Current Perspectives on In Vivo Noninvasive Tracking of Extracellular Vesicles with Molecular Imaging

Gangadaran, Prakash,Hong, Chae Moon,Ahn, Byeong-Cheol Hindawi Publishing Corporation 2017 BioMed research international Vol.2017 No.-

<P>Clinical and preclinical in vivo tracking of extracellular vesicles (EVs) are a crucial tool for the development and optimization of EV-based diagnosis and treatment. EVs have gained interest due to their unique properties that make them excellent candidates for biological applications. Noninvasive in vivo EV tracking has allowed marked progress towards elucidating the mechanisms and functions of EVs in real time in preclinical and clinical studies. In this review, we summarize several molecular imaging methods that deal with EVs derived from different cells, which have allowed investigations of EV biodistribution, as well as their tracking, delivery, and tumor targeting, to determine their physiological functions and to exploit imaging-derived information for EV-based theranostics.</P>

Extracellular vesicles from mesenchymal stem cells activates VEGF receptors and accelerates recovery of hindlimb ischemia

Gangadaran, Prakash,Rajendran, Ramya Lakshmi,Lee, Ho Won,Kalimuthu, Senthilkumar,Hong, Chae Moon,Jeong, Shin Young,Lee, Sang-Woo,Lee, Jaetae,Ahn, Byeong-Cheol Elsevier Science Publishers 2017 Journal of controlled release Vol.264 No.-

<P><B>Abstract</B></P> <P>Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are potential therapies for various diseases, but their angiogenic mechanisms of therapeutic efficacy remain unclear. Here, we describe how MSC-EVs, activates VEGF receptors and downstream angiogenesis pathways. Mouse MSC-EVs were isolated from cell culture medium and characterized using transmission electron microscopy, nanoparticle analysis, and western blotting. <I>In vitro</I> migration, proliferation, and tube formation assays using endothelial cells were used to assess the angiogenic potential of MSC-EVs, and revealed higher levels of cellular migration, proliferation, and tube formation after treatment. qRT-PCR and western blotting (WB) revealed higher protein and mRNA expression of the angiogenic genes VEGFR1 and VEGFR2 in mouse SVEC-4 endothelial cells after MSC-EVs treatment. Additionally, other vital pro-angiogenic pathways (SRC, AKT, and ERK) were activated by <I>in vitro</I> MSC-EV treatment. WB and qRT-PCR revealed enriched presence of VEGF protein and miR-210-3p in MSC-EV. The hindlimb ischemia mouse model was established and MSC-EVs with or without Matrigel (EV-MSC+Gel) were injected into the ischemic area and blood reperfusion was monitored using molecular imaging techniques. The <I>in vivo</I> administration of MSC-EVs increased both blood reperfusion and the formation of new blood vessels in the ischemic limb, with the addition of matrigel enhancing this effect further by releasing EVs slowly. MSC-EVs enhance angiogenesis in ischemic limbs, most likely <I>via</I> the overexpression of VEGFR1 and VEGFR2 in endothelial cells. These findings reveal a novel mechanism of activating receptors by MSC-EVs influence the angiogenesis.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Treatment Effect of Combining Lenvatinib and Vemurafenib for BRAF Mutated Anaplastic Thyroid Cancer

홍채문,오지민,GANGADARAN PRAKASH,Ramya Lakshmi Rajendran,안병철 대한갑상선학회 2021 International Journal of Thyroidology Vol.14 No.2

Background and Objectives: Even though most of the thyroid cancer shows good prognosis, de-differentiatedthyroid cancer is still refractory to conventional treatments. Recently, kinase inhibitors including multi-kinase andBRAF inhibitors are widely used for treatment of de-differentiated thyroid cancers, but resistant to single kinaseinhibitor treatment eventually encountered. Therefore, combination therapy may have better therapeutic effectthan single therapy for thyroid cancer. In this study, we evaluated therapeutic effect of multi-kinase and BRAFinhibitor combination to anaplastic thyroid cancer cell lines with and without BRAF mutation. Materials andMethods: We used anaplastic thyroid cancer cell lines with BRAFV600E mutation (8505C) and with NRAS mutation(HTh7). Both cell lines were treated with various concentration of multi-kinase inhibitor (lenvatinib) and BRAFinhibitor (vemurafenib). And combination of various concentration of both kinase inhibitors were used to treatboth cell lines. Cytotoxic effect was assessed with cell counting kit-8 and therapeutic effect of single kinaseinhibitor therapy and the combination therapy was compared. Results: Anti-proliferative effect of vemurafenibon 8505C BRAFV600E-mutated cells was demonstrated from 0.25 μM concentration. However, HTh7 cells withNRAS mutation represented drug resistance up to 4 μM of vemurafenib. In case of lenvatinib treatment as amulti-kinase inhibitor, 8505C and HTh7 cells showed decreased cell viability dose-dependent manner. Combinationtreatment with vemurafenib and lenvatinib showed synergistic cytotoxic effect in BRAF mutated 8505C cell line,even at lower concentrations. Conclusion: Combination treatment with multi-kinase inhibitor and BRAF inhibitorshowed promising therapeutic results in BRAF mutated anaplastic thyroid cancer cell line.

