HDAC3 genetic and pharmacologic inhibition radiosensitizes fusion positive rhabdomyosarcoma by promoting DNA double-strand breaks.

Radiotherapy (RT) plays a critical role in the management of rhabdomyosarcoma (RMS), the prevalent soft tissue sarcoma in childhood. The high risk PAX3-FOXO1 fusion-positive subtype (FP-RMS) is often resistant to RT. We have recently demonstrated that inhibition of class-I histone deacetylases (HDACs) radiosensitizes FP-RMS both in vitro and in vivo. However, HDAC inhibitors exhibited limited success on solid tumors in human clinical trials, at least in part due to the presence of off-target effects. Hence, identifying specific HDAC isoforms that can be targeted to radiosensitize FP-RMS is imperative. We, here, found that only HDAC3 silencing, among all class-I HDACs screened by siRNA, radiosensitizes FP-RMS cells by inhibiting colony formation. Thus, we dissected the effects of HDAC3 depletion using CRISPR/Cas9-dependent HDAC3 knock-out (KO) in FP-RMS cells, which resulted in Endoplasmatic Reticulum Stress activation, ERK inactivation, PARP1- and caspase-dependent apoptosis and reduced stemness when combined with irradiation compared to single treatments. HDAC3 loss-of-function increased DNA damage in irradiated cells augmenting H2AX phosphorylation and DNA double-strand breaks (DSBs) and counteracting irradiation-dependent activation of ATM and DNA-Pkcs as well as Rad51 protein induction. Moreover, HDAC3 depletion hampers FP-RMS tumor growth in vivo and maximally inhibits the growth of irradiated tumors compared to single approaches. We, then, developed a new HDAC3 inhibitor, MC4448, which showed specific cell anti-tumor effects and mirrors the radiosensitizing effects of HDAC3 depletion in vitro synergizing with ERKs inhibition. Overall, our findings dissect the pro-survival role of HDAC3 in FP-RMS and suggest HDAC3 genetic or pharmacologic inhibition as a new promising strategy to overcome radioresistance in this tumor.

Antitumour effects of SFX-01 molecule in combination with ionizing radiation in preclinical and in vivo models of rhabdomyosarcoma.

BACKGROUND: Despite a multimodal approach including surgery, chemo- and radiotherapy, the 5-year event-free survival rate for rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in childhood, remains very poor for metastatic patients, mainly due to the selection and proliferation of tumour cells driving resistance mechanisms. Personalised medicine-based protocols using new drugs or targeted therapies in combination with conventional treatments have the potential to enhance the therapeutic effects, while minimizing damage to healthy tissues in a wide range of human malignancies, with several clinical trials being started. In this study, we analysed, for the first time, the antitumour activity of SFX-01, a complex of synthetic d, l-sulforaphane stabilised in alpha-cyclodextrin (Evgen Pharma plc, UK), used as single agent and in combination with irradiation, in four preclinical models of alveolar and embryonal RMS. Indeed, SFX-01 has shown promise in preclinical studies for its ability to modulate cellular pathways involved in inflammation and oxidative stress that are essential to be controlled in cancer treatment. METHODS: RH30, RH4 (alveolar RMS), RD and JR1 (embryonal RMS) cell lines as well as mouse xenograft models of RMS were used to evaluate the biological and molecular effects induced by SFX-01 treatment. Flow cytometry and the modulation of key markers analysed by q-PCR and Western blot were used to assess cell proliferation, apoptosis, autophagy and production of intracellular reactive oxygen species (ROS) in RMS cells exposed to SFX-01. The ability to migrate and invade was also investigated with specific assays. The possible synergistic effects between SFX-01 and ionising radiation (IR) was studied in both the in vitro and in vivo studies. Student's t-test or two-way ANOVA were used to test the statistical significance of two or more comparisons, respectively. RESULTS: SFX-01 treatment exhibited cytostatic and cytotoxic effects, mediated by G2 cell cycle arrest, apoptosis induction and suppression of autophagy. Moreover, SFX-01 was able to inhibit the formation and the proliferation of 3D tumorspheres as monotherapy and in combination with IR. Finally, SFX-01, when orally administered as single agent, displayed a pattern of efficacy at reducing the growth of tumour masses in RMS xenograft mouse models; when combined with a radiotherapy regime, it was observed to act synergistically, resulting in a more positive outcome than would be expected by adding each exposure alone. CONCLUSIONS: In summary, our results provide evidence for the antitumour properties of SFX-01 in preclinical models of RMS tumours, both as a standalone treatment and in combination with irradiation. These forthcoming findings are crucial for deeper investigations of SFX-01 molecular mechanisms against RMS and for setting up clinical trials in RMS patients in order to use the SFX-01/IR co-treatment as a promising therapeutic approach, particularly in the clinical management of aggressive RMS disease.

