Mock-transfected Cells Research Articles

Inducible Caspase9-mediated suicide gene for MSC-based cancer gene therapy

Cellular therapies based on mesenchymal stromal/stem cells (MSC) are promising strategies in regenerative medicine and oncology. Despite encouraging results, there is still some level of concerns on inoculating MSC in cancer patients. To face this issue, one possibility resides in engineering MSC by incorporating a suicide gene in order to control their fate once infused. Strategies based on Herpes Simplex Virus Thymidine Kinase (HSV-TK) and the Cytosine Deaminase genes have been developed and more recently a novel suicide gene, namely, iCasp9, has been proposed. This approach is based on a variant of human Caspase9 that binds with high affinity to a synthetic, bioinert small molecule (AP20187) leading to cell death. Based on this technology so far marginally applied to MSC, we tested the suitability of iCasp9 suicide strategy in MSC to further increase their safety. MSC have been transfected by a lentiviral vector carrying iCasp9 gene and then tested for viability after AP20187 treatment in comparison with mock-transfected cells. Moreover, accounting our anti-tumor approaches based on MSC expressing potent anti-cancer ligand TNF-Related Apoptosis-Inducing Ligand (TRAIL), we generated adipose MSC co-expressing iCasp9 and TRAIL successfully targeting an aggressive sarcoma type. These data show that anti-cancer and suicide mechanisms can coexist without affecting cells performance and hampering the tumoricidal activity mediated by TRAIL. In conclusion, this study originally indicates the suitability of combining a MSC-based anti-cancer gene approach with iCasp9 demonstrating efficiency and specificity.

Immobilized fibrinogen activates human platelets through glycoprotein VI.

Glycoprotein VI, a major platelet activation receptor for collagen and fibrin, is considered a particularly promising, safe antithrombotic target. In this study, we show that human glycoprotein VI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen was abolished in platelets from glycoprotein VI- deficient patients suggesting that fibrinogen activates platelets through glycoprotein VI. While mouse platelets failed to spread on fibrinogen, human-glycoprotein VI-transgenic mouse platelets showed full spreading and increased Ca2+ signaling through the tyrosine kinase Syk. Direct binding of fibrinogen to human glycoprotein VI was shown by surface plasmon resonance and by increased adhesion to fibrinogen of human glycoprotein VI-transfected RBL-2H3 cells relative to mock-transfected cells. Blockade of human glycoprotein VI with the Fab of the monoclonal antibody 9O12 impaired platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human glycoprotein VI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow.

Expression of the desmosome-related molecule periplakin is associated with advanced stage and poor prognosis of esophageal squamous cell carcinoma

Background: Our previous report showed that periplakin (PPL), a member of the plakin family of proteins, was expressed in all the normal esophageal squamous cells, except the Ki67+ basal cell layer; however, PPL-negative cells were observed in esophageal squamous cell carcinoma (ESCC) tissue samples and the proportion of PPL-positive areas in tumors showed considerable variation. In this study, we analyzed the relationships between PPL expression in tumors and the clinicopathological features of ESCC. Methods: PPL expression was evaluated by immunostaining ESCC samples from 70 patients who underwent surgery and we classified the samples into PPL-negative and PPL-positive groups based on the proportion of PPL-positive areas in tumors, and the relationships among PPL expression, clinical features, and the ESCC prognosis were analyzed. ESCC cell line KYSE270 cells were stably transfected with the PPL expression vector and their growth was assessed by colony formation assay and subcutaneous xenografts in athymic mice. Results: As found in our previous study, decreased PPL staining intensity was observed in all of the cancer tissues analyzed, compared with that observed in paired normal esophageal mucosae; however, the PPL-positive group (the percentage of PPL-positive tumor cells was >20% on immunostaining) exhibited positive associations with the pT classification (larger primary tumor) (P=0.029), pN classification (lymph node metastasis) (P=0.0462), and advanced stages of cancer (P=0.0253). High PPL expression was observed in all of 26 lymph node metastases analyzed. Furthermore, patients with PPL-positive tumors showed poor postoperative prognosis compared to those with PPL-negative ones. Forced expression of PPL in the KYSE270 ESCC cell line induced a stratified structure and colony formation. In addition, PPL-transfected cells formed larger tumors in nude mice than mock-transfected cells, suggesting that relatively high PPL expression in tumors may facilitate cell-cell adhesion by desmosome formation and eventual cell growth in vivo. Conclusions: PPL expression was generally reduced in ESCC compared with paired non-cancer tissue; however, relatively high levels of PPL expression in tumors correlated with tumor progression, lymph node metastasis, advanced stage cancer, and a poor prognosis.

