U87 Tumor Research Articles

Maritoclax induces apoptosis in acute myeloid leukemia cells with elevated Mcl-1 expression

Acute myeloid leukemia (AML) is one of the deadliest leukemias for which there is an urgent and unmet need for the development of novel treatment strategies. Multiple drug resistance mechanisms mediate poor drug response and relapse in patients, and a selective Mcl-1 inhibitor has been speculated to be a promising agent in the treatment of AML. Here, we describe that maritoclax, a small molecule Mcl-1 inhibitor, induces Mcl-1 proteasomal degradation without transcriptional downregulation. Maritoclax killed AML cell lines and primary cells with elevated Mcl-1 levels through selective Mcl-1 downregulation, and synergized with ABT-737 to overcome Mcl-1-mediated ABT-737 resistance. Maritoclax was more effective than daunorubicin at inducing leukemic cell death when co-cultured with HS-5 bone marrow stroma cells, while being less toxic than daunorubicin against HS-5 stroma cells, primary mouse bone marrow cells, and hematopoietic progenitor cells. Moreover, maritoclax administration at 20 mg/kg/d intraperitoneally caused significant U937 tumor shrinkage, as well as 36% tumors remission rate in athymic nude mice, without apparent toxicity to healthy tissue or circulating blood cells. In summary, our studies suggest that maritoclax belongs to a novel class of Mcl-1 inhibitors that has the potential to be developed for the treatment of AML.

Effect of the STAT3 inhibitor STX-0119 on the proliferation of a temozolomide-resistant glioblastoma cell line.

Glioblastoma multiforme (GBM) is one of the most malignant and aggressive tumors and has a very poor prognosis, with a median survival time of less than 2 years. Once recurrence develops, there are few therapeutic approaches to control the growth of glioblastoma. In particular, temozolomide (TMZ)-resistant (TMZ-R) GBM is very difficult to treat, and a novel approach to overcome resistance is eagerly awaited. Previously, we reported a novel small molecule inhibitor of STAT3 dimerization, STX-0119, as a cancer therapeutic. In the current study, the efficacy of STX-0119 was evaluated against our established TMZ-resistant U87 cell line using quantitative PCR-based gene expression analysis, in vitro assay and animal experiments. The growth inhibitory effect of STX-0119 on U87 and TMZ-R U87 cells was moderate (IC₅₀, 34 and 45 µM, respectively). In particular, STX-0119 did not show significant inhibition of U87 tumor growth; however, it suppressed the growth of the TMZ-R U87 tumor in nude mice by more than 50%, and prolonged the median survival time compared to the control group. Quantitative PCR revealed that YKL-40, MAGEC1, MGMT, several EMT genes, mesenchymal genes and STAT3 target genes were upregulated, but most of those genes were downregulated by STX-0119 treatment. Furthermore, the invasive activity of TMZ-R U87 cells was significantly inhibited by STX-0119. YKL-40 levels in TMZ-R U87 cells and their supernatants were significantly decreased by STX-0119 administration. These results suggest that STX-0119 is an efficient therapeutic to overcome TMZ resistance in recurrent GBM tumors, and could be the next promising compound leading to survival prolongation, and YKL-40 may be a possible surrogate marker for STAT3 targeting.

Preclinical Pharmacological Evaluation of a Novel Multiple Kinase Inhibitor, ON123300, in Brain Tumor Models

ON123300 is a low molecular weight multikinase inhibitor identified through a series of screens that supported further analyses for brain tumor chemotherapy. Biochemical assays indicated that ON123300 was a strong inhibitor of Ark5 and CDK4, as well as growth factor receptor tyrosine kinases such as β-type platelet-derived growth factor receptor (PDGFRβ). ON123300 inhibited U87 glioma cell proliferation with an IC(50) 3.4 ± 0.1 μmol/L and reduced phosphorylation of Akt, yet it also unexpectedly induced Erk activation, both in a dose- and time-dependent manner that subsequently was attributed to relieving Akt-mediated C-Raf S259 inactivation and activating a p70S6K-initiated PI3K-negative feedback loop. Cotreatment with the EGFR inhibitor gefitinib produced synergistic cytotoxic effects. Pursuant to the in vitro studies, in vivo pharmacokinetic and pharmacodynamic studies of ON123300 were completed in mice bearing intracerebral U87 tumors following intravenous doses of 5 and 25 mg/kg alone, and also at the higher dose concurrently with gefitinib. ON123300 showed high brain and brain tumor accumulation based on brain partition coefficient values of at least 2.5. Consistent with the in vitro studies, single agent ON123300 caused a dose-dependent suppression of phosphorylation of Akt as well as activation of Erk in brain tumors, whereas addition of gefitinib to the ON123300 regimen significantly enhanced p-Akt inhibition and prevented Erk activation. In summary, ON123300 demonstrated favorable pharmacokinetic characteristics, and future development for brain tumor therapy would require use of combinations, such as gefitinib, that mitigate its Erk activation and enhance its activity.

Distribution of the phosphatidylinositol 3-kinase inhibitors Pictilisib (GDC-0941) and GNE-317 in U87 and GS2 intracranial glioblastoma models-assessment by matrix-assisted laser desorption ionization imaging.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and the limited available treatment options have not meaningfully impacted patient survival in the past decades. Such poor outcomes can be at least partly attributed to the inability of most drugs tested to cross the blood-brain barrier and reach all areas of the glioma. The objectives of these studies were to visualize and compare by matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry the brain and tumor distribution of the phosphatidylinositol 3-kinase (PI3K) inhibitors pictilisib (GDC-0941, 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine) and GNE-317 [5-(6-(3-methoxyoxetan-3-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-2-yl)pyrimidin-2-amine] in U87 and GS2 orthotopic models of GBM, models that exhibit differing blood-brain barrier characteristics. Following administration to tumor-bearing mice, pictilisib was readily detected within tumors of the contrast-enhancing U87 model whereas it was not located in tumors of the nonenhancing GS2 model. In both GBM models, pictilisib was not detected in the healthy brain. In contrast, GNE-317 was uniformly distributed throughout the brain in the U87 and GS2 models. MALDI imaging revealed also that the pictilisib signal varied regionally by up to 6-fold within the U87 tumors whereas GNE-317 intratumor levels were more homogeneous. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses of the nontumored half of the brain showed pictilisib had brain-to-plasma ratios lower than 0.03 whereas they were greater than 1 for GNE-317, in agreement with their brain penetration properties. These results in orthotopic models representing either the contrast-enhancing or invasive areas of GBM clearly demonstrate the need for whole-brain distribution to potentially achieve long-term efficacy in GBM.

The impact of a chlorotoxin-modified liposome system on receptor MMP-2 and the receptor-associated protein ClC-3

Currently, it is unknown whether a receptor-associated protein will be affected when a ligand modified delivery system interacts with its receptor. Besides, chlorotoxin (ClTx)-modified liposomes can target to glioma cells, but the target molecule is not clear: MMP-2, ClC-3 or both? Here a comparative study of ClTx-conjugated liposomes was conducted on two types of tumor cells: U87, a human glioma cell line with high expression of both MMP-2 and ClC-3, and A549, a human lung cancer cell line with expression of only MMP-2. ClTx-modified liposomes behaved similarly in these two cancer cells in terms of in vitro cell uptake, endocytosis pathway, intracellular trafficking and in vivo targeting efficacy, though the two tested cell lines were very different in ClC-3 expression. These results revealed that the targeted delivery of ClTx modified liposomes to U87 tumor was MMP-2-mediated and not correlated with the chloride channel ClC-3. On the other hand, ClTx modified on the liposomes did activate the receptor-associated protein ClC-3 via the binding with MMP-2, leading to the inhibition on cell migration and chloride currents. This is significant because cell migration is a key step in tumor metastasis. Interestingly, higher in vitro cellular uptake and lower in vivo tumor accumulation of liposomal systems was found in U87 compared to the A549 model, possibly due to the biological differences between in vitro and in vivo models. In general, ClTx-modified delivery systems may potentially target to tumors other than glioma that express a high level of MMP-2, and its effect on ClC-3 may help prevent tumor metastasis.

