Ve Cells Research Articles

SL-401, a Novel IL-3Rα/CD123-Directed Agent Targets Stem-like Cells in Multiple Myeloma

Introduction Multiple myeloma (MM) remains incurable despite novel therapies, highlighting the need for further identification of factors mediating disease progression and resistance. One such factor is the development of a drug-resistant stem cell-like subpopulation. Previous studies have shown that MM side population cells (MM-SPs) exhibit stem-cell like features and contribute to relapse of MM. Recent research efforts, which have focused the biology of MM stem-like cells in order to derive specific therapy, have shown that stem-cell transcription factor Oct-4 is linked to stemness and chemoresistance. Here we examined the effect of enforced expression of Oct4 in MM cells and MM-SPs on the development of stem cell-like characteristics and drug-resistance in MM. These studies allow us to establish stable MM cell lines with characteristic stem-cell like features, which in turn facilitate screening of novel agents that effectively target this cell population in MM.Methods MM-SPs were isolated from RPMI-8226 cells by flow-cytometry based Hoechst 33342 staining. RPMI-8226 and RPMI-8226-SP cells were transfected with a phOct4-GFP construct (Gerrard et al., Stem Cell 2005, 23:124-133), and selected with G418 (0.5 mg/ml) to derive stable RPMI-8226-Oct4 and RPMI-8226-SP-Oct4 cell lines. Oct-4 expression was confirmed using FACS. Cell viability was analyzed by WST assays. SL-401 is a targeted therapy directed to IL-3Rα/CD123, comprised of recombinant human IL-3 fused to truncated diphtheria toxin. Drug and reagent source: SL-401 was obtained from Stemline Therapeutics; Bortezomib and flow antibodies were purchased from Selleck Chemicals and BD Biosciences, respectively. Statistical significance was derived using GraphPad Prism.Results 1) RPMI-8226 and RPMI-8226-Oct4 cells were analyzed for the expression of surface markers associated with stem cells (CD123/IL-3Rα, CD133 and CD27) by multicolor flow analysis. Oct-4 transfection does not affect the overall CD123 expression in RPMI-8226 cells, as the % MFI-CD123hi in RPMI-8226 versus RPMI-8226-Oct4 remains unchanged. However, the stable selection makes RPMI-8226-Oct4 more clonal in nature (%CD123hi : RPMI8226; 14.9% vs RPMI-8226-Oct4; 60%). 2) A significant increase in the frequency of CD133+ve cells was observed in RPMI-8226-SP-Oct4-tranfected cells versus either RPMI-8226-SP cells or RPMI-8226-Oct-4 cells [RPMI-8226-SP: 6.3%; RPMI-8226-Oct4: 27.8%; RPMI-8226-SP-Oct4: 40%; p< 0.05]. 3) Analysis of CD27 surface marker showed highest expression in RPMI-8226-SP-Oct4 cells compared to RPMI-8226-Oct4, RPMI-8226-SP, or RPMI-8226 cells (% MFI: RPMI-8226-SP-Oct4 > RPMI-8226-Oct4 > RPMI-8226-SP > RPMI-8226 cells). 4) Treatment of RPMI-8226 and RPMI-8226-Oct4 cells with proteasome inhibitor bortezomib decreased the viability of RPMI-8226 cells; in contrast, bortezomib did not significantly alter the viablity of RPMI-8226-Oct4 cells [% Viability after bortezomib: RPMI-8226; <50% versus RPMI-8226-Oct4; 95%]. Finally, 5) SL-401 significantly decreased the viability of RPMI-8226-Oct4 cells [IC50: RPMI-8226-Oct4 cells: 75 pM; RPMI-8226-SP cells: 350 pM; RPMI-8226 cells: 1367 pM]. We have previously shown anti-MM activity of SL-401 by an additional mechanism of targeting IL-3Rα-expressing plasmacytoid dendritic cells (pDCs) localized in the tumor microenvironment and blocking pDC-induced MM cell growth.Conclusions Our data show that stem-like cells in MM are relatively resistant to proteasome inhibitor therapy. Importantly, a novel agent SL-401 effectively targets these cells. Oct4-driven stable RPMI-8226 MM cell line serves as a novel tool to screen and develop newer agents targeting stem-like cells in MM. Overall, we show the ability of SL-401 to target a drug-resistant stem-like cell population in MM, and provide an additional rationale for clinical evaluation of SL-401 to improve patient outcome. A clinical trial of SL-401 in MM is currently ongoing (NCT02661022). DisclosuresMacri:Stemline Therapeutics, Inc.: Employment. Chen:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Chauhan:Oncopeptide AB: Consultancy; Epicent Rx: Consultancy; C4 Therapeutics: Equity Ownership; Stemline Therapeutics, Inc.: Consultancy. Anderson:Celgene: Membership on an entity’s Board of Directors or advisory committees; Sonofi Aventis: Membership on an entity’s Board of Directors or advisory committees; Acetylon: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Gilead: Membership on an entity’s Board of Directors or advisory committees; Onyx: Membership on an entity’s Board of Directors or advisory committees.