The botanical drug PBI-05204, a supercritical CO2 extract of Nerium oleander, sensitizes alveolar and embryonal rhabdomyosarcoma to radiotherapy in vitro and in vivo.

Treatment of rhabdomyosarcoma (RMS), the most common a soft tissue sarcoma in childhood, provides intensive multimodal therapy, with radiotherapy (RT) playing a critical role for local tumor control. However, since RMS efficiently activates mechanisms of resistance to therapies, despite improvements, the prognosis remains still largely unsatisfactory, mainly in RMS expressing chimeric oncoproteins PAX3/PAX7-FOXO1, and fusion-positive (FP)-RMS. Cardiac glycosides (CGs), plant-derived steroid-like compounds with a selective inhibitory activity of the Na+/K+-ATPase pump (NKA), have shown antitumor and radio-sensitizing properties. Herein, the therapeutic properties of PBI-05204, an extract from Nerium oleander containing the CG oleandrin already studied in phase I and II clinical trials for cancer patients, were investigated, in vitro and in vivo, against FN- and FP-RMS cancer models. PBI-05204 induced growth arrest in a concentration dependent manner, with FP-RMS being more sensitive than FN-RMS, by differently regulating cell cycle regulators and commonly upregulating cell cycle inhibitors p21Waf1/Cip1 and p27Cip1/Kip1. Furthermore, PBI-05204 concomitantly induced cell death on both RMS types and senescence in FN-RMS. Notably, PBI-05204 counteracted in vitro migration and invasion abilities and suppressed the formation of spheroids enriched in CD133+ cancer stem cells (CSCs). PBI-05204 sensitized both cell types to RT by improving the ability of RT to induce G2 growth arrest and counteracting the RT-induced activation of both Non-Homologous End-Joining and homologous recombination DSBs repair pathways. Finally, the antitumor and radio-sensitizing proprieties of PBI-05204 were confirmed in vivo. Notably, both in vitro and in vivo evidence confirmed the higher sensitivity to PBI-05204 of FP-RMS. Thus, PBI-05204 represents a valid radio-sensitizing agent for the treatment of RMS, including the intrinsically radio-resistant FP-RMS.

Translational Implications for Radiosensitizing Strategies in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence that includes FP-RMS, harboring the fusion oncoprotein PAX3/7-FOXO1 and FN-RMS, often mutant in the RAS pathway. Risk stratifications of RMS patients determine different prognostic groups and related therapeutic treatment. Current multimodal therapeutic strategies involve surgery, chemotherapy (CHT) and radiotherapy (RT), but despite the deeper knowledge of response mechanisms underpinning CHT treatment and the technological improvements that characterize RT, local failures and recurrence frequently occur. This review sums up the RMS classification and the management of RMS patients, with special attention to RT treatment and possible radiosensitizing strategies for RMS tumors. Indeed, RMS radioresistance is a clinical problem and further studies aimed at dissecting radioresistant molecular mechanisms are needed to identify specific targets to hit, thus improving RT-induced cytotoxicity.

Radioresistance in rhabdomyosarcomas: Much more than a question of dose.

Management of rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, frequently accounting the genitourinary tract is complex and requires a multimodal therapy. In particular, as a consequence of the advancement in dose conformity technology, radiation therapy (RT) has now become the standard therapeutic option for patients with RMS. In the clinical practice, dose and timing of RT are adjusted on the basis of patients' risk stratification to reduce late toxicity and side effects on normal tissues. However, despite the substantial improvement in cure rates, local failure and recurrence frequently occur. In this review, we summarize the general principles of the treatment of RMS, focusing on RT, and the main molecular pathways and specific proteins involved into radioresistance in RMS tumors. Specifically, we focused on DNA damage/repair, reactive oxygen species, cancer stem cells, and epigenetic modifications that have been reported in the context of RMS neoplasia in both in vitro and in vivo studies. The precise elucidation of the radioresistance-related molecular mechanisms is of pivotal importance to set up new more effective and tolerable combined therapeutic approaches that can radiosensitize cancer cells to finally ameliorate the overall survival of patients with RMS, especially for the most aggressive subtypes.

