully exploit such data there is an increasing need for software that allows user-friendly composition of efficient, 3D models of vascularized tissues, and comparison of predictions with in vivo or in vitro experiments and alternative computational formulations. Microvessel Chaste can be used to build simulations of vessel growth and adaptation in response to mechanical and chemical stimuli; intra- and extravascular transport of nutrients, growth factors and drugs; and cell proliferation in complex 3D geometries. In addition, it can be used to develop custom software for integrating modeling with experimental data processing workflows, facilitated by a comprehensive Python interface to solvers implemented in C++. This article links to two reproducible example problems, showing how the library can be used to build simulations of tumor growth and angiogenesis with realistic vessel networks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2017.03.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425404PMC
May 2017

Neutrophils promote hepatic metastasis growth through fibroblast growth factor 2-dependent angiogenesis in mice.

Hepatology 2017 06 2;65(6):1920-1935. Epub 2017 May 2.

CRUK/MRC Oxford Institute for Radiation Oncology, Oxford, United Kingdom.

Hepatic metastases are amenable to ablation; however, many patients are not suitable candidates for such therapy and recurrence is common. The tumor microenvironment is known to be essential for metastatic growth, yet identification of plausible targets for cancer therapy in the microenvironment has proven elusive. We found that human colorectal cancer liver metastases and murine gastrointestinal experimental liver metastases are infiltrated by neutrophils. Plasticity in neutrophils has recently been shown to lead to both protumor and antitumor effects. Here, neutrophils promoted the growth of hepatic metastases, given that depletion of neutrophils in already established, experimental, murine liver metastases led to diminished metastatic growth. Decreased growth was associated with reductions in vascular density and branching suggestive of vessel normalization. Metastasis-associated neutrophils expressed substantially more fibroblast growth factor 2 (FGF2) than naïve neutrophils, indicating neutrophil polarization by the tumor microenvironment. Administration of FGF2 neutralizing antibody to mice bearing experimental liver metastases phenocopied neutrophil depletion by reducing liver metastatic colony growth, vascular density, and branching.

Conclusion: Here, we show, using FGF2 as an example, that identification of factors responsible for the protumoral effects of infiltrating myeloid cells can be used to target established liver metastases. Such therapies could be utilized to limit disease progression and potentiate the effects of standard ablative therapies. (Hepatology 2017;65:1920-1935).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.29088DOI Listing
June 2017

Predicting the Influence of Microvascular Structure On Tumor Response to Radiotherapy.

IEEE Trans Biomed Eng 2017 03 8;64(3):504-511. Epub 2016 Sep 8.

Mathematical Institute, University of Oxford.

Objective: The purpose of this study is to investigate how theoretical predictions of tumor response to radiotherapy (RT) depend on the morphology and spatial representation of the microvascular network.

Methods: A hybrid multiscale model, which couples a cellular automaton model of tumor growth with a model for oxygen transport from blood vessels, is used to predict the viable fraction of cells following one week of simulated RT. Both artificial and biologically derived three-dimensional (3-D) vessel networks of well vascularized tumors are considered and predictions compared with 2-D descriptions.

Results: For literature-derived values of the cellular oxygen consumption rate there is little difference in predicted viable fraction when 3-D network representations of biological or artificial vessel networks are employed. Different 2-D representations are shown to either over- or under-estimate viable fractions relative to the 3-D cases, with predictions based on point-wise descriptions shown to have greater sensitivity to vessel network morphology.

Conclusion: The predicted RT response is relatively insensitive to the morphology of the microvessel network when 3-D representations are adopted, however, sensitivity is greater in certain 2-D representations.

Significance: By using realistic 3-D vessel network geometries this study shows that real and artificial network descriptions and assumptions of spatially uniform oxygen distributions lead to similar RT response predictions in relatively small tissue volumes. This suggests that either a more detailed description of oxygen transport in the microvasculature is required or that the oxygen enhancement ratio used in the well known linear-quadratic RT response model is relatively insensitive to microvascular structure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1109/TBME.2016.2606563DOI Listing
March 2017

Estimating oxygen distribution from vasculature in three-dimensional tumour tissue.

J R Soc Interface 2016 Mar;13(116)

Cancer Research UK/MRC Oxford Institute for Radiation Oncology, Gray Laboratory, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford OX3 7DQ, UK.

Regions of tissue which are well oxygenated respond better to radiotherapy than hypoxic regions by up to a factor of three. If these volumes could be accurately estimated, then it might be possible to selectively boost dose to radio-resistant regions, a concept known as dose-painting. While imaging modalities such as 18F-fluoromisonidazole positron emission tomography (PET) allow identification of hypoxic regions, they are intrinsically limited by the physics of such systems to the millimetre domain, whereas tumour oxygenation is known to vary over a micrometre scale. Mathematical modelling of microscopic tumour oxygen distribution therefore has the potential to complement and enhance macroscopic information derived from PET. In this work, we develop a general method of estimating oxygen distribution in three dimensions from a source vessel map. The method is applied analytically to line sources and quasi-linear idealized line source maps, and also applied to full three-dimensional vessel distributions through a kernel method and compared with oxygen distribution in tumour sections. The model outlined is flexible and stable, and can readily be applied to estimating likely microscopic oxygen distribution from any source geometry. We also investigate the problem of reconstructing three-dimensional oxygen maps from histological and confocal two-dimensional sections, concluding that two-dimensional histological sections are generally inadequate representations of the three-dimensional oxygen distribution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1098/rsif.2016.0070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4843681PMC
March 2016

Nitroxoline impairs tumor progression in vitro and in vivo by regulating cathepsin B activity.

Oncotarget 2015 Aug;6(22):19027-42

Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia.

Cathepsin B is a ubiquitously expressed lysosomal cysteine protease that participates in protein turnover within lysosomes. However, its protein and activity levels have been shown to be increased in cancer. Cathepsin B endopeptidase activity is involved in the degradation of extracellular matrix, a process that promotes tumor invasion, metastasis and angiogenesis. Previously, we reported an established antibiotic nitroxoline as a potent and selective inhibitor of cathepsin B. In the present study, we elucidated its anti-tumor properties in in vitro and in vivo tumor models. Tumor and endothelial cell lines with high levels of active cathepsin B were selected for functional analysis of nitroxoline in vitro. Nitroxoline significantly reduced extracellular DQ-collagen IV degradation by all evaluated cancer cell lines using spectrofluorimetry. Nitroxoline also markedly decreased tumor cell invasion monitored in real time and reduced the invasive growth of multicellular tumor spheroids, used as a 3D in vitro model of tumor invasion. Additionally, endothelial tube formation was significantly reduced by nitroxoline in an in vitro angiogenesis assay. Finally, nitroxoline significantly abrogated tumor growth, angiogenesis and metastasis in vivo in LPB fibrosarcoma and MMTV-PyMT breast cancer mouse models. Overall, our results designate nitroxoline as a promising drug candidate for anti-cancer treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4662473PMC
http://dx.doi.org/10.18632/oncotarget.3699DOI Listing
August 2015

Modeling of microvascular permeability changes after electroporation.

PLoS One 2015 20;10(3):e0121370. Epub 2015 Mar 20.

University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Biocybernetics, Trzaska cesta 25, SI-1000 Ljubljana, Slovenia.

Vascular endothelium selectively controls the transport of plasma contents across the blood vessel wall. The principal objective of our preliminary study was to quantify the electroporation-induced increase in permeability of blood vessel wall for macromolecules, which do not normally extravasate from blood into skin interstitium in homeostatic conditions. Our study combines mathematical modeling (by employing pharmacokinetic and finite element modeling approach) with in vivo measurements (by intravital fluorescence microscopy). Extravasation of fluorescently labeled dextran molecules of two different sizes (70 kDa and 2000 kDa) following the application of electroporation pulses was investigated in order to simulate extravasation of therapeutic macromolecules with molecular weights comparable to molecular weight of particles such as antibodies and plasmid DNA. The increase in blood vessel permeability due to electroporation and corresponding transvascular transport was quantified by calculating the apparent diffusion coefficients for skin microvessel wall (D [μm2/s]) for both molecular sizes. The calculated apparent diffusion coefficients were D = 0.0086 μm2/s and D = 0.0045 μm2/s for 70 kDa and 2000 kDa dextran molecules, respectively. The results of our preliminary study have important implications in development of realistic mathematical models for prediction of extravasation and delivery of large therapeutic molecules to target tissues by means of electroporation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121370PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4368817PMC
February 2016

Endoglin silencing has significant antitumor effect on murine mammary adenocarcinoma mediated by vascular targeted effect.

Curr Gene Ther 2015 ;15(3):228-44

Institute of Oncology Ljubljana, Zaloska 2, 1000 Ljubljana, Slovenia.

New targets and therapeutic approaches for vascular targeted strategies in oncology are continuously explored. Endoglin, a co-receptor of TGF-β, is a known target, however, its silencing with vector-based RNA interference technology has not been evaluated yet. Therefore, in our study, we assembled plasmid DNA coding for shRNA against endoglin, and used gene electrotransfer as a delivery method to determine its antitumor and vascular targeted effects. In vitro and in vivo data provide evidence of vascular targeted effects of endoglin silencing. The vascular targeted action of endoglin silencing could be described as a result of two separated effect; antiangiogenic and vascular disrupting effect. This was first supported by in vitro data; predominantly by reduction of proliferation and tube formation of endothelial cells. In the TS/A murine mammary carcinoma model, in which the tumor cells do not express endoglin, reduced tumor growth and number of vessels were observed. Quick destruction of existing activated blood vessels at the site of tumor cells' injection and sustained growth of tumors afterwards was observed in tumors that were growing in dorsal window chamber by intravital microscopy. This observation supports both vascular disrupting and antiangiogenic action. In conclusion, the results of our study provide evidence of endoglin as a valid target for cancer therapy and support further development of plasmid shRNA delivery, which have prolonged antitumor effect, especially in combined schedules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1566523215666150126115501DOI Listing
January 2016

Mcam Silencing With RNA Interference Using Magnetofection has Antitumor Effect in Murine Melanoma.

Mol Ther Nucleic Acids 2014 Oct 28;3:e205. Epub 2014 Oct 28.

Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia.

The melanoma cell adhesion molecule (MCAM) is involved in melanoma development and its progression, including invasiveness, metastatic potential and angiogenesis. Therefore, MCAM represents a potential target for gene therapy of melanoma, whose expression could be hindered with posttranscriptional specific gene silencing with RNA interference technology. In this study, we constructed a plasmid DNA encoding short hairpin RNA against MCAM (pMCAM) to explore the antitumor and antiangiogenic effects. The experiments were performed in vitro on murine melanoma and endothelial cells, as well as in vivo on melanoma tumors in mice. The antiproliferative, antimigratory, antiangiogenic and antitumor effects were examined after gene therapy with pMCAM. Gene delivery was performed by magnetofection, and its efficacy compared to gene electrotransfer. Gene therapy with pMCAM has proved to be an effective approach in reducing the proliferation and migration of melanoma cells, as well as having antiangiogenic effect in endothelial cells and antitumor effect on melanoma tumors. Magnetofection as a developing nonviral gene delivery system was effective in the transfection of melanoma cells and tumors with pMCAM, but less efficient than gene electrotransfer in in vivo tumor gene therapy due to the lack of antiangiogenic effect after silencing Mcam by magnetofection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mtna.2014.56DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4217080PMC
October 2014

Inhibitor of endocytosis impairs gene electrotransfer to mouse muscle in vivo.

Bioelectrochemistry 2015 Jun 29;103:111-9. Epub 2014 Aug 29.

Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia; Department of Natural and Medical Subjects, Faculty of Health Sciences, University of Primorska, Polje 42, SI-6310 Izola, Slovenia. Electronic address:

Application of electric pulses (electroporation/electropermeabilization) is an effective method for gene transfer (i.e. gene electrotransfer (GET)) in vitro and in vivo. Currently, the mechanisms by which the DNA enters the cell are not yet fully understood. Experimental evidence is building up that endocytosis is the main mechanism by which the DNA, which is later expressed, enters the cell. Therefore the aim of our study was to elucidate whether inhibitors of endocytosis, methyl-β-cyclodextrin (MβCD), Concanavalin A (ConA) and Dynasore, can impair the transfection efficacy of GET in vitro in B16F1 murine melanoma and in vivo in m. tibialis cranialis in mice. We show that MβCD--general inhibitor of endocytosis--can almost prevent GET of EGFP-N1 plasmid in vitro, that ConA--inhibitor of clathrin mediated endocytosis--also abrogates GET but to a lesser extent, and when using Dynasore--reversible inhibitor of dynamin--there is no effect on GET efficacy, if endocytosis is blocked for only 5 min after GET. Moreover, MβCD also reduced GET efficacy in vivo in m. tibialis cranialis and this effect was long lasting. The results of this study show that endocytosis is probably the main mechanism of entrance of DNA after GET in vitro and also in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bioelechem.2014.08.020DOI Listing
June 2015