Advances in Molecular Imaging Strategies for <i> In Vivo</i> Tracking of Immune Cells

Lee, Ho Won,Gangadaran, Prakash,Kalimuthu, Senthilkumar,Ahn, Byeong-Cheol Hindawi Publishing Corporation 2016 BioMed research international Vol.2016 No.-

<P>Tracking of immune cells<I> in vivo</I> is a crucial tool for development and optimization of cell-based therapy. Techniques for tracking immune cells have been applied widely for understanding the intrinsic behavior of immune cells and include non-radiation-based techniques such as optical imaging and magnetic resonance imaging (MRI), radiation-based techniques such as computerized tomography (CT), and nuclear imaging including single photon emission computerized tomography (SPECT) and positron emission tomography (PET). Each modality has its own strengths and limitations. To overcome the limitations of each modality, multimodal imaging techniques involving two or more imaging modalities are actively applied. Multimodal techniques allow integration of the strengths of individual modalities. In this review, we discuss the strengths and limitations of currently available preclinical<I> in vivo</I> immune cell tracking techniques and summarize the value of immune cell tracking in the development and optimization of immune cell therapy for various diseases.</P>

In Vivo Tracking of Chemokine Receptor CXCR4-Engineered Mesenchymal Stem Cell Migration by Optical Molecular Imaging

Kalimuthu, Senthilkumar,Oh, Ji Min,Gangadaran, Prakash,Zhu, Liya,Lee, Ho Won,Rajendran, Ramya Lakshmi,Baek, Se hwan,Jeon, Yong Hyun,Jeong, Shin Young,Lee, Sang-Woo,Lee, Jaetae,Ahn, Byeong-Cheol Hindawi 2017 Stem cells international Vol.2017 No.-

<P>CXCR4, the stromal cell-derived factor-1 receptor, plays an important role in the migration of hematopoietic progenitor/stem cells to injured and inflamed areas. Noninvasive cell tracking methods could be useful for monitoring cell fate. Therefore, in this study, we evaluated the efficacy of an intravenous infusion of genetically engineered mesenchymal stem cells (MSCs) overexpressing CXC chemokine receptor 4 (CXCR4) to home to the tumor, by optical imaging. We constructed a retroviral vector containing CXCR with dual reporter genes, <I>eGFP</I> and <I>Fluc2</I>, under the control of an EF1<I>α</I> promoter (pBABE-EF1<I>α</I>-CXCR4-eGFP-IRES-Fluc2). We also developed an eGFP-Fluc2 construct in the Retro-X retroviral vector (Retro-X-eGFP-Fluc2). MSCs were transduced with retroviruses to generate CXCR4-overexpressing MSCs (MSC-CXCR4/Fluc2) and MSCs (MSC/Fluc2). CXCR4 mRNA and protein expression was confirmed by RT-PCR and Western blotting, respectively, and it was higher in MSC-CXCR4/Fluc2 than in naive MSCs. eGFP expression was confirmed by confocal microscopy. The transfected MSC-CXCR4/Fluc2 cells showed higher migratory capacity than naive MSCs observed in Transwell migration assay. The in vivo migration of CXCR4-overexpressing MSCs to MDAMB231/Rluc tumor model by BLI imaging was also confirmed. Intravenous delivery of genetically modified MSCs overexpressing CXCR4 with a <I>Fluc2</I> reporter gene may be a useful, noninvasive BLI imaging tool for tracking cell fate.</P>

Regulated Mesenchymal Stem Cells Mediated Colon Cancer Therapy Assessed by Reporter Gene Based Optical Imaging

Kalimuthu, Senthilkumar,Zhu, Liya,Oh, Ji Min,Lee, Ho Won,Gangadaran, Prakash,Rajendran, Ramya Lakshmi,Baek, Se Hwan,Jeon, Yong Hyun,Jeong, Shin Young,Lee, Sang-Woo,Lee, Jaetae,Ahn, Byeong-Cheol MDPI 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.4

<P>Colorectal cancer is the most common cancer in both men and women and the second most common cause of cancer-related deaths. Suicide gene-based therapy with suicide gene-transduced mesenchymal stem cells (MSCs) is a promising therapeutic strategy. A tetracycline-controlled Tet-On inducible system used to regulate gene expression may be a useful tool for gene-based therapies. The aim of this study was to develop therapeutic MSCs with a suicide gene that is induced by an artificial stimulus, to validate therapeutic gene expression, and to monitor the MSC therapy for colon cancer using optical molecular imaging. For our study, we designed the Tet-On system using a retroviral vector and developed a response plasmid RetroX-TRE (tetracycline response element) expressing a mutant form of herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Bone marrow-derived MSCs were transduced using a RetroX-Tet3G (Clontech, CA, USA) regulatory plasmid and RetroX-TRE-HSV1-sr39TK-eGFP-IRES-Fluc2, for a system with a Tet-On (MSC-Tet-TK/Fluc2 or MSC-Tet-TK) or without a Tet-On (MSC-TK/Fluc2 or MSC-TK) function. Suicide gene engineered MSCs were co-cultured with colon cancer cells (CT26/Rluc) in the presence of the prodrug ganciclovir (GCV) after stimulation with or without doxycycline (DOX). Treatment efficiency was monitored by assessing Rluc (CT26/Rluc) and Fluc (MSC-Tet-TK and MSC-TK) activity using optical imaging. The bystander effect of therapeutic MSCs was confirmed in CT26/Rluc cells after GCV treatment. Rluc activity in CT26/Rluc cells decreased significantly with GCV treatment of DOX(+) cells (<I>p</I> < 0.05 and 0.01) whereas no significant changes were observed in DOX(−) cells. In addition, Fluc activity in also decreased significantly with DOX(+) MSC-Tet-TK cells, but no signal was observed in DOX(−) cells. In addition, an MSC-TK bystander effect was also confirmed. We assessed therapy with this system in a colon cancer xenograft model (CT26/Rluc). We successfully transduced cells and developed a Tet-On system with the suicide gene HSV1-sr39TK. Our results confirmed the therapeutic efficiency of a suicide gene with the Tet-On system for colon cancer. In addition, our results provide an innovative therapeutic approach using the Tet-On system to eradicate tumors by administration of MSC-Tet-TK cells with DOX and GCV.</P>