The Effect of Thymoquinone, α7 Receptor Agonist and α7 Receptor Allosteric Modulator on the Cerebral Cortex in Experimentally Induced Alzheimer's Disease in Relation to MSCs Activation.

Background and ObjectivesAlzheimer’s disease (AD) is the most common form of dementia among older persons. Thymoquinone (TQ) has anti-inflammatory, anticonvulsant and antioxidant activity. A novel α7 nicotinic acetyl choline receptor (α7 nAChR ) agonist (PNU- 282987) have been identified to enhance the cognitive performance. An alternative treatment strategy via compounds known as nicotinic “positive allosteric modulators” (PAMs) has been reported. This study was designed to investigate the combination of PAM of α7 nAChRs with PNU- 282987 or with TQ as a possible treatment for AD in rat.Methods48 male albino rats were divided into 4 groups. Group I (Control), Group II received lipopolysaccharide, 0.8 mg/kg by intraperitoneal injection (IPI) once, Group III received TQ 10 mg/kg by IPI, Group IV received PNU-120596 1 mg/kg by IPI, in addition to PNU-282987 1 mg/kg by IPI in subgroup IVa and TQ in subgroup b. All treatment drugs were given for 5 days.ResultsAcidophilic masses, deformed neurons, Congo red +ve masses and reduced Phospho-CREB immunoexpression were seen in group II. All changes regressed by treatment. Some CD44 +ve cells were noticed in group II and few +ve cells in subgroup IVa, that became multiple in group III and subgroup IVb. The histological, histochemical and immunohistochemical changes were confirmed statistically and significant differences were recorded.ConclusionsTQ or α7 nAChR agonist combined with PAM can have an important role in treatment of AD that is superior to thymoquinone alone. Exceptionally, TQ single or combined with PAM proved activation of MSC.

Coexistence of inversion 16 in chronic myeloid leukaemia in blast crisis

Chronic myeloid leukaemia (CML) is consistently associated with the BCR-ABL1 fusion gene located on the derivative chromosome 22 (Philadelphia chromosome, Ph + ve) as a result of a t(9;22)(q34;q11.2) translocation. We describe a 36-year-old male in blast phase CML presenting with acute myelomonocytic leukaemia and eosinophilia. Five months after initial disease diagnosis and despite being treated with standard imatinib therapy (400 mg daily) conventional cytogenetic analysis (CCA) demonstrated the evolution of a chromosome 16 pericentric inversion (inv(16)(p13q22)) within the already Ph + ve cells. Bone marrow aspiration and biopsy revealed 60% blasts, positive for CD34, HLADR, CD33 and CD13. Fluorescence in situ hybridization (FISH) and reverse transcription–polymerase chain reaction (RT-PCR) confirmed presence of a CBFβ-MHY11 rearrangement whilst next generation sequencing (NGS) detected an E255K point mutation within the BCR-ABL1 tyrosine kinase domain. Neither the CBFβ-MHY11 rearrangement nor the E225K point mutation were present at diagnosis. The patient received a combination of daunorubicin (60 mg/m2) and cytarabine (100 mg/m2) and had his tyrosine kinase inhibitor (TKI) changed to nilotinib before undergoing a male unrelated donor (MUD) stem cell transplant (SCT). The t(9;22) and inv(16) rearrangements persisted at day 100 post MUD SCT and his TKI was further switched to dasatinib. The patient presented with pleural effusions and had subsequent CNS relapse and passed away from relapsed disease and infective complications, 19-month post MUD allograft.

Resistance to Trastuzumab in Clinical Her2 Breast Cancer: Possible Relation to Molecular Subtype and Cancer Stem Cells

Molecular breast cancer (BC) subtypes and cancer stem cells (CSCs) can determine sensitivity to trastuzumab. A better predictivevalue of clinical human epidermal growth factor receptor2 (cHER2) BC can be provided, by incorporating CSCs heterogeneityinto clinical decisions. The study aimed at investigating involvement of transition of CSCs biology and the molecular subtype indevelopment of trastuzumab resistance in HER2 BC, recommending the use of trastuzumab combinations/modifications able tosuppress CSCs and overcome trastuzumab resistance. The study was conducted on 40 BC patients age range 27-63 years, 20served as control group and 20 as metastatic (predicted resistance) group. Clinicopathological analysis included HER2, ER andPR status, chemotherapy regimen and number of herceptin cycles. Histological analysis included, molecular subtype markers(HER2 and cytokeratin (CK) 5/6), CSCs marker CD44 and morphometric studies. Quantitative polymerase chain reaction (qPCR)was performed. In the metastatic group significant decrease in the area% of HER2 positive (+ve) immunoexpression (IE),significant increase in the area% of CK5/6 IE and CD44 +ve cells was detected. qPCR values of HER2 gene confirmed asignificant decrease. It was concluded that resistance to trastuzumab in the metastatic group of HER2 BC is related to basal likesubtype and overexpression of CD44 +ve CSCs. This nictitates IHC assessment of molecular subtype and type of CSCs to allowthe choice of most appropriate treatment regimen giving best expected prognosis.

Inactivation of TGFβ receptors in stem cells drives cutaneous squamous cell carcinoma.

Melanoma patients treated with oncogenic BRAF inhibitors can develop cutaneous squamous cell carcinoma (cSCC) within weeks of treatment, driven by paradoxical RAS/RAF/MAPK pathway activation. Here we identify frequent TGFBR1 and TGFBR2 mutations in human vemurafenib-induced skin lesions and in sporadic cSCC. Functional analysis reveals these mutations ablate canonical TGFβ Smad signalling, which is localized to bulge stem cells in both normal human and murine skin. MAPK pathway hyperactivation (through BrafV600E or KrasG12D knockin) and TGFβ signalling ablation (through Tgfbr1 deletion) in LGR5+ve stem cells enables rapid cSCC development in the mouse. Mutation of Tp53 (which is commonly mutated in sporadic cSCC) coupled with Tgfbr1 deletion in LGR5+ve cells also results in cSCC development. These findings indicate that LGR5+ve stem cells may act as cells of origin for cSCC, and that RAS/RAF/MAPK pathway hyperactivation or Tp53 mutation, coupled with loss of TGFβ signalling, are driving events of skin tumorigenesis.

Differential Modulation of Circulating Endothelial Progenitor Cells in Peripheral Blood Monocyte Population of an Early Stage of Streptozotocin-Induced Diabetic Retinopathy

Objective: To enumerate the circulating endothelial progenitor cells (EPCs) in peripheral blood mononuclear cells (PBMC) in streptozotocin (STZ) induced diabetic retinopathy (DR) rat model. Materials and Methods: Retinal microvascular complications in STZ induced diabetic Wistar rats were validated after 4 months using trypsin digestion assay of retinal vasculature, histological analysis of retina, glial fibrillary acidic protein (GFAP) immunostaining. Subsequently, circulating EPCs were quantified in PBMCs from diabetes induced rats after 4 months using markers such as CD-34, CD-31, CD-146, VE-Cadherin and Flk-1 by flow cytometry. Results: After 4 months of diabetes induction, pericyte loss, acellular capillaries, retinal layer thickness, microglial activation and increased nitric oxide (NO) levels were observed. While the EPC markers such as CD-34, CD-31, VE-Cadherin were decreased, the Flk-1 +ve cells were up-regulated in the endogenous EPC pool. Conclusion: The quantity of EPCs are modulated in diabetes which may provide an insight into the use of EPCs as a biomarker for the progression of diabetes induced microvascular complication. Key words: Diabetic retinopathy, Endothelial progenitor cells, FlK-1, GFAP, Streptozotocin.

Abstract 637: Allograft Inflammatory Factor 1 Promotes Key Macrophage Functions that Limit Plaque Destabilization

Objective: Allograft inflammatory factor-1 (Aif-1) is best characterized as a cytoplasmic protein involved in actin bundling and phagocytosis. We investigated Aif-1 function in macrophage biology and the pathogenesis of atherosclerosis. Hypothesis: Aif-1-mediated potentiation of NFκB signaling promotes key macrophage functions and supports characteristics of plaque stability. Methods: We generated apoE -/- and aif-1 -/- ; apoE -/- mice (SKO and DKO, respectively), fed them a high fat diet for 18 weeks, and analyzed lesion burden in en face aorta, aortic root, and brachiocephalic arteries (BCA). We used immunofluorescence to assess marker expression and FACS analysis for proliferation and efferocytosis. Results: Lesion burden was not significantly different in aortic roots (DKO vs. SKO; 1.385e+006 ± 2065 vs 1.957e+006 ± 558, total lesion area), BCAs (DKO vs. SKO; 63 vs.65, % of Oil red O staining) and en face aortas (9 vs. 10, % of Oil red O staining). On the other hand, aortic root lesions from DKO mice showed more necrosis (DKO vs. SKO; 0.65 vs 0.4) and decreased fibrous cap thickness (DKO vs. SKO; 0.3 vs. 0.45, p0.05 respectively) normalized to lesion size. We also observed less macrophage staining (DKO vs. SKO; 0.6 vs. 2.2 % area/aortic root) and increased apoptosis (14 vs. 6 %Tunel+ cells), which correlated with decreased lesional P-NFκB p65 (DKO vs. SKO; 4 vs. 7 +nuclei/aortic root) activation. In vitro , aif-1 -/- macrophages showed decreases in oxidative stress-induced survival (40 vs. 80%), phagocytosis (30-60% decrease in zymosan particle uptake) and efferocytosis (40 vs.60 %F4/80;Tamra double +ve cells), and interleukin-6 release (1.2 vs 3 ng/ml in conditioned media). Mechanistically, these increased key macrophage functions correlated with P-NFκB p65 activation, which was impaired in aif-1 -/- macrophages. Conclusion: Our in vivo and in vitro data point to an essential role of Aif-1, via NFκB activation, in support of key macrophage functions, including survival and efferocytosis, that limit necrotic core size and increase atherosclerotic plaque stability.

CD66 and CD49f expressing cells are associated with distinct neoplastic phenotypes and progression in human cervical cancer

BackgroundIn this study, building on our recent work identifying a subset of CD66+ve cells with distinctive tumourigenic properties in human cervical cancers, we examine patterns of expression and function of these cells; to generate insights into the process of metastasis. MethodsOur broad approach in this study has been to compare the expression and function of two subsets marked by CD66 and CD49f. We use a combination of histopathology, immunostaining and flow cytometry, functional analysis of an established cervical cancer cell line and a retrospective analysis of a cohort of cervical cancer. ResultsWe noted CD66 expression associated with clusters of cells which are spindle shaped, SMA+ve, podoplanin+ve, phalloidin high, fibronectin high, plakoglobin low, ki67−ve and CK10+ve at the migratory phase along with features of partial EMT. Further, TGFβ1 a well known regulator of EMT, positively correlated with CD66 expression. The additional CD49f+ve subset at the leading invading front of migration was SMA−ve, phalloidin low, fibronectin low, plakoglobin high, Ki67+ve and CK14+ve. These data are consistent with a role for CD66 cells in metastatic invasion with a collective cell migration process co-opting the CD49f subset. Our retrospective analysis of a cohort is consistent with a role for CD66 in metastasis. However, the broad analysis of CD66, CD49f and TