scholarly journals Partial Sperm beta1 Integrin Subunit Deletion Proves Its Involvement in Mouse Gamete Adhesion/Fusion

2020 ◽  
Vol 21 (22) ◽  
pp. 8494
Author(s):  
Virginie Barraud-Lange ◽  
Côme Ialy-Radio ◽  
Céline Chalas ◽  
Isabelle Holtzmann ◽  
Jean-Philippe Wolf ◽  
...  
Keyword(s):  
Western Blot ◽  
Germ Cells ◽  
Blot Analysis ◽  
Integrin Subunit ◽  
Perivitelline Space ◽  
Male Germ Cells ◽  
Beta1 Integrin ◽  
First Time

We have previously shown, using antibodies, that the sperm alpha6beta1 integrin is involved in mouse gamete fusion in vitro. Here we report the conditional knockdown of the sperm Itgb1 gene. It induced a drastic failure of sperm fusogenic ability with sperm accumulation in the perivitelline space of in vitro inseminated oocytes deleted or not for the Itgb1 gene. These data demonstrate that sperm, but not oocyte, beta1 integrin subunit is involved in gamete adhesion/fusion. Curiously, knockdown males were fertile in vivo probably because of the incomplete Cre-mediated deletion of the sperm Itgb1 floxed gene. Indeed, this was shown by Western blot analysis and confirmed by both the viability and litter size of pups obtained by mating partially sperm Itgb1 deleted males with females producing completely deleted Itgb1 oocytes. Because of the total peri-implantation lethality of Itgb1 deletion in mice, we assume that sperm that escaped the Itgb1 excision seemed to be preferentially used to fertilize in vivo. Here, we showed for the first time that the deletion, even partial, of the sperm Itgb1 gene makes the sperm unable to normally fertilize oocytes. However, to elucidate the question of the essentiality of its role during fertilization, further investigations using a mouse expressing a recombinase more effective in male germ cells are necessary.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Interfering With Hyaluronic Acid Metabolism Suppresses Glioma Cell Proliferation by Regulating Autophagy

2020 ◽  
Author(s):  
Tao Yan ◽  
Xin Chen ◽  
Hua Zhan ◽  
Penglei Yao ◽  
Ning Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hyaluronic Acid ◽  
Tumor Microenvironment ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cell ◽  
Glioma Cells

Abstract BackgroundThe tumor microenvironment plays an important role in tumor progression. Hyaluronic acid (HA), an important component of the extracellular matrix in the tumor microenvironment, abnormally accumulates in a variety of tumors. Whereas the role of abnormal HA metabolism in glioma remains unclear. MethodsThe expression level of hyaluronic acid (HA) was analyzed by ELISA assay and proteins such as HAS3, CD44, P62, LC3, CCND1 and CCNB1 were measured with Western blot analysis. The cell viability and proliferation were measured by MTT and KI67 immunofluorescence staining respectively. Autophagic vesicles and autophagosomes were quantified by transmission electron microscopy (TEM) and GFP-RFP-LC3 fluorescence analysis respectively. Cell cycle was analyzed by flowcytometry and Western blot analysis. Immunohistochemical (IHC) staining was used to detect expression levels of HA, Ki67, HAS3 and CD44 in human and mouse tumor tissues. Lentivirus constructed HAS3 and CD44 knockout stable glioma cells were transplanted to BALB/C nude mice for in vivo experiments. 4-Methylumbelliferone (4MU) was also used to treat glioma bearing mice for verifing its anti-tumor ability. The expression curve of HAS3, CD44 and the disease-free survival (DFS) curves for HAS3, CD44 in patients with LGG and GBM was performed based on TCGA database. ResultsAs shown in the present study, HA, hyaluronic acid synthase 3 (HAS3) and a receptor of HA named CD44 are expressed at high levels in human glioma tissues and negatively correlated with the prognosis of patients with glioma. Silencing HAS3 or blocking CD44 inhibited the proliferation of glioma cells in vitro and in vivo. The underlying mechanism was attributed to the inhibition of autophagy flux and further maintaining glioma cell cycle arrest in G1 phase. More importantly, 4-Methylumbelliferone (4-MU), a small competitive inhibitor of UDP with the ability to penetrate the blood-brain barrier (BBB), also inhibited the proliferation of glioma cells in vitro and in vivo. ConclusionApproaches that interfere with HA metabolism by altering the expression of HAS3 and CD44 and the administration of 4-MU potentially represent effective strategies for glioma treatment.