Synthesis and radiolabeling of 111In-core-cross linked polymeric micelle-octreotide for near-infrared fluoroscopy and single photon emission computed tomography imaging

The objective of this study was the development of a dual-modality imaging device, namely 111In-core-cross-linked polymeric micelle (CCPM)-octreotide, for neuroendocrine tumor detection, using near-infrared fluoroscopy (NIRF) and single photon emission computed tomography (SPECT). The tumor targeting ability of the 111In-labeled CCPM-octreotide was evaluated in a tumor mouse model. SPECT/CT, NIRF and gamma imaging results showed high tumor uptake of 111In-labeled CCPM-octreotide. In contrast, there was a much lower signal in the same mouse model injected with 111In-labeled CCPM. The high accumulation of 111In-labeled CCPM-octreotide in U87 tumor was reduced after co-injection with an excess amount of CCPM-octreotide. These results suggested CCPM-octreotide’s potential applications in tumor diagnosis, drug delivery and molecular imaging.

Thiophene-Based Compounds as Fluorescent Tags to Study Mesenchymal Stem Cell Uptake and Release of Taxanes

Human mesenchymal stem cells (hMSC) are multipotent cells that display the unique ability to home and engraft in tumor stroma. This remarkable tumor tropic property has generated a great deal of interest in many clinical settings. Recently, we showed that hMSC represent an excellent base for cell-mediated anticancer therapy since they are able to internalize paclitaxel (PTX) and to release it in an amount sufficient to inhibit tumor cell proliferation. In order to shed light on the dynamics of drug uptake and release, in the present paper we describe the synthesis of two novel thiophene-based fluorophore-paclitaxel conjugates, namely PTX-F32 and PTX-F35, as tools for in vitro drug tracking. We aimed to study the ability of these novel derivatives to be efficiently internalized by hMSC and, in a properly engineered coculture assay, to be released in the medium and taken up by tumor cells. In order to ensure better stability of the conjugates toward enzymatic hydrolysis, the selected oligothiophenes were connected to the taxol core at the C7 position through a carbamate linkage between PTX and the diamino linker. Antiproliferative experiments on both tumor cells and stromal cells clearly indicate that, in good correlation with the parent compound, cells are sensitive to nanomolar concentrations of the fluorescent conjugates. Moreover, in the coculture assay we were able to monitor, by fluorescence microscopy, PTX-F32 trafficking from hMSC toward glioblastoma U87 tumor cells. Our work paves the way for novel possibilities to perform extensive and high quality fluorescence-based analysis in order to better understand the cellular mechanisms involved in drug trafficking, such as microvescicle/exosome mediated release, in hMSC vehicle cells.

Mediterranien cuttlefish sepia officinalis squid ink is cytotoxic but does not inhibit Glioblastoma U87 tumor cells proliferation, with high nutritional values of edible viscera

This study aims to identify nutritional values of some viscera’s cuttlefish Sepia officinalis commonly edible in Tunisia (Nidamental gland, hepatopancreas, male gonad, squid ink) and to evaluate the role of cuttlefish ink, on viability and proliferation of human glioblastoma cells U87.Biochemical analysis shows that solid and liquid viscera are rich in water, in Protein and in total Ash. They contain less fat and Carbohydrates. Energy values are substantial. Sex and Cooking over low have no significant effect on these nutritional values. These results lead us to encourage the use of these marine co-products (generally rejected), not only for food but for prevent and reduce the incidence of certain diseases. In fact our results show also a cytotoxic effect of cuttlefish ink at increasing doses on human cells cancer Glioblastoma U87 for an incubation time of 5 hours with a concentration higher than 20μg/ml. The IC50 value is 25μg/ml which may be due to tyrosinase, responsible for the cephalopod ink toxic. Supernatant of cuttlefish ink Sepia offinalis does not block proliferation of cancer cells U87 during 4 days of incubation. Further analyses are warranted and necessary to substantiate and complete our findings such as the study of the effect of this ink in adhesion and migration of Glioblastoma cells U87.