Reconstruction of Toll-like receptor 9-mediated responses in HEK-Blue hTLR9 cells by transfection of human macrophage scavenger receptor 1 gene

We used human Toll-like receptor 9 (hTLR9)-expressing HEK-Blue hTLR9 cells, which release secreted embryonic alkaline phosphatase (SEAP) upon response to CpG DNA, to evaluate the immunological properties of nucleic acid drug candidates. Our preliminary studies showed that phosphodiester CpG DNA hardly induced any SEAP secretion in HEK-Blue hTLR9 cells. In the current study, therefore, we developed HEK-Blue hTLR9 cells transduced with human macrophage scavenger receptor-1 (hMSR1), a cell-surface DNA receptor, and determined whether HEK-Blue hTLR9/hMSR1 cells respond to phosphorothioate (PS) CpG DNA and phosphodiester (PO) CpG DNA. We selected PS CpG2006, a single-stranded PO CpG DNA (ssCpG), and a tetrapod-like structured DNA (tetrapodna) containing ssCpG (tetraCpG) as model TLR9 ligands. Alexa Fluor 488-labeled ligands were used for flow cytometry. Unlike the mock-transfected HEK-Blue hTLR9 cells, the HEK-Blue hTLR9/hMSR1 cells efficiently took up all three CpG DNAs. SEAP release was almost proportional to the uptake. Treatment of HEK-Blue hTLR9/hMSR1 cells with an anti-hMSR1 antibody significantly reduced the uptake of ssCpG and tetraCpG. Collectively, reconstruction of TLR9-mediated responses to CpG DNA in HEK-Blue hTLR9 cells can be used to evaluate the toxicity of nucleic acid drug candidates with diverse physicochemical properties.

DNA hypermethyation and silencing of PITX1 correlated with advanced stage and poor postoperative prognosis of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is associated with the accumulation of genetic and epigenetic changes in the background mucosa. Dysregulated DNA methylation is known to lead to the inactivation of tumor suppressor genes and the activation of oncogenes. To identify the genes whose expression is perturbed by abnormal DNA methylation in ESCC, integrative transcriptomics by serial analysis of gene expression (SAGE) and methylome sequencing by methyl-DNA immunoprecipitation (MeDIP) analysis were performed. We found 159 genes with significantly decreased expression in ESCC compared to that in noncancerous esophageal mucosa. MeDIP-seq analysis identified hypermethylation in the promoter region of 56 of these genes. Using surgically resected tissues of 40 cases, we confirmed that the paired-like homeodomain 1 (PITX1) gene was hypermethylated in ESCC compared to that in normal tissues (P < 0.0001) by pyrosequencing. PITX1 overexpression in ESCC cell lines inhibited cell growth and colony formation, whereas PITX1 knockdown accelerated cell growth. A PITX1-transfected ESCC cell line, KYSE30, formed smaller tumors in nude mice than in mock-transfected cells. Hypermethylation of PITX1 was associated with tumor depth (P = 0.0011) and advanced tumor stage (P = 0.0052) and predicted poor survival in ESCC (hazard ratio, 0.1538; 95% confidence interval, 0.03159–0.7488; P = 0.0169). In this study, we found a novel tumor suppressor gene of ESCC, PITX1, which is silenced by DNA hypermethylation. Downregulation of PITX1 contributes to the growth and progression of ESCC. Hypermethylation of the PITX1 in ESCC correlated with tumor progression and advanced stage cancer, and may predict a poor prognosis.

Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins.

Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.

Cartilage oligomeric matrix protein promotes prostate cancer progression by enhancing invasion and disrupting intracellular calcium homeostasis.

Cartilage oligomeric matrix protein (COMP) was recently implicated in the progression of breast cancer. Immunostaining of 342 prostate cancer specimens in tissue microarrays showed that COMP expression is not breast cancer-specific but also occurs in prostate cancer. The expression of COMP in prostate cancer cells correlated with a more aggressive disease with faster recurrence. Subcutaneous xenografts in immunodeficient mice showed that the prostate cancer cell line DU145 overexpressing COMP formed larger tumors in vivo as compared to mock-transfected cells. Purified COMP bound to and enhanced the invasion of DU145 cells in vitro in an integrin-dependent manner. In addition, intracellular COMP expression interfered with cellular metabolism by causing a decreased level of oxidative phosphorylation with a concurrent upregulation of lactate production (Warburg effect). Further, expression of COMP protected cells from induction of apoptosis via several pathways. The effect of COMP on metabolism and apoptosis induction was dependent on the ability of COMP to disrupt intracellular Ca2+ signalling by preventing Ca2+ release from the endoplasmic reticulum. In conclusion, COMP is a potent driver of the progression of prostate cancer, acting in an anti-apoptotic fashion by interfering with the Ca2+ homeostasis of cancer cells.

Abstract P318: Signaling Pathways Triggered by the Mas Receptor

Mas receptor (MasR) is a class A Orphan G-protein-coupled receptor (GPCR). Although angiotensin-(1-7) [Ang-(1-7)] has been reported as its putative ligand, the intracellular signaling pathways activated by the MasR remain only partially characterized. In this study we examined MasR-dependent activation of G protein-mediated and ERK mediated signaling pathways. With that aim in human lung carcinoma A549 cells we evaluated Gq-coupled activity by monitoring changes in intracellular Ca 2+ levels after Ang(1-7) stimulation. Incubation with 100nM Ang-(1-7) failed to increase Ca 2+ levels either in endogenously expressing or MasR overexpressing A549 cells. On the other hand, transfection of HEK293T cells with a wild-type MasR (wt-MasR) construct resulted in a significant decrease in basal cAMP levels (p&lt;0.05) that depended on the amount of wt-MasR protein expressed but was not observed when increasing amounts of mutant MasR lacking the PDZ binding motif were expressed. Pretreatment of wt-MasR expressing cells with pertussis toxin restored basal cAMP levels (p&lt;0.05) whereas no effect was observed in mock-transfected cells. Also, cAMP production after forskolin stimulation was lower in cells expressing wt-MasR than in control cells. These results indicate a high level of constitutive receptor activity towards cAMP modulation involving Gαi-protein. Treatment with Ang-(1-7) increased p-ERK levels in a concentration-dependent manner in both wt-MasR and in mock-transfected HEK293T cells. In view of these results we are analyzing whether ERK activation is mediated through Ang-(1-7) binding to endogenously expressed receptors different from MasR. As MasR has been suggested to participate in cardiovascular and renal functions, comprehensive pharmacological characterization of MasR signaling is essential for developing clinical therapeutics targeting MasR function.

Abstract 5756: Effects of Epstein-Barr virus latent membrane protein 1 (LMP1) on cell invasiveness and expression of endogenous microRNAs in human cells in vitro

Abstract Infection by the Epstein-Barr virus (EBV) is associated with a variety of human cancers, notably Burkitt lymphoma and nasopharyngeal carcinoma (NPC). Most malignant cells within EBV-associated cancers are latently infected, and some viral products expressed during the latent phase of the viral cycle have well-described carcinogenicity, including the EBV latent membrane protein 1 (LMP1). Distinct viral genotypes described so far include the B95.8, the most studied viral strain originally found in an elderly patient with transfusion-induced infectious mononucleosis, and the M81 strain, isolated from a NPC case from a Chinese patient. Compared to B95.8, the EBV M81 strain infects epithelial cells more readily, and it is more likely to induce spontaneous viral lytic reactivation. However, whether these EBV strains have distinct biological features relevant for the progression of EBV-associated cancers remains to be elucidated. Thus, this study aimed to evaluate whether the expression of B95.1 and M81 variants of EBV LMP1 have different impact on cell migration and invasiveness of human cells lines in vitro, as well as on the expression of selected endogenous microRNAs (miRs) that were previously reported to have a role on cancer progression. Expression of EBV LMP1 was achieved in 293 and NP69 cells transfected with vectors encoding its B95.8 or M81 variants. In vitro migration and invasion rates were assessed by the scratch wound healing assay and the Boyden chamber assay, respectively. The expression of 91 selected miRs were evaluated by a customized qPCR-array approach. Cells expressing EBV LMP1 showed increased migration and invasion rates compared with mock-transfected cells. However, the differences observed between B95.8 and M81 variants did not achieve statistical significance. Conversely, 293 cells expressing the M81 variant of LMP1 showed hyperexpression of human miRs 497-5p, 17-3p, and 34a-5p, compared to B95.8. The in silico target prediction for these miRs showed enrichment of gene targets involved in the MAPK, NF-κB, and PI3K/AKT intracellular signaling pathways. This would reflect in negative regulation of cell proliferation and cell motility/adhesion, as well as higher levels of apoptosis. Based on these data, it is plausible to suggest that EBV LMP1 from M81 viral strain is somewhat less effective in terms of carcinogenicity, and this might be a reflect of the improved ability of EBV M81 to induce lytic viral infection compared to the B95.8 strain, as previously reported. In conclusion, the results indicate that cells expressing LMP1 from EBV isolates B95.8 and M81 showed differences in terms of miR expression signatures, which ultimately might impact the pathogenesis and progression of EBV-associated cancers. Citation Format: Bárbara Grasiele Müller-Coan, Ethel Cesarman, Deilson Elgui De Oliveira. Effects of Epstein-Barr virus latent membrane protein 1 (LMP1) on cell invasiveness and expression of endogenous microRNAs in human cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5756. doi:10.1158/1538-7445.AM2017-5756