Multimodal evaluation of 19F-BPA internalization in pancreatic cancer cells for boron capture and proton therapy potential applications.

PURPOSE: One of the obstacles to the application of Boron Neutron Capture Therapy (BNCT) and Proton Boron Fusion Therapy (PBFT) concerns the measurement of borated carriers' biodistribution. The objective of the present study was to evaluate the in vitro internalization of the 19F-labelled p-boronophenylalanine (19F-BPA) in the human cancer pancreatic cell line (PANC-1) for the potential application of BNCT and PBFT in pancreatic cancer. The 19F-BPA carrier has the advantage that its bio-distribution may be monitored in vivo using 19F-Nuclear Magnetic Resonance (19F NMR). MATERIALS AND METHODS: The 19F-BPA internalization in PANC-1 cells was evaluated using three independent techniques on cellular samples left in contact with growing medium enriched with 13.6 mM 19F-BPA corresponding to a 11B concentration of 120 ppm: neutron autoradiography, which quantifies boron; liquid chromatography hyphenated to tandem mass spectrometry and UV-Diode Array Detection (UV-DAD), which quantifies 19F-BPA molecule; and 19F NMR spectroscopy, which detects fluorine nuclei. RESULTS: Our studies suggested that 19F-BPA is internalized by PANC-1 cells. The three methods provided consistent results of about 50% internalization fraction at 120 ppm of 11B. Small variations (less than 15%) in internalization fraction are mainly dependent on the proliferation state of the cells. CONCLUSIONS: The ability of 19F NMR spectroscopy to study 19F-BPA internalization was validated by well-established independent techniques. The multimodal approach we used suggests 19F-BPA as a promising BNCT/PBFT carrier for the treatment of pancreatic cancer. Since the quantification is performed at doses useful for BNCT/PBFT, 19F NMR can be envisaged to monitor 19F-BPA bio-distribution during the therapy.

MS-275 (Entinostat) Promotes Radio-Sensitivity in PAX3-FOXO1 Rhabdomyosarcoma Cells.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. About 25% of RMS expresses fusion oncoproteins such as PAX3/PAX7-FOXO1 (fusion-positive, FP) while fusion-negative (FN)-RMS harbors RAS mutations. Radiotherapy (RT) plays a crucial role in local control but metastatic RMS is often radio-resistant. HDAC inhibitors (HDACi) radio-sensitize different cancer cells types. Thus, we evaluated MS-275 (Entinostat), a Class I and IV HDACi, in combination with RT on RMS cells in vitro and in vivo. MS-275 reversibly hampered cell survival in vitro in FN-RMS RD (RASmut) and irreversibly in FP-RMS RH30 cell lines down-regulating cyclin A, B, and D1, up-regulating p21 and p27 and reducing ERKs activity, and c-Myc expression in RD and PI3K/Akt/mTOR activity and N-Myc expression in RH30 cells. Further, MS-275 and RT combination reduced colony formation ability of RH30 cells. In both cell lines, co-treatment increased DNA damage repair inhibition and reactive oxygen species formation, down-regulated NRF2, SOD, CAT and GPx4 anti-oxidant genes and improved RT ability to induce G2 growth arrest. MS-275 inhibited in vivo growth of RH30 cells and completely prevented the growth of RT-unresponsive RH30 xenografts when combined with radiation. Thus, MS-275 could be considered as a radio-sensitizing agent for the treatment of intrinsically radio-resistant PAX3-FOXO1 RMS.

Romidepsin (FK228) fails in counteracting the transformed phenotype of rhabdomyosarcoma cells but efficiently radiosensitizes, in vitro and in vivo, the alveolar phenotype subtype.