Functional comparison of Notch ligands in tumour angiogenesis

IntroductionDelta-like 4 (DLL4) and Jagged1 (JAG1) are two key Notch ligands that are implicated in tumour angiogenesis. DLL4-Notch signalling inhibits sprouting but promotes tumour growth through better perfused vessels. In contrast to, very little is known about JAG1- Notch signalling in tumour angiogenesis and its influence on tumour growth and progression. ObjectiveTo study the functional difference between DLL4- and JAG1- Notch signalling in vitro and in vivo. MethodsEndothelial cells (ECs) were cultured on DLL4 or JAG1 coated plates and subjected to quantitative PCR to study the regulation of Notch target genes. Angiogenic assays were employed to study the functional effects of these two ligands in ECs. U87 cell lines over-expressing murine DLL4 (mDLL4) or murine JAG1 (mJAG1) were generated via lentiviral transduction. The growth of these cells in vitro or as subcutaneous tumours in mice was compared to that of control wild-type U87 cell line. Changes in vascular morphology, proliferation (MIB-1), necrosis (H&E), and markers of hypoxia (CA9) were then assessed. Results & DiscussionJAG1 was less potent than DLL4 in stimulation of Notch target genes in ECs. JAG1- and DLL4- Notch signaling had different effects on vessel formation. The growth of U87 mDLL4 and U87 mJAG1 cells was slower compared to wild-type U87 cell line in vitro, however mDLL4 and mJAG1 promoted U87 tumour growth compared to wild type control cells in vivo. Both ligands did not have any effect on U87 cell proliferation but significantly reduced tumoural necrosis compared to control tumours. U87 mDLL4 tumours were less hypoxic compared to control tumours. Interestingly, tumours over- expressing mDLL4 had less but larger vessels compared to control, whereas mJAG1 produced more yet functional vessels. ConclusionJAG1- and DLL4-Notch signalling have different effects on vessel formation, which impacted on the growth of the tumours in vivo.

Deciphering the Systems Biology of mTOR Inhibition by Integrative Transcriptome Analysis

The mTOR signaling plays an integral role in cellular homeostasis controlling the transition between the catabolic and anabolic states. Originally approved as immunosuppressive agents preventing allograft rejection, inhibitors of mTOR signaling have recently entered the arena of cancer therapy. Using rapamycin derivative (RAD001) as a prototype inhibitor, we aimed to systematically analyze the molecular mechanisms underlying the pleiotropic effects of mTOR signaling. Using proliferation- and clonogenic survival assays, a preferential sensitivity of microvascular endothelial cells (HDMVEC) followed by fibroblasts and U87 gliblastoma to RAD001 treatment was found. In contrast, lung- and prostate tumor cells demonstrated relative resistance against RAD001 treatment. In co-culture with fibroblasts, RAD001 exerted potent antiangiogenic effects by inhibiting endothelial cell tube formation. Further, RAD001 treatment efficiently prevented tumor growth in U87 tumor xenografts. Integrative transcriptome analysis was performed to decipher the molecular mechanism underlying RAD001 -induced anti-tumor and antiangiogenic effects. The predominant expression pattern was downregulation of genes after RAD001 treatment in all three sensitive cell types. Among the RAD001 downregulated genes, a transcriptional network was discovered enriched for genes related to angiogenesis processes and extracellular matrix remodeling, e.g., VEGF, HIF1A, CXCLs, IL6, FN, PAI-1 or NRP1. Of note, key components of PI3K upstream (PDK1) as well as mTORC2 downstream signaling (SGK1, NDRG) were downregulated by RAD001. Decreased expression of IMPDH and 139 common gene targets between mycophenolic acid and RAD001 suggested in part shared mechanisms underlying their antiangiogenic and immunosuppressive effects. In summary, key genetic participants governing anti-tumor and anti-angiogenic effects of mTOR inhibition were identified.