Novel variant of OCT4B4 is differentially expressed in human embryonic stem and embryonic carcinoma cells

POU domain proteins are an important family of transcription factors that regulates cell type-specific gene expression. One of the most crucial members of this family that maintains pluripotency and self-renewal of embryonic stem cells is POU5F1/OCT4. The OCT4 gene can generate several variants under different situations/cell types includes OCT4A that is the major factor sustains pluripotency in embryonic stem and embryonic carcinoma cells, and also OCT4B and OCT4B1, which are transcribed from a different potential promoter located in intron1 and are expressed in various tissues and cell types.In present study, during expression check of OCT4B1 in embryonic carcinoma cells (NT2), we discovered a novel OCT4 transcript for the first time and designated it as OCT4B4. This variant is expressed in various human pluripotent cells and its expression is down-regulated upon induction of differentiation. Moreover, knocking down of OCT4B4 by shRNA resulted in increased accumulation of transfected cells in G0/G1 phase compared to the mock-transfected control cells.

Mitochondrial ND5 mutation mediated elevated ROS regulates apoptotic pathway epigenetically in a P53 dependent manner for generating pro-cancerous phenotypes

We have previously observed concomitant events of mutations in mitochondrial and nuclear genes, along with elevated reactive oxygen species (ROS) and differential methylation within the promoters of nuclear genes in tumors and in vitro experiments of tumorigenesis. These observations have made it pertinent to replicate and understand the role of acquired mitochondrial condition in tuning a cell to accomplish a pro-cancerous state. Using a codon optimized vector system for exogenous over-expression and mitochondrial localization; we have characterized here the role of over-expressed wild type mtND5 and one of its non-synonymous somatic mutation, ND5:P265H. The ectopically over-expressed ND5:P265H in mitochondria resulted in a reduced Complex I activity, generation of higher ADP/ATP ratio, reactive oxygen species (ROS) and carbonylation of proteins as compared to mock-transfected cells. Cells over-expressing mtND5 variant produced both peroxide as well as super-oxide ROS; the generation of which was dependent on the functional status of P53; modulating epigenetically the expression of key apoptosis pathway genes. The pro-cancerous phenotypes, of anchorage dependent and independent growth; increased glucose uptake and lactate production, were selectively observed only in P53 non-functional cells over-expressing mutant ND5:P265H. We propose that somatic mutation in mtND5 resulting in down-regulated complex I enzyme activity, elevated ROS and up-regulation of a set of nuclear anti-apoptotic genes epigenetically in the P53 dysfunctional cellular background, has provided a unique understanding of the molecular mechanism of mitochondrial mutation; and the concomitant existence of somatically acquired mitochondrial and nuclear p53 mutations, in cancer progression and promotion.