scholarly journals In Vitro and In Vivo Effects of Fermented Oyster-Derived Lactate on Exercise Endurance Indicators in Mice

2020 ◽  
Vol 17 (23) ◽  
pp. 8811
Author(s):  
Storm N. S. Reid ◽  
Joung-Hyun Park ◽  
Yunsook Kim ◽  
Yi Sub Kwak ◽  
Byeong Hwan Jeon
Keyword(s):  
Lactate Dehydrogenase ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Biochemical Analysis ◽  
Positive Control ◽  
Exercise Endurance

Exogenous lactate administration has more recently been investigated for its various prophylactic effects. Lactate derived from potential functional foods, such as fermented oyster extract (FO), may emerge as a practical and effective method of consuming exogenous lactate. The current study endeavored to ascertain whether the lactate derived from FO may act on muscle cell biology, and to what extent this may translate into physical fitness improvements. We examined the effects of FO in vitro and in vivo, on mouse C2C12 cells and exercise performance indicators in mice, respectively. In vitro, biochemical analysis was carried out to determine the effects of FO on lactate content and muscle cell energy metabolism, including adenosine triphosphate (ATP) activity. Western blot analysis was also utilized to measure the protein expression of total adenosine monophosphate-activated protein kinase (AMPK), p-AMPK (Thr172), lactate dehydrogenase (LDH), succinate dehydrogenase (SDHA) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in response to FO administration. Three experimental groups were formed: a positive control (PC) treated with 1% horse serum, FO10 treated with 10 μg/mL and FO50 treated with 50 μg/mL. In vivo, the effects of FO supplementation on exercise endurance were measured using the Rota-rod test, and Western blot analysis measured myosin heavy-chain 2 (MYH2) to assess skeletal muscle growth, alongside p-AMPK, total-AMPK, PGC-1α, cytochrome C and UCP3 protein expression. Biochemical analysis was also performed on muscle tissue to measure the changes in concentration of liver lactate, lactate dehydrogenase (LDH), glycogen and citrate. Five groups (n = 10/per group) consisted of a control group (CON), exercise group (Ex), positive control treated with Ex and 500 mg/kg Taurine (Ex-Tau), Ex and 100 mg/kg FO supplementation (Ex-FO100) and Ex and 200 mg/kg FO supplementation (Ex-FO200) orally administered over the 4-week experimental period.FO50 significantly increased PGC-1α expression (p < 0.001), whereas both FO10 and FO50 increased the expression of p-AMPK (p < 0.001), in C2C12 muscle cells, showing increased signaling important for mitochondrial metabolism and biogenesis. Muscle lactate levels were also significantly increased following FO10 (p < 0.05) and FO50 (p < 0.001). In vivo, muscle protein expression of p-AMPK (p < 0.05) and PGC-1α were increased, corroborating our in vitro results. Cytochrome C also significantly increased following FO200 intake. These results suggest that the effects of FO supplementation may manifest in a dose-response manner. FO administration, in vitro, and supplementation, in vivo, both demonstrate a potential for improvements in mitochondrial metabolism and biogenesis, and even for potentiating the adaptive effects of endurance exercise. Mechanistically, lactate may be an important molecule in explaining the aforementioned positive effects of FO.

scholarly journals Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress

2009 ◽  
Vol 296 (3) ◽  
pp. G499-G509 ◽  
Author(s):  
Mallikarjuna R. Metukuri ◽  
Donna Beer-Stolz ◽  
Rajaie A. Namas ◽  
Rajeev Dhupar ◽  
Andres Torres ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ischemia Reperfusion ◽  
Redox Stress ◽  
No Donor ◽  
Interacting Protein

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1–4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.