PURPOSE: Herein we describe the in vitro and in vivo activity of FK228 (Romidepsin), an inhibitor of class I HDACs, in counteracting and radiosensitizing embryonal (ERMS, fusion-negative) and alveolar (ARMS, fusion-positive) rhabdomyosarcoma (RMS). METHODS: RH30 (ARMS, fusion-positive) and RD (ERMS, fusion-negative) cell lines and human multipotent mesenchymal stromal cells (HMSC) were used. Flow cytometry analysis, RT-qPCR, western blotting and enzymatic assays were performed. Irradiation was delivered by using an x-6 MV photon linear accelerator. FK228 (1.2 mg/kg) in vivo activity, combined or not with radiation therapy (2 Gy), was assessed in murine xenografts. RESULTS: Compared to HMSC, RMS expressed low levels of class I HDACs. In vitro, FK228, as single agents, reversibly downregulated class I HDACs expression and activity and induced oxidative stress, DNA damage and a concomitant growth arrest associated with PARP-1-mediated transient non-apoptotic cell death. Surviving cells upregulated the expression of cyclin A, B, D1, p27, Myc and activated PI3K/Akt/mTOR and MAPK signaling, known to be differently involved in cancer chemoresistance. Interestingly, while no radiosensitizing effects were detected, in vitro or in vivo, on RD cells, FK228 markedly radiosensitized RH30 cells by impairing antioxidant and DSBs repair pathways in vitro. Further, FK228 when combined with RT in vivo significantly reduced tumor mass in mouse RH30 xenografts. CONCLUSION: FK228 did not show antitumor activity as a single agent whilst its combination with RT resulted in radiosensitization of fusion-positive RMS cells, thus representing a possible strategy for the treatment of the most aggressive RMS subtype.

Clinically relevant radioresistant rhabdomyosarcoma cell lines: functional, molecular and immune-related characterization.

BACKGROUND: The probability of local tumor control after radiotherapy (RT) remains still miserably poor in pediatric rhabdomyosarcoma (RMS). Thus, understanding the molecular mechanisms responsible of tumor relapse is essential to identify personalized RT-based strategies. Contrary to what has been done so far, a correct characterization of cellular radioresistance should be performed comparing radioresistant and radiosensitive cells with the same isogenic background. METHODS: Clinically relevant radioresistant (RR) embryonal (RD) and alveolar (RH30) RMS cell lines have been developed by irradiating them with clinical-like hypo-fractionated schedule. RMS-RR cells were compared to parental isogenic counterpart (RMS-PR) and studied following the radiobiological concept of the "6Rs", which stand for repair, redistribution, repopulation, reoxygenation, intrinsic radioresistance and radio-immuno-biology. RESULTS: RMS-RR cell lines, characterized by a more aggressive and in vitro pro-metastatic phenotype, showed a higher ability to i) detoxify from reactive oxygen species; ii) repair DNA damage by differently activating non-homologous end joining and homologous recombination pathways; iii) counteract RT-induced G2/M cell cycle arrest by re-starting growth and repopulating after irradiation; iv) express cancer stem-like profile. Bioinformatic analyses, performed to assess the role of 41 cytokines after RT exposure and their network interactions, suggested TGF-ß, MIF, CCL2, CXCL5, CXCL8 and CXCL12 as master regulators of cancer immune escape in RMS tumors. CONCLUSIONS: These results suggest that RMS could sustain intrinsic and acquire radioresistance by different mechanisms and indicate potential targets for future combined radiosensitizing strategies.

Modulating the dose-rate differently affects the responsiveness of human epithelial prostate- and mesenchymal rhabdomyosarcoma-cancer cell line to radiation.

Purpose: Radiation therapy (RT), by using ionizing radiation (IR), destroys cancer cells inducing DNA damage. Despite several studies are continuously performed to identify the best curative dose of IR, the role of dose-rate, IR delivered per unit of time, on tumor control is still largely unknown.Materials and methods: Rhabdomyosarcoma (RMS) and prostate cancer (PCa) cell lines were irradiated with 2 or 10 Gy delivered at dose-rates of 1.5, 2.5, 5.5 and 10.1 Gy/min. Cell-survival rate and cell cycle distribution were evaluated by clonogenic assays and flow cytometry, respectively. The production of reactive oxygen species (ROS) was detected by cytometry. Quantitative polymerase chain reaction assessed the expression of anti-oxidant-related factors including NRF2, SODs, CAT and GPx4 and miRNAs (miR-22, -126, -210, -375, -146a, -34a). Annexin V and caspase-8, -9 and -3 activity were assessed to characterize cell death. Senescence was determined by assessing ß-galactosidase (SA-ß-gal) activity. Immunoblotting was performed to assess the expression/activation of: i) phosphorylated H2AX (γ-H2AX), markers of DNA double strand breaks (DSBs); ii) p19Kip1/Cip1, p21Waf1/Cip1 and p27Kip1/Cip1, senescence-related-markers; iii) p62, LC3-I and LC3-II, regulators of autophagy; iv) ATM, RAD51, DNA-PKcs, Ku70 and Ku80, mediators of DSBs repair.Results: Low dose-rate (LDR) more efficiently induced apoptosis and senescence in RMS while high dose-rate (HDR) necrosis in PCa. This paralleled with a lower ability of LDR-RMS and HDR-PCa irradiated cells to activate DSBs repair. Modulating the dose rate did not differently affect the anti-oxidant ability of cancer cells.Conclusion: The present results indicate that a stronger cytotoxic effect was induced by modulating the dose-rate in a cancer cell-dependent manner, this suggesting that choose the dose-rate based on the individual patient's tumor characteristics could be strategic for effective RT exposures.