Human SR-BII mediates SAA uptake and contributes to SAA pro-inflammatory signaling in vitro and in vivo

Serum amyloid A (SAA) is an acute phase protein with cytokine-like and chemotactic properties, that is markedly up-regulated during various inflammatory conditions. Several receptors, including FPRL-1, TLR2, TLR4, RAGE, class B scavenger receptors, SR-BI and CD36, have been identified as SAA receptors. This study provides new evidence that SR-BII, splice variant of SR-BI, could function as an SAA receptor mediating its uptake and pro-inflammatory signaling. The uptake of Alexa Fluor488 SAA was markedly (~3 fold) increased in hSR-BII-expressing HeLa cells when compared with mock-transfected cells. The levels of SAA-induced interleukin-8 secretion by hSR-BII-expressing HEK293 cells were also significantly (~3–3.5 fold) higher than those detected in control cells. Moderately enhanced levels of phosphorylation of all three mitogen-activated protein kinases, ERK1/2, and p38 and JNK, were observed in hSR-BII-expressing cells following SAA stimulation when compared with control wild type cells. Transgenic mice with pLiv-11-directed liver/kidney overexpression of hSR-BI or hSR-BII were used to assess the in vivo role of each receptor in SAA-induced pro-inflammatory response in these organs. Six hours after intraperitoneal SAA injection both groups of transgenic mice demonstrated markedly higher (~2-5-fold) expression levels of inflammatory mediators in the liver and kidney compared to wild type mice. Histological examinations of hepatic and renal tissue from SAA-treated mice revealed moderate level of damage in the liver of both transgenic but not in the wild type mice. Activities of plasma transaminases, biomarkers of liver injury, were also moderately higher in hSR-B transgenic mice when compared to wild type mice. Our findings identify hSR-BII as a functional SAA receptor that mediates SAA uptake and contributes to its pro-inflammatory signaling via the MAPKs-mediated signaling pathways.

Regulation of intestinal myofibroblasts by KRas-mutated colorectal cancer cells through heparin-binding epidermal growth factor-like growth factor.

In colorectal cancer, gain-of-function mutations in KRas play a critical role in malignant transformation. Tumor growth in colorectal cancer is known to be promoted by the intestinal myofibroblasts (IMFs) that localize adjacent to the cancer cells, but the mechanisms of interaction between KRas-mutated cancer cells and the myofibroblasts remain unclear. Here, we investigated the effects of KRas-mutated cells on the behavior of myofibroblasts by using mouse primary IMFs and cells of an IMF cell line (LmcMF) and a mouse colon epithelial cell line (aMoC1). Conditioned medium (CM) was collected from aMoC1 cells overexpressing a control vector or KRasV12 vector (KRasV12-CM), and the effects of KRasV12-CM on IMFs were analyzed by performing proliferation assays, wound-healing assays, Boyden chamber assays, and western blotting. Whereas KRasV12-CM exerted little effect on the differentiation and proliferation of primary IMFs, the CM promoted migration of both primary IMFs and LmcMF cells. In KRasV12-overexpressing aMoC1 cells, mRNA expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was higher than in mock-transfected aMoC1 cells, and HB-EGF promoted the migration of primary IMFs and LmcMF cells. Moreover, KRasV12-CM-induced IMF migration was suppressed by dacomitinib, an inhibitor of HB-EGF receptors. Notably, in LmcMF cells, both KRasV12-CM and HB-EGF activated extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK), whereas KRasV12-CM-induced migration of IMFs was suppressed following treatment with either an ERK inhibitor (FR180204) or a JNK inhibitor (SP600125). These results suggest that HB-EGF secreted from KRas-mutated colorectal cancer cells promotes IMF migration through ERK and JNK activation, which, in turn, could support cancer progression.

VEPH1 expression decreases vascularisation in ovarian cancer xenografts and inhibits VEGFA and IL8 expression through inhibition of AKT activation

Background:VEPH1 is amplified in several cancers including ovarian but its impact on tumour progression is unknown. Previous work has shown that VEPH1 inhibits TGFβ signalling while its Drosophila ortholog increases tissue growth, raising the possibility that VEPH1 could impact tumour growth or progression.Methods:A CRISPR approach was used to disrupt VEPH1 expression in ovarian cancer ES-2 cells, while VEPH1-negative SKOV3 cells were stably transfected with VEPH1 cDNA. The impact of altered VEPH1 expression was assessed using in vitro and in vivo assays and mechanistic studies were performed in vitro.Results:VEPH1 expression in SKOV3 cells resulted in a reduced tumour growth rate associated with increased necrotic area, and decreased microvessel density and VEGF-A levels relative to tumours formed by mock-transfected cells. VEPH1 expression also decreased VEGFA and IL8 expression in SKOV3 cells and was associated with decreased activated AKT levels. These effects were not observed in ES-2 cells, which bear a BRAFV600E activating mutation that leads to constitutively increased IL8 and VEGFA expression.Conclusions:VEPH1 expression in SKOV3 ovarian cancer cells inhibits AKT activation to decrease VEGFA and IL8 expression, which leads to decreased tumour vascularisation and progression.

Mitigated NSAID-induced apoptotic and autophagic cell death with Smad7 overexpression.

Non-steroidal anti-inflammatory drugs damaged gastrointestinal mucosa in cyclooxygenase-dependent and -independent pathway, among which apopototic or autophagic cell death in gastrointestinal cells might be one of key cytotoxic mechanisms responsible for NSAID-induced damages. Therefore, alleviating this cell death after NSAIDs can be a rescuing strategy. In this study, we explored the role of Smad7 on NSAID-induced cytotoxicity in gastric epithelial cells. Using RGM1 cells, we have compared biological changes between mock-transfected and Smad7-overexpressed cells. As results, significantly decreased cytotoxicity accompanied with decreased levels of cleaved caspase-3 and poly (ADP-ribose) polymerase, Bax, and autophagic vesicles concurrent with decreased expressions of autophagy protein 5 and microtubule-associated protein light chain 3B-II were noted in Smad7-overexpressed cells with indomethacin administration compared to mock-transfected cells. Contrast to mitigated apoptotic execution, anti-apoptotic Bcl-2 and Beclin-1 were significantly increased in Smad7-overexpressed cells compared to mock-transfected cells. Smad7 siRNA significantly reversed these protective actions of Smad7 against indomethacin, in which p38 mitogen-activated protein kinase was significantly intervened. Furthermore, indomethacin-induced Smad7 degradation through ubiquitin-proteasome pathway was relevant to increased cytotoxicity, while chloroquine as autophagy inhibitor significantly attenuated indomethacin-induced cytotoxicity through Smad7 preservation via repressed ubiquitination. Conclusively, either genetic overexpression or pharmacological induction of Smad7 significantly attenuated indomethacin-induced gastric cell damages.

Real - time monitoring of circulation tumor cells in a surgical orthotopic implantation model of human metastatic prostate cancer

Objective To study the relationship of circulation tumor cells (CTCs) and metastatic process, we use subcutaneous model and surgical orthotopic implantation model of human metastatic prostate cancer and detected by in vivo flow cytometry (IVFC). Methods The human prostate cancer cell line PC3 cells were transduced with pLVX-EGFP-3FLAG lentivirus vector containing the green fluorescent protein (GFP) and puromycin resistance gene. PC3 cells, high-expression of GFP, were obtained by screening of stably transfected clones. PC3-GFP cells or unlabeled tumor cells were implanted into the right armpit of 10 nude mice to build up subcutaneous xenograft prostate cancer mode1, which provided tumor fragments to implant the orthotopic prostate tumor mode1. Tumor tissue with high fluorescent from the subcutaneous tumor was harvested and cut into pieces of 1 mm3, and grafted into the anterior prostates of 10 nude mice. The number of CTCs monitored by IVFC real-time detecting fluorescent signal noninvasively at 5 d, 10 d and 15 d after tumor established. Results A PC3-GFP cell line was established which had the same growth pattern as well as tumorigenesis ability as mock-transfected PC3 cells. IVFC detected 2.37, 6.25, 99.75 CTCs/h at 5, 10, and 15 days. Conclusion The orthotopic prostate cancer models are better to assess the morphology, growth, metastatic pattern and development characteristics of clinical disease. They are also more representative of a primary tumor with respect to tumor site and metastasis, especially CTCs dynamics. The in vivo flow cytometry technique combined with orthotopic tumor models might provide insights into tumor hematogenous metastasis and guidance to cancer therapy. Key words: Prostate cancer; Metastasis; Circulating tumor cells; In vivo flow cytometry

Brincidofovir Is Not a Substrate for the Human Organic Anion Transporter 1: A Mechanistic Explanation for the Lack of Nephrotoxicity Observed in Clinical Studies.

Brincidofovir (BCV) is an orally bioavailable lipid conjugate of cidofovir (CDV) with increased in vitro potency relative to CDV against all 5 families of double-stranded DNA viruses that cause human disease. After intravenous (IV) administration of CDV, the organic anion transporter 1 (OAT1) transports CDV from the blood into the renal proximal tubule epithelial cells with resulting dose-limiting nephrotoxicity. To study whether OAT1 transports BCV and to evaluate the pharmacokinetic and renal safety profile of oral BCV compared with IV CDV. The cellular uptake of BCV and its major metabolites was assessed in vitro. Renal function at baseline and during and after treatment in subjects in BCV clinical studies was examined. In OAT1-expressing cells, uptake of BCV and its 2 major metabolites (CMX103 and CMX064) was the same as in mock-transfected control cells and was not inhibited by the OAT inhibitor probenecid. In human pharmacokinetic studies, BCV administration at therapeutic doses resulted in detection of CDV as a circulating metabolite; peak CDV plasma concentrations after oral BCV administration in humans were <1% of those observed after IV CDV administration at therapeutic doses. Analysis of renal function and adverse events from 3 BCV clinical studies in immunocompromised adult and pediatric subjects indicated little to no evidence of associated nephrotoxicity. Over 80% of subjects who switched from CDV or foscarnet to BCV experienced an improvement in renal function as measured by maximum on-treatment estimated glomerular filtration rate. The lack of BCV uptake through OAT1, together with lower CDV concentrations after oral BCV compared with IV CDV administration, likely explains the superior renal safety profile observed in immunocompromised subjects receiving BCV compared with CDV.

Abstract B028: Blockade of the CD47 “Do not eat” signal by TTI-621 (SIRPαFc) leads to enhanced antitumor CD8+ T cell responses in vitro

Abstract High expression of the CD47 “do-not-eat” signal is a mechanism commonly used by tumor cells to escape phagocytosis by macrophages. CD47 transmits an inhibitory signal upon binding to its receptor signal-regulatory protein α (SIRPα) on the surface of macrophages. We have previously shown that TTI-621, a soluble SIRPαFc fusion protein, neutralizes the suppressive effects of CD47 and triggers macrophage-mediated phagocytosis of tumor cells in vitro, and effectively controls tumor growth in vivo. Using a human culture system, we investigated whether this increase in macrophage-mediated phagocytosis results in augmented T cell responses. To evaluate the T cell response to a model tumor antigen, Jurkat, a human leukemia cell line, was stably transfected with a construct containing the human cytomegalovirus phosphoprotein pp65 (CMV-Jurkat). Consistent with our previous studies, blockade of CD47 on CMV-Jurkat cells using TTI-621 led to a dramatic increase in tumor cell phagocytosis by primary macrophages derived from peripheral blood monocytes of healthy, HLA-A2+ donors. Using a high-affinity soluble TCR multimer that specifically recognizes an immunodominant epitope from pp65 complexed with HLA-A2, we found that TTI-621-mediated phagocytosis of CMV-Jurkat resulted in increased pp65 antigen presentation on the surface of macrophages. In cultures treated with a control Fc fragment, where macrophages only exhibited a low level of phagocytosis, no presentation of pp65 peptide could be detected. Moreover, mock-transfected tumor cells were efficiently phagocytosed in the presence of TTI-621, yet did not result in presentation of CMV pp65 peptide. To assess whether this increase in antigen presentation results in augmented T cell responses, macrophages from the phagocytosis assay were co-cultured with autologous CD8+ T cells for five days. We observed robust proliferation of CMV pp65-specific CD8+ T cells following co-culture with macrophages that had phagocytosed CMV-Jurkat in the presence of TTI-621 compared to control Fc treatment. In contrast, no proliferation of CMV pp65-specific CD8+ T cells occurred when macrophages had phagocytosed mock-transfected tumor cells, suggesting that the proliferation of CMV-specific CD8+ T cells is a tumor antigen-specific response. In addition, preliminary data indicate that primed CMV-specific CD8+ T cells are fully functional and are capable of exhibiting cytotoxicity against CMV-Jurkat. Collectively, our study demonstrates for the first time that in a human culture system, blockade of the CD47 “do not eat” signal results in increased phagocytosis, augmented tumor antigen presentation and enhanced anti-tumor CD8+ T cell responses. These results provide further support for the concept that CD47 lies at the intersection of the innate and adaptive immune systems. TTI-621, which is currently in a Phase I trial in patients with advanced hematological malignancies (NCT02663518), may thus be able to harness the anti-tumor activity of both macrophages and T cells. Citation Format: Natasja Nielsen Viller, Tran Truong, Emma Linderoth, Lisa D. Johnson, Stephane Viau, Gloria H. Y. Lin, Mark Wong, Xinli Pang, Penka S. Petrova, Robert A. Uger. Blockade of the CD47 “Do not eat” signal by TTI-621 (SIRPαFc) leads to enhanced antitumor CD8+ T cell responses in vitro [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B028.

Hepatic aberrant glycosylation by N-acetylglucosaminyltransferase V accelerates HDL assembly.

Glycosylation is involved in various pathophysiological conditions. N-Acetylglucosaminyltransferase V (GnT-V), catalyzing β1-6 branching in asparagine-linked oligosaccharides, is one of the most important glycosyltransferases involved in cancer and the immune system. Recent findings indicate that aberrant N-glycan structure can modify lipid metabolism. In this study, we investigated the effects of aberrant glycosylation by GnT-V on high-density lipoprotein cholesterol (HDL) assembly. We used GnT-V transgenic (Tg) mice and GnT-V Hep3B cell (human hepatoma cell line) transfectants. The study also included 96 patients who underwent medical health check-ups. Total serum cholesterol levels, particularly HDL-cholesterol (HDL-C) levels, were significantly increased in Tg vs. wild-type (WT) mice. Hepatic expression of apolipoprotein AI (ApoAI) and ATP-binding cassette subfamily A member 1 (ABCA1), two important factors in HDL assembly, were higher in Tg mice compared with WT mice. ApoAI and ABCA1 were also significantly elevated in GnT-V transfectants compared with mock-transfected cells. Moreover, ApoAI protein in the cultured media of GnT-V transfectants was significantly increased. Finally, we found a strong correlation between serum GnT-V activity and HDL-C concentration in human subjects. Multivariate logistic analyses demonstrated that GnT-V activity was an independent and significant determinant for serum HDL-C levels even adjusted with age and gender differences. Further analyses represented that serum GnT-V activity had strong correlation especially with the large-size HDL particle concentration. These findings indicate that enhanced hepatic GnT-V activity accelerated HDL assembly and could be a novel mechanism for HDL synthesis.

Autoantibodies against glutamate receptor δ2 after allogenic stem cell transplantation.

Objective:To report on a Caucasian patient who developed steroid-responsive transverse myelitis, graft vs host disease of the gut, and anti-GluRδ2 after allogenic stem cell transplantation.Methods:Histoimmunoprecipitation (HIP) with the patient's serum and cryosections of rat and porcine cerebellum followed by mass spectrometry was used to identify the autoantigen. Correct identification was verified by indirect immunofluorescence using recombinant GluRδ2 expressed in HEK293 cells.Results:The patient's serum produced a granular staining of the cerebellar molecular layer (immunoglobulin G1 and immunoglobulin G3; endpoint titer: 1:1,000) but did not react with other CNS tissues or 28 established recombinant neural autoantigens. HIP revealed a unique protein band at ∼110 kDa that was identified as GluRδ2. The patient's serum also stained GluRδ2 transfected but not mock-transfected HEK293 cells. Control sera from 38 patients with multiple sclerosis, 85 patients with other neural autoantibodies, and 205 healthy blood donors were negative for anti-GluRδ2. Preadsorption with lysate from HEK293-GluRδ2 neutralized the patient's tissue reaction whereas control lysate had no effect. In addition to anti-GluRδ2, the patient's serum contained immunoglobulin G autoantibodies against the pancreatic glycoprotein CUZD1, which are known to be markers of Crohn disease.Conclusions:In the present case, the development of anti-GluRδ2 was associated with transverse myelitis, which was supposedly triggered by the stem cell transplantation. Similar to encephalitis in conjunction with anti-GluRδ2 reported in a few Japanese patients, the patient's neurologic symptoms ameliorated after steroid therapy.