ADAMTS13 Binds VWF Via its C-Terminal CUB2 Domain.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 126-126 ◽  
Author(s):  
Weirui Zhang ◽  
David Motto ◽  
David Ginsburg
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Expression Vector ◽  
Full Length ◽  
Mammalian Expression ◽  
Partial Denaturation

Abstract Thrombotic thrombocytopenic purpura (TTP) is a life threatening illness due to a deficiency of the VWF-cleaving protease, ADAMTS13. The ADAMTS13 protein is composed of a propeptide, followed by a typical zinc metalloprotease domain. The C-terminal 2/3 of the molecule contains disintegrin-like, cystine-rich, and spacer domains, as well as a total of eight TSP1 motifs and two CUB domains. The function of this C-terminal portion of the molecule and its composite motifs is unknown, though TSP1 and CUB domains of other proteins have been shown to mediate protein-protein interactions. To further explore the interaction between ADAMTS13 and VWF, we cloned full length human cDNAs for both ADAMTS13 and VWF into the mammalian expression vector pcDNA3.1. These constructs were transiently transfected into 293T cells and COS cells respectively, and conditioned media collected for analysis. Using an anti-myc antibody, myc-tagged VWF co-immunoprecipitated (co-IP) with ADAMTS13, as demonstrated by western blot analysis using antisera raised against a C-terminal peptide derived from the predicted ADAMTS13 sequence. This direct interaction required partial denaturation of VWF in 1M urea, with no co-IP observed in the absence of urea. To map the segment within ADAMTS13 responsible for VWF binding, we cloned a series of overlapping ADAMTS13 fragments into the bacterial expression vector, Pet44b. Fusion proteins were purified by binding of the included His-tag to Ni-NTA beads and incubated with recombinant myc-VWF in the presence of 1M urea. Association with VWF was analyzed by co-IP with anti-myc followed by western blot analysis using an antibody to the C-terminal HSV-tag present in each fusion protein. The CUB2 (Glu1298- Thr1427) fusion protein co-IP’d with full-length VWF and also demonstrated concentration-dependent competition with full-length ADAMTS13 for VWF binding. In summary, we have demonstrated a direct protein-protein interaction between VWF and ADAMTS13. Binding requires partial denaturation of VWF and appears to be mediated primarily through contacts with the ADAMTS13 CUB2 domain. This interaction may account for the previously observed co-purification of VWF and ADAMTS13 from human plasma. Furthermore, the requirement for 1M urea suggests that this interaction may only occur physiologically under conditions of high shear. Though others have shown that the C-terminal domains of ADAMTS13, including CUB2, are not required for VWF cleavage in vitro, our data, together with several C-terminal mutations previously reported in TTP patients, suggest that interactions between VWF and the ADAMTS13 CUB2 domain may be important in vivo.

scholarly journals The Immunoexpression of FSH-R in the Ductuli Efferentes and the Epididymis of Men and Rat: Effect of FSH on the Morphology and Steroidogenic Activity of Rat Epididymal Epithelial Cells In Vitro

2010 ◽  
Vol 2010 ◽  
pp. 1-8 ◽  
Author(s):  
Małgorzata Świder-Al-Amawi ◽  
Agnieszka Kolasa ◽  
Andrzej Sikorski ◽  
Mariola Marchlewicz ◽  
Irena Baranowska-Bosiacka ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Sertoli Cells ◽  
Blot Analysis ◽  
Target Organ ◽  
Fsh Receptor ◽  
Ductuli Efferentes ◽  
Estradiol Synthesis ◽  
First Time