Wharton's jelly mesenchymal stromal cells have contrasting effects on proliferation and phenotype of cancer stem cells from different subtypes of lung cancer.

Studies on the role of multipotent mesenchymal stromal cells (MSC) on tumor growth have reported both a tumor promoting and a suppressive effect. The aim of the present study was to determine the effect of MSC isolated from Wharton's jelly of umbilical cord (WJMSC) on lung cancer stem cells (LCSC) derived from human lung tumors: two adenocarcinomas (AC) and two squamous cell carcinomas (SCC). LCSC derived from SCC and AC expressed, to varying extents, the more relevant stem cell markers. The effect of WJMSC on LCSC was investigated in vitro using conditioned medium (WJ-CM): a proliferation increase in AC-LCSC was observed, with an increase in the ALDH+ and in the CD133+ cell population. By contrast, WJ-CM hampered the growth of SCC-LCSC, with an increase in the pre-G1 phase indicating the induction of apoptosis. Furthermore, the ALDH+ and CD133+ population was also reduced. In vivo, subcutaneous co-transplantation of AC-LCSC/WJMSC generated larger tumors than AC-LCSC alone, characterized by an increased percentage of CD133+ and CD166+ cells. By contrast, co-transplantation of WJMSC and SCC-LCSC did not affect the tumor size. Our results strongly suggest that WJMSC exert, both in vitro and in vivo, contrasting effects on LCSC derived from different lung tumor subtypes.

Key role of MEK/ERK pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem-like cell population.

BACKGROUND: The identification of signaling pathways that affect the cancer stem-like phenotype may provide insights into therapeutic targets for combating embryonal rhabdomyosarcoma. The aim of this study was to investigate the role of the MEK/ERK pathway in controlling the cancer stem-like phenotype using a model of rhabdospheres derived from the embryonal rhabdomyosarcoma cell line (RD). METHODS: Rhabdospheres enriched in cancer stem like cells were obtained growing RD cells in non adherent condition in stem cell medium. Stem cell markers were evaluated by FACS analysis and immunoblotting. ERK1/2, myogenic markers, proteins of DNA repair and bone marrow X-linked kinase (BMX) expression were evaluated by immunoblotting analysis. Radiation was delivered using an x-6 MV photon linear accelerator. Xenografts were obtained in NOD/SCID mice by subcutaneously injection of rhabdosphere cells or cells pretreated with U0126 in stem cell medium. RESULTS: MEK/ERK inhibitor U0126 dramatically prevented rhabdosphere formation and down-regulated stem cell markers CD133, CXCR4 and Nanog expression, but enhanced ALDH, MAPK phospho-active p38 and differentiative myogenic markers. By contrast, MAPK p38 inhibition accelerated rhabdosphere formation and enhanced phospho-active ERK1/2 and Nanog expression. RD cells, chronically treated with U0126 and then xeno-transplanted in NOD/SCID mice, delayed tumor development and reduced tumor mass when compared with tumor induced by rhabdosphere cells. U0126 intraperitoneal administration to mice bearing rhabdosphere-derived tumors inhibited tumor growth . The MEK/ERK pathway role in rhabdosphere radiosensitivity was investigated in vitro. Disassembly of rhabdospheres was induced by both radiation or U0126, and further enhanced by combined treatment. In U0126-treated rhabdospheres, the expression of the stem cell markers CD133 and CXCR4 decreased and dropped even more markedly following combined treatment. The expression of BMX, a negative regulator of apoptosis, also decreased following combined treatment, which suggests an increase in radiosensitivity of rhabdosphere cells. CONCLUSIONS: Our results indicate that the MEK/ERK pathway plays a prominent role in maintaining the stem-like phenotype of RD cells, their survival and their innate radioresistance. Thus, therapeutic strategies that target cancer stem cells, which are resistant to traditional cancer therapies, may benefit from MEK/ERK inhibition combined with traditional radiotherapy, thereby providing a promising therapy for embryonal rhabdomyosarcoma.

Cord blood CD34+ cells expanded on Wharton's jelly multipotent mesenchymal stromal cells improve the hematopoietic engraftment in NOD/SCID mice.

OBJECTIVE: This study aims to investigate the capability of Wharton's jelly multipotent mesenchymal stromal cells (WJ-MSC) to support the in vitro expansion of hematopoietic stem/progenitor cells (HSPC) derived from cord blood (CB) in the absence of exogenous cytokines, and the effect on engraftment of the expanded cells in a mouse model. METHODS: CB-CD34+ cells were seeded on WJ-MSC layer and cultured in HP01 serum-free medium. Day-7 and day-13 expanded cells were transplanted in NOD/SCID mice. After 8 wk, engraftment was evaluated in mouse bone marrow as percentage of human CD45+ cells. RESULTS: CD34+ population was expanded without increasing the differentiation rate. Co-culture increased the expansion of the CD34+ cells by 2.0 and 7.3 times after 7 and 13 d, respectively, and maintained the CD34+ cells up to day 20. In particular, earlier CD34+/CD90+ and CD34+/CD33- subtypes were increased. An advantage of the day-7 co-cultured HSPC in respect of HSPC at day 0 in the engraftment of NOD/SCID mice was obtained both as percentage of mice engrafted (100% vs. 75%) and as percentage of chimerism. CONCLUSIONS: Although the increase in hematopoietic progenitors is not dramatic as in the presence of added cytokines, this study demonstrates the effectiveness of the WJ-MSC not only to preserve the CD34+ population but also to improve the repopulating efficacy of the amplified HSPC, also in the absence of added cytokines and growth factors.

Induction of dopaminergic neurons from human Wharton's jelly mesenchymal stem cell by forskolin.

The purpose of this study was to investigate the Wharton's jelly mesenchymal stem cells differentiation ability toward neuronal fate. Human Wharton's jelly mesenchymal stem cells (hWJMSC) have been isolated from human umbilical cord of full-term births and characterized by flow cytometry analysis for their stem mesenchymal properties through specific surface markers expression (CD73, CD90, and CD105). hWJMSC mesodermal lineage differentiation ability and karyotype analysis were assessed. The trans-differentiation of hWJMSC into neural lineage was investigated in presence of forskolin, an agent known to increase the intracellular levels of cAMP. A molecular profile of differentiated hWJMSC was performed by microarray technology which revealed 1,532 statistically significant modulated genes respect to control cells. Most of these genes are mainly involved in functional neuronal signaling pathways and part of them are specifically required for the neuronal dopaminergic induction. The acquisition of the dopaminergic phenotype was evaluated via immunocytochemistry and Western blot analysis revealed the significant induction of Nurr1, NeuroD1, and TH proteins expression in forskolin-induced hWJMSC. Moreover, the treatment with forskolin promoted, in hWJMSC, a strong upregulation of the neurotrophin Trk receptors related to the high release of brain-derived neurotrophic factor. Taken together these findings show that hWJMSC may be represent an optimal therapeutic strategy for neurological diseases.

Valproic acid affects the engraftment of TPO-expanded cord blood cells in NOD/SCID mice.

Hematopoietic stem and progenitor cells (HSPC) can improve the long-term outcome of transplanted individuals and reduce the relapse rate. Valproic acid (VPA), an inhibitor of histone deacetylase, when combined with different cytokine cocktails, induces the expansion of CD34+ cell populations derived from cord blood (CB) and other sources. We evaluated the effect of VPA, in combination with thrombopoietin (TPO), on the viability and expansion of CB-HSPCs and on short- and long-term engraftability in the NOD/SCID mouse model. In vitro, VPA+TPO inhibited HSPC differentiation and preserved the CD34+ cell fraction; the self-renewal of the CD34+ TPO+VPA-treated cells was suggested by the increased replating efficiency. In vivo, short- and long-term engraftment was determined after 6 and 20 weeks. After 6 weeks, the median chimerism percentage was 13.0% in mice transplanted with TPO-treated cells and only 1.4% in those transplanted with TPO+VPA-treated cells. By contrast, after 20 weeks, the engraftment induced by the TPO+VPA-treated cells was three times more effective than that induced by TPO alone, and over ten times more effective compared to the short-term engraftment induced by the TPO+VPA-treated cells. The in vivo results are consistent with the higher secondary plating efficiency of the TPO+VPA-treated cells in vitro.

Italian national survey of blood donors: external quality assessment (EQA) of syphilis testing.

The detection of syphilis among blood donors may reveal high-risk sexual behavior, which can go unreported at the time of donor selection and compromise the safety of the donated blood. In Italy, blood is collected, tested, and distributed by transfusion services (TSs), which also perform outpatient transfusions. Although the TSs must screen for syphilis by law, there are no indications of the specific type of method to be used, generating discrepancies in the results obtained by the different TSs. To determine the proficiency of the TSs in screening for syphilis, we performed an external quality assessment (EQA). The EQA was based on two shipments of serum panels; 133 and 118 of the 326 existing TSs participated in the first and second shipments, respectively. Each panel consisted of both positive and negative serum samples. The results confirmed that the use of a single nontreponemal test (the Venereal Disease Research Laboratory [VDRL] and the rapid plasma reagin [RPR] tests) is the least sensitive means of identifying samples that are positive for syphilis antibodies. We also found that the interpretation of the results of manual techniques, such as the RPR test, the VDRL test, the Treponema pallidum hemagglutination (TPHA) assay, and the T. pallidum particle agglutination (TPPA) assay, can vary greatly among different TSs and operators. Total Ig enzyme immunoassays (EIAs) are the most sensitive. However, the determination of syphilis on the basis of the results of a single test is not sufficient for an accurate screening; and all blood units should thus be assessed by two distinct treponemal tests, that is, a total Ig EIA and the TPHA or the TPPA assay.

Tumorigenic potential of olfactory bulb-derived human adult neural stem cells associates with activation of TERT and NOTCH1.

BACKGROUND: Multipotent neural stem cells (NSCs) have been isolated from neurogenic regions of the adult brain. Reportedly, these cells can be expanded in vitro under prolonged mitogen stimulation without propensity to transform. However, the constitutive activation of the cellular machinery required to bypass apoptosis and senescence places these cells at risk for malignant transformation. METHODOLOGY/PRINCIPAL FINDINGS: Using serum-free medium supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), we established clonally derived NS/progenitor cell (NS/PC) cultures from the olfactory bulb (OB) of five adult patients. The NS/PC cultures obtained from one OB specimen lost growth factor dependence and neuronal differentiation at early passage. These cells developed glioblastoma tumors upon xenografting in immunosuppressed mice. The remaining NS/PC cultures were propagated either as floating neurospheres or as adherent monolayers with maintenance of growth factor dependence and multipotentiality at late passage. These cells were engrafted onto the CNS of immunosuppressed rodents. Overall, the grafted NS/PCs homed in the host parenchyma showing ramified morphology and neuronal marker expression. However, a group of animals transplanted with NS/PCs obtained from an adherent culture developed fast growing tumors histologically resembling neuroesthesioblastoma. Cytogenetic and molecular analyses showed that the NS/PC undergo chromosomal changes with repeated in vitro passages under mitogen stimulation, and that up-regulation of hTERT and NOTCH1 associates with in vivo tumorigenicity. CONCLUSIONS/SIGNIFICANCE: Using culturing techniques described in current literature, NS/PCs arise from the OB of adult patients which in vivo either integrate in the CNS parenchyma showing neuron-like features or initiate tumor formation. Extensive xenografting studies on each human derived NS cell line appear mandatory before any use of these cells in the clinical setting.

Nucleic acid testing (NAT) for HCV RNA in Italian transfusion centres: an external quality assessment.

BACKGROUND: We conducted an external quality assessment of the results obtained in Italian transfusion centre laboratories employing nucleic acid testing (NAT) for detection of HCV RNA in donated blood. STUDY DESIGN: Of 110 transfusions centres in Italy, 101 voluntarily participated. Each laboratory received seven separate shipments of samples for HCV RNA testing by NAT. Each shipment contained 8 plasma samples for a total of 23 negative and 33 positive samples with viral loads ranging from 25 to 1000 IU/mL. RESULTS: Of the 2080 HCV RNA-negative samples, 14 (0.7%) were reported as positive. The highest percent of false-negative results (6.9%) was found on samples from the first shipment with viral loads from 75 to 100 IU/mL. In subsequent shipments, the highest false-negative percentage ranged from 0.6% for samples with viral loads of 170-1000 IU/mL to 3.4% for samples with viral loads of 35-50 IU/mL. A false-negative rate of 4.9% occurred in samples in the sixth shipment with the lowest viral load (25IU/mL). Five (4.9%) centres were identified as having laboratories with low-performance. There were no significant differences among genotypes 1b, 2c and 3a with respect to percent of false-negative results reported. CONCLUSIONS: Overall, the accuracy of NAT observed in this study of Italian transfusion centre laboratories was excellent for all HCV genotypes tested, even for samples with low HCV RNA titres.

Long-term platelet production assessed in NOD/SCID mice injected with cord blood CD34+ cells, thrombopoietin-amplified in clinical grade serum-free culture.

OBJECTIVE: Delayed platelet recovery post-cord blood (CB) transplantation might be due to CB characteristics: low maturity of stem cell compartment, poor production of CD34+/CD41+ cells when induced to differentiate along the megakaryocytic (MK) lineage, retention of a low ploidy in the expanded MKs. Ex vivo expansion of CB hematopoietic progenitor cells for reconstitution of different human hematopoietic lineages has already been developed in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. However, optimal conditions for MK-progenitor engraftment to reduce hemorrhaging risk still to be developed. This study assesses the hypothesis that CB-CD34+ amplification with thrombopoietin (TPO) can be applied to a portion of a CB transplant unit to stimulate recovery along MK differentiation program. MATERIALS AND METHODS: Human CB-CD34+ cells were amplified in a serum-free, clinical grade medium with 100 ng/mL TPO alone and in addition to other cytokines (Kit ligand, interleukin-6, and Flt-3 ligand). Seven-day cultured cells were transplanted into irradiated NOD/SCID mice and engraftment, megakaryocytopoiesis, and platelet production were assessed. RESULTS: Platelet release was successful and continuously present for at least 8 weeks in NOD/SCID mice transplanted with CB cells stimulated by TPO. Thrombocytopoiesis was more effective with transplanted TPO-amplified cells than with the cytokine cocktails. CONCLUSION: Platelet number obtained is within the minimum level considered sufficient for hemostasis. Furthermore, amplified cells maintain their self-renewal capacity and multilineage potential differentiation. Thus, transplantation of TPO-expanded CB cells has the potential favoring both platelet recovery and human engraftment.

HDAC inhibition is associated to valproic acid induction of early megakaryocytic markers.

Valproic acid (VPA), a histone deacetylase inhibitor, causes differentiation in different cell lines and in a cell-specific manner; yet, its effect on megakaryocytic (MK) differentiation has not been studied. We evaluated whether VPA induces MK differentiation in a UT-7 cell line through histone acetylation in the GpIIIa gene region and activation of the ERK pathway. UT-7 cells, derived from megakaryoblastic leukemia, were treated with VPA at various concentrations, and the expression of differentiation markers as well as the gene expression profile was assessed. Flow cytometry, immunoblot analysis, and RT-PCR demonstrated that VPA induced the expression of the early MK markers GpIIIa (CD61) and GpIIb/IIIa (CD41) in a dose-dependent manner. The VPA-treated cells showed hyperacetylation of the histones H3 and H4; in particular, histone acetylation was found to have been associated with CD61 expression, in that the GpIIIa promoter showed H4 hyperacetylation, as demonstrated by the chromatin immunoprecipitation assay. Furthermore, activation of the ERK pathway was involved in VPA-mediated CD61/CD41 expression and in cell adhesion, as demonstrated by using the MEK/ERK inhibitor U0126. In conclusion, the capacity of VPA to commit UT-7 cells to MK differentiation is mediated by its inhibitory action on HDAC and the long-lived activation of ERK1/2.