Histone Deacetylase Inhibitors with Enhanced Enzymatic Inhibition Effects and Potent in vitro and in vivo Antitumor Activities

In the present work, a series of small molecules were designed and synthesized based on structural optimization. A significant improvement in the enzyme inhibitory activity of these compounds was discovered. Moreover, the tested compounds have moderate preference for class I HDACs over HDAC6, as demonstrated by enzyme selectivity assays. In vitro antiproliferation assay results show that representative compounds can selectively inhibit the growth of non-solid lymphoma and leukemic cells such as U937, K562, and HL60. In the in vivo antitumor assay, (S)-4-(2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-phenylacetamido)-N-hydroxybenzamide (D17) showed better performance than SAHA in blocking U937 tumor growth. Western blot analysis revealed that representative molecules can block the function of both class I HDACs and HDAC6. More importantly, our western blot results reveal that the levels of some oncogenic proteins (p-Akt in the PI3K/AKT/mTOR signal pathway, c-Raf and p-Erk in the MAPK signal pathway) were dramatically down-regulated by our compounds in the U937 cell line rather than MDA-MB-231 cells. This distinction in cellular mechanism might be an important reason why the U937 cell line was found to more sensitive to our HDAC inhibitors than the MDA-MB-231 cell line.

Ruthenium(II)/4,6-dimethyl-2-mercaptopyrimidine complexes: Synthesis, characterization, X-ray structures and in vitro cytotoxicity activities on cancer cell lines

The complexes [RuCl(SpymMe2-N,S)2(NO)] (1) and [Ru(dppb)(SpymMe2-N,S)2] (2) (dppb=1,4-bis(diphenylphosphino)butane, SpymMe2=deprotonated 4,6-dimethyl-2-mercaptopyrimidine) were obtained and characterized by elemental analysis, spectroscopic techniques including X-ray crystallography, conductimetry and cyclic voltammetry. Energy Decomposition Analysis (EDA) calculation indicated strong intermolecular interaction between monomers of complex 1, as suggested by the 1H NMR spectrum of this complex, in chloroform solution. Additionally, preliminary tests were carried out, in order to evaluate the cytotoxicity activity on cancer cell lines of the new complexes, and the free ligands, against MDA-MB-231 (breast cancer), Hela (cervical cancer), U251 (glioma cancer) tumor cell lines and the V79 (Chinese lung fibroblast hamster) cell line. The best result was obtained for the complex 1 with IC50 value of 0.11±0.02, against MDA-MB-231 tumor cells.

Glioma-homing peptide with a cell-penetrating effect for targeting delivery with enhanced glioma localization, penetration and suppression of glioma growth

Tumor-targeted delivery systems are useful in enhancing drug delivery and increasing anti-tumor effects. Cell-penetrating peptides have been widely used for this purpose but have been hampered by the poor selectivity between neoplastic and non-neoplastic cells. As a peptide derived from interleukin-13, interleukin-13 peptide (IL-13p) is specifically targeted to IL13Rα2, a tumor-restricted receptor. More interestingly, IL-13p possesses cell-penetrating properties that can specifically enhance the uptake by tumor cells compared with endothelial cells. Thus, we anchored IL-13p onto nanoparticles (ILNPs) for glioma-targeting delivery. The uptake of ILNPs by U87 cells was higher than that of unmodified nanoparticles (NPs). However, there was no significant difference in the uptake by human umbilical vein endothelial cells. In addition, free IL-13p could also enhance the uptake of both NPs and ILNPs by U87 cells. Anchoring with IL-13p could enhance the penetration of particles into the core of spheroids. In vivo, the fluorescence intensity of ILNPs in tumors was 2.96-fold higher than that of NPs. The modification with IL-13p also significantly improved the speed and rate of penetration from vessels to tumor cells. The enhanced tumor localization of ILNPs was mostly attributable to the elevated tumor cell internalization of ILNPs, whereas most NPs were colocalized with microvessels or macrophages. Correspondingly, docetaxel-loaded NPs effectively suppressed the growth of subcutaneous U87 tumors. The average tumor volume of the ILNP group was only 31.4% that of the control, which was significantly smaller than that of the docetaxel and NP groups. In conclusion, the modification of IL-13p selectively enhanced tumor cell uptake, improved the penetration effect of NPs and improved the glioma localization ability, which led to a better tumor-suppression effect.

Dynamic Quantitative Intravital Imaging of Glioblastoma Progression Reveals a Lack of Correlation between Tumor Growth and Blood Vessel Density

The spatiotemporal and longitudinal monitoring of cellular processes occurring in tumors is critical for oncological research. We focused on glioblastoma multiforme (GBM), an untreatable highly vascularized brain tumor whose progression is thought to critically depend on the oxygen and metabolites supplied by blood vessels. We optimized protocols for orthotopic GBM grafting in mice that were able to recapitulate the biophysical constraints normally governing tumor progression and were suitable for intravital multiphoton microscopy. We repeatedly imaged tumor cells and blood vessels during GBM development. We established methods for quantitative correlative analyses of dynamic imaging data over wide fields in order to cover the entire tumor. We searched whether correlations existed between blood vessel density, tumor cell density and proliferation in control tumors. Extensive vascular remodeling and the formation of new vessels accompanied U87 tumor cell growth, but no strong correlation was found between local cell density and the extent of local blood vessel density irrespective of the tumor area or time points. The technique moreover proves useful for comparative analysis of mice subjected either to Bevacizumab anti-angiogenic treatment that targets VEGF or to AMD3100, an antagonist of CXCR4 receptor. Bevacizumab treatment massively reduced tumoral vessel densities but only transiently reduced U87 tumor growth rate. Again, there was no correlation between local blood vessel density and local cell density. Moreover, Bev applied only prior to tumor implantation inhibited tumor growth to the same extent as post-grafting treatment. AMD3100 achieved a potent inhibition of tumor growth without significant reduction in blood vessel density. These results indicate that in the brain, in this model, tumor growth can be sustained without an increase in blood vessel density and suggest that GBM growth is rather governed by stromal properties.

Dual-Modality Micro-Positron Emission Tomography/Computed Tomography and Near-Infrared Fluorescence Imaging of EphB4 in Orthotopic Glioblastoma Xenograft Models

In glioblastoma, EphB4 receptors, a member of the largest family of receptor tyrosine kinases, are overexpressed in both tumor cells and angiogenic blood vessels. The purpose of this study was to examine whether the EphB4-binding peptide TNYL-RAW labeled with both (64)Cu and near-infrared fluorescence dye Cy5.5 could be used as a molecular imaging agent for dual-modality positron emission tomography/computed tomography [PET/CT] and optical imaging of human glioblastoma in orthotopic brain tumor models. TNYL-RAW was conjugated to Cy5.5 and the radiometal chelator 1,4,7,10-tetraazadodecane-N,N',N″,N‴-tetraacetic acid. The conjugate was then labeled with (64)Cu for in vitro binding and in vivo dual μPET/CT and optical imaging studies in nude mice implanted with EphB4-expressing U251 and EphB4-negative U87 human glioblastoma cells. Tumors and brains were removed at the end of the imaging sessions for immunohistochemical staining and fluorescence microscopic examinations. μPET/CT and near-infrared optical imaging clearly showed specific uptake of the dual-labeled TNYL-RAW peptide in both U251 and U87 tumors in the brains of the nude mice after intravenous injection of the peptide. In U251 tumors, the Cy5.5-labeled peptide colocalized with both tumor blood vessels and tumor cells; in U87 tumors, the tracer colocalized only with tumor blood vessels, not with tumor cells. Dual-labeled EphB4-specific peptide could be used as a noninvasive molecular imaging agent for PET/CT and optical imaging of glioblastoma owing to its ability to bind to both EphB4-expressing angiogenic blood vessels and EphB4-expressing tumor cells.

Mapping the Extracellular and Membrane Proteome Associated with the Vasculature and the Stroma in the Embryo

In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments.

Properties and Evaluation of Quaternized Chitosan/Lipid Cation Polymeric Liposomes for Cancer-Targeted Gene Delivery

Development of high-stability and efficient nonviral vectors with low cytoxicity is important for targeted tumor gene therapy. In this study, cationic polymeric liposomes (CPLs), with similar lipid bilayer structure and high thermal stability, were prepared from polymeric surfactants of quaternized (carboxymethyl)chitosan with different carbon chains (dodecyl, tetradecyl, hexadecyl, and octadecyl). By comparing different factors that influence gene delivery, tetradecyl-quaternized (carboxymethy)chitosan (TQCMC) CPLs, with suitable size (184.4 ± 17.1 nm), ζ potentials (27.5 ± 4.9 mV), and productivity for synthesis TQCMC (weight yield 13.1%), were selected for gene transfection evaluation in various cancer cell lines. Although TQCMC CPLs have lower gene transfection efficiency compared with cationic liposomes (Lipofectamine 2000) in vitro, they displayed higher reporter gene delivery ability for cancer tissues (bearing U87 and SMMC-7721 tumors) in vivo after intravenous injection. TQCMC CPLs also have lower cell cytotoxicity and lower cytokine production or liver injury for BALB/c mice. We conclude that the CPLs are promising gene delivery systems that may be used to target various cancers.

Differential biodistribution of intravenously administered endothelial progenitor and cytotoxic T-cells in rat bearing orthotopic human glioma

BackgroundA major challenge in the development of cell based therapies for glioma is to deliver optimal number of cells (therapeutic dose) to the tumor. Imaging tools such as magnetic resonance imaging (MRI), optical imaging, positron emission tomography (PET) and single-photon emission computed tomography (SPECT) has been used in cell tracking and/or biodistribution studies. In this study, we evaluate the dynamic biodistribution of systemic injected labeled cells [human cord blood derived endothelial progenitor cells (EPCs) and cytotoxic T-cells (CTLs)] in rat glioma model with in vivo SPECT imaging.MethodsHuman cord blood EPCs, T-cells and CD14+ cells (monocytes/dendritic cells) were isolated using the MidiMACS system. CD14+ cells were converted to dendritic cells (DC) and also primed with U251 tumor cell line lysate. T-cells were co-cultured with irradiated primed DCs at 10:1 ratio to make CTLs. Both EPCs and CTLs were labeled with In-111-oxine at 37°C in serum free DMEM media. Glioma bearing animals were randomly assigned into three groups. In-111 labeled cells or In-111 oxine alone were injected through tail vein and SPECT imaging was performed on day 0, 1, and 3. In-111 oxine activity in various organs and tumor area was determined. Histochemical analysis was performed to further confirm the migration and homing of injected cells at the tumor site.ResultsEPCs and CTLs showed an In-111 labeling efficiency of 87.06 ± 7.75% and 70.8 ± 12.9% respectively. Initially cell migration was observed in lung following inravenous administration of In-111 labeled cells and decreased on day 1 and 3, which indicate re-distribution of labeled cells from lung to other organs. Relatively higher In-111 oxine activity was observed in tumor areas at 24 hours in animals received In-111 labeled cells (EPCs or CTLs). Histiological analysis revealed iron positive cells in and around the tumor area in animals that received labeled cells (CTLs and EPCs).ConclusionWe observed differential biodistribution of In-111-oxine labeled EPCs and CTLs in different organs and intracranial glioma. This study indicates In-111 oxine based SPECT imaging is an effective tool to study the biodistribution of therapeutically important cells.

SU‐F‐500‐10: Targeted Delivery of Microbeam Irradiation and Initial Mouse Brain Tumor Model Studies Using a Table Top MRT System

Purpose: Microbeam radiation therapy (MRT) is an experimental and preclinical radiotherapy method for cancer treatment that has been shown, in animal studies, to have the capability to selectively eradicate tumor without damaging normal tissue functions. The reliance of MRT on synchrotron radiation has prevented widespread research in this field and is a major roadblock for future clinical applications. Our goal is to develop a compact image‐guided MRT system based on high power distributed x‐ray source array and explore the radiobiology behind MRT using various mouse brain tumor models. Methods: We developed a novel protocol of combined MRI/x‐ray radiograph image‐guided MRT which enables irradiating two mice simultaneously. This protocol proceeds as follows: tumor localization with MRI, landmark identification with x‐ray projections, registration between the two images to determine the relative location of the tumor in the RT coordinate space, and treatment with the microbeam irradiator. We evaluated the targeting accuracy and tumor cell response of the protocol with U87 MG human glioma tumor bearing mice using γ‐H2AX staining. A survival study of U87 human glioma tumor bearing mice treated with three‐microbeam MRT (32Gy/beam) was carried out using this protocol. Results: The average targeting accuracy measured in the γ‐H2AX stained slices was about 280 μrn which is well below our beamwidth (350 μrn). In the survival study, U87 tumor mice treated using this MRT protocol showed an average survival time of 45 days, an extension of 6 days compared with 39 days in the control group. Conclusion: The initial tumor mice studies have demonstrated the feasibility of MRI/x‐ray radiograph guided MRT treatment in animal research. We believe this image‐guide MRT protocol could enable more efficient animal experiments with various brain tumor models to better understand MRT mechanisms.

The SOX2-Interactome in Brain Cancer Cells Identifies the Requirement of MSI2 and USP9X for the Growth of Brain Tumor Cells

Medulloblastomas and glioblastomas, the most common primary brain tumors in children and adults, respectively, are extremely difficult to treat. Efforts to identify novel proteins essential for the growth of these tumors may help to further our understanding of the biology of these tumors, as well as, identify targets for future therapies. The recent identification of multiple transcription factor-centric protein interaction landscapes in embryonic stem cells has identified numerous understudied proteins that are essential for the self-renewal of these stem cells. To identify novel proteins essential for the fate of brain tumor cells, we examined the protein interaction network of the transcription factor, SOX2, in medulloblastoma cells. For this purpose, Multidimensional Protein Identification Technology (MudPIT) identified >280 SOX2-associated proteins in the medulloblastoma cell line DAOY. To begin to understand the roles of SOX2-associated proteins in brain cancer, we focused on two SOX2-associated proteins, Musashi 2 (MSI2) and Ubiquitin Specific Protease 9x (USP9X). Recent studies have implicated MSI2, a putative RNA binding protein, and USP9X, a deubiquitinating enzyme, in several cancers, but not brain tumors. We demonstrate that knockdown of MSI2 significantly reduces the growth of DAOY cells as well as U87 and U118 glioblastoma cells. We also demonstrate that the knockdown of USP9X in DAOY, U87 and U118 brain tumor cells strongly reduces their growth. Together, our studies identify a large set of SOX2-associated proteins in DAOY medulloblastoma cells and identify two proteins, MSI2 and USP9X, that warrant further investigation to determine whether they are potential therapeutic targets for brain cancer.