The Sertoli cells were regarded as the only target for FSH in male reproductive system. The expression of FSH receptor (FSH-R) was detected also in epithelial cells of the caput epididymis of rat and monkey. We showed in the immunohistochemistry study the expression of FSH-R in rat and human ductuli efferentes and the caput, corpus, and cauda epididymis, moreover, by Western blot analysis in the caput and cauda epididymis of rat. Additionally, we presented that the morphology of rat epididymal epithelial cells in vitro was affected by FSH, and FSH stimulation resulted in the increase of 17β-estradiol synthesis by rat caput epididymal cells in dose-depended manner. In conclusion, the identification of FSH receptors in human and rat epididymides supports our results that the epididymis is a target organ not only for LH but additionally for FSH. On the basis of the results we showed for the first time that morphology of epididymal epithelial cells and epididymal steroidogenesis can be regulated by FSH.

scholarly journals Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

2012 ◽  
Vol 14 (4) ◽  
pp. 574-579 ◽  
Author(s):  
Yong Zhu ◽  
Hong-Liang Hu ◽  
Peng Li ◽  
Shi Yang ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Ips Cells ◽  
In Vivo Study ◽  
Male Germ Cells ◽  
Induced Pluripotent

scholarly journals Bauhiniastatin-1 alleviates diet induced obesity and lipid accumulation through modulating PPAR-γ/AMPK expressions: In-vitro, in-vivo and in-silico studies.

2021 ◽  
Author(s):  
Karunakaran Reddy Sankaran ◽  
Lokanatha Oruganti ◽  
Muni Swamy Ganjayi ◽  
Venkataramaiah Chintha ◽  
Muni Kesavulu Muppuru ◽  
...  
Keyword(s):  
Body Weight ◽  
Weight Gain ◽  
Western Blot ◽  
Blot Analysis ◽  
Body Weight Gain ◽  
Liver Lipid ◽  
Ppar Γ ◽  
Diet Induced Obesity

Abstract Background: Consumption of energy dense foods and sedentary lifestyles have led to high prevalence of obesity and associated disorders. Intensive research efforts have focussed to develop effective alternative therapeutics from plant sources. Bauhiniastatins have been reported to possess antineoplastic activity. In the present study, Bauhiniastatin-1 (BSTN1) was isolated and purified from Bauhinia purpurea and evaluated for its therapeutic efficacy against adipogenesis and obesity using high fat diet (HFD)-induced obese rodent model and 3T3-L1 cells.Methods: We performed in-vitro experiments like MTT assay, Oil Red O (ORO) stain, cellular lipid content, glycerol release and RT-PCR analysis in 3T3-L1 cells. In-vivo parameters like body weight gain, body composition, plasma adipokines, serum & liver lipid profiles, liver marker enzymes, western blot analysis and histopathological examination were conducted in rat model. In addition, molecular docking studies were also performed to understand interaction of BSTN1 with peroxisome proliferator-activated gamma receptor (PPAR-γ) and AMP-activated protein kinase (AMPK) which supported our experimental results.Results: BSTN1 at 20 μM significantly (p<0.001) inhibited cell differentiation and lipid accumulation of 3T3-L1 adipocytes. Mechanistic studies showed that mRNA expression of key adipogenic markers, PPAR-γ, fatty acid synthase (FAS) and sterol-regulatory element-binding protein-1 (SREBP1) were down-regulated while AMPK was up-regulated by BSTN1. Oral administration of BSTN1 (5 mg/kg. b.wt.) to HFD-induced obese rats substantially decreased body weight gain, fat mass, serum and liver lipid levels and promoted integrity of hepatic and adipose tissue architecture compared to HFD-control rats. In BSTN1 administered groups, decreased serum aspartate transaminase (AST) and alanine aminotransferase (ALT) levels, decreased plasma leptin but increased adiponectin levels were noted. Western blot analysis of adipose and hepatic tissues collected from BSTN1 treated rats showed decreased expression level of PPAR-γ but increase in AMPK expression relative to the untreated group. In-silico studies showed strong binding interactions of BSTN1 against PPAR-γ and AMPK, the key molecules of adipogenesis and obesity.Conclusions: Taken together, the results suggest that BSTN1 could be promising molecule for the treatment of diet-induced obesity and non-alcoholic fatty liver disease (NAFLD).

Effect of trametinib in combination with panitumumab and trastuzumab on tumor growth in an orthotopic xenograft model of human pancreatic cancer.

2013 ◽  
Vol 31 (4_suppl) ◽  
pp. 190-190
Author(s):  
James M. Lindberg ◽  
Sara J Adair ◽  
Timothy E. Newhook ◽  
Alison Kim ◽  
J Thomas Parsons ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Growth ◽  
Western Blot ◽  
Growth Inhibition ◽  
Triple Therapy ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ras Pathway

190 Background: Aberrant MAPK and EGFR family signaling are key drivers of pancreatic ductal adenocarcinoma(PDAC). We hypothesized that combination trametinib(MEK1/2 inhibitor), panitumumab(EGFR inhibitor) and trastuzumab(Her2 inhibitor) would more effectively suppress tumor growth than any of these monotherapies. Methods: Patient-derived PDAC cell line MAD09-366 was exposed to trametinib, panitumumab, trastuzumab, and combination therapies in vitro. Western blot analysis was performed on treated cell lysates. Athymic, nude mice were orthotopically implanted with patient-derived PDAC xenografts(MAD09-366, 08-608, and 08-738). Established murine tumors were treated with control, trametinib (0.3mg/kg, qDay), panitumumab (500ug, BIW), trastuzumab (200ug, BIW) or in combination. MRI was used to assess tumor response. Results: Two of 3 PDACs were Kras mutant, 2 of 3 demonstrated increased Her2 activity, and all 3 showed increased EGFR activity. In vitro studies showed increased growth inhibition of triple-therapy-treated cells relative to control or each inhibitor alone. Western blot analysis revealed that EGF stimulation increased Ras pathway signaling in this Kras-mutant cell line. With EGF stimulation, the greatest Ras pathway signaling inhibition was seen in triple-therapy-treated cells. In vivo studies in all PDAC xenografts revealed that triple therapy significantly decreased tumor growth rate relative to control, trametinib alone, panitumumab alone, or panitumumab plus trastuzumab. In 2 of 3 PDACs assessed, triple therapy was superior to trametinib plus panitumumab. Average tumor size in MAD08-738 triple-therapy-treated mice decreased by 9.3%. Conclusions: Triple therapy with trametinib, panitumumab, and trastuzumab demonstrated the greatest in vitro Ras signaling blockade. In vivo, this combination produced significant tumor growth inhibition or regression in all PDAC tumors studied. This regimen should be considered for a future clinical trial in pancreatic cancer patients.

Potent In Vivo Anti-Tumor Activity Of Extracellular Vesicles Isolated From Genetically Engineered Primary Mesenchymal Stromal Cells Expressing The Trans-Membrane TNF-Related Apoptosis-Inducing Ligand (TRAIL)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1658-1658
Author(s):  
Stefano Buttiglieri ◽  
Carmelo Carlo-Stella ◽  
Tiziana Spatola ◽  
Roberta Pulito ◽  
Luigi Naldini ◽  
...  
Keyword(s):  
Western Blot ◽  
Tumor Cells ◽  
Stromal Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cytotoxic Effect ◽  
Genetically Engineered ◽  
Nsg Mice

Abstract Introduction TNF-related apoptosis-inducing ligand (TRAIL) is a protein functioning as a ligand that induces the process of cell death. TRAIL has been shown to kill in vitro a wide variety of tumor cells with minimal effects on normal cells. Despite its in vitro activity, recombinant soluble TRAIL has so far shown limited efficacy in vivo. In contrast, recent reports have shown that significant apoptosis can be observed both in vitro and in vivo when TRAIL is expressed on the cell membrane (mTRAIL). A further innovation might be the delivery of bioactive proapoptotic TRAIL through its expression by extracellular vescicles (EVs), the nanovesicular organelles secreted by cells. In fact, EVs are viewed as an effective tool for intercellular cross-talk and receptor discharge. The trans-membrane expression of TRAIL ligand within the double layer exosomal membrane may induce a more potent death signal when compared with the soluble molecule. Material and Methods Mesenchymal Stromal Cells (MSC) from bone marrow were cultured in vitro and used for EVs production. Cultured MSC in 75 cm2 flasks, at 80% confluence were infected with a lentivector encoding TRAIL, maintained in culture, and cell-supernatants repeatedly collected over several days, ultracentrifugated, with EVs-containing pellet harvested in PBS. EVs were produced also from uninfected MSC as control (EVs-CTRL). EVs were characterized by flow cytometry for expression of MSC markers and mTRAIL, EV size was evaluated by NanoSight technology. Total protein concentration was used to quantify EVs, Western Blot analysis was performed to characterize membrane-bound TRAIL. In vitro analysis was performed on SU-DHL-4 (human B cell lymphoma) and MEL-1300 (human melanoma) cell lines, exposed for 24 hours to 20-100 μg/ml EVs-TRAIL or EVs-CTRL. Annexin/propidium iodide assay was used to quantify apoptotic/necrotic cells. For the in vivo assessments, SU-DHL-4 and MEL-1300 cells were transduced with Luc-Lentiviral particles to obtain Luciferase positive cell lines. These cells were used to engraft NOD scid gamma (NSG) mice (2x106 SU-DHL-4 and 3x105 MEL-1300 cells for each subcutaneous injection point). To visualize tumor cells, mice were injected intraperitoneum with luciferin and analyzed with the Xenogen system. Mice bearing subcutaneous tumor nodules received single intravenous injections of 100, 200, 300 µg or multiple (x 3) 200 µg injections of either EVs-TRAIL or EVs-CTRL. Results FACS analysis showed strong TRAIL expression on EVs from TRAIL-infected MSC compared to EVs-CTRL, with a high proportion of positive particles (median 85%, range 78-93). In addition, EVs-TRAIL displayed MSC membrane markers, i.e. CD 105, CD 90, CD73 and CXCR4. Western Blot analysis under non-reducing conditions showed the presence of TRAIL ligand, with strong prevalence of dimeric TRAIL isoform (barely detectable the trimeric isoform, undetectable monomeric isoforms). NanoSight analysis revealed that EVs had a variable size, up to approximately 400 nm in diameter, with a predominant peak at 273 nm. A strong and dose-dependent cytotoxic effect was observed on SU-DHL-4 cells exposed to EVs-TRAIL (annexin/PI+ve cells: up to 87% for 100 μg/ml EVs-TRAIL), compared to EVs-CTRL exposure (15% Annexin/PI+ve cells for 100 μg/ml EVs-TRAIL). A similar, albeit less pronounced in vitro cytotoxic effect of EVs-TRAIL was observed on the melanoma MEL-1300 cell line. The anti-tumor effect was remarkably strong when EVs-TRAIL were injected in vivo in mice bearing either SU-DHL-4 or MEL-1300 nodules. A marked reduction of the tumor luminescence from 1.2x1010 photon/sec to <108 photon/sec was observed at seven days since a single EVs-TRAIL injection at 200 and 300 μg. Multiple administrations of 200 μg EVs-TRAIL induced the strongest luminescence reduction, as observed in MEL-1300 bearing NSG mice. Histological examination of nodules from EVs treated mice showed necrosis areas along with extensive intra-tumor vascular disruption. Conclusion EVs isolated from genetically engineered TRAIL-expressing MSC: i. do express mTRAIL; ii. display potent antitumor activity, inducing extensive apoptosis/necrosis both in vitro and in vivo in animal models bearing lymphoma and melanoma nodules. Thus, EVs-TRAIL may represent a promising strategy for delivering pro-apoptotic signals to tumor cells. Moreover, the Results could pave the way to the use of EVs for therapeutic purposes. Disclosures: No relevant conflicts of interest to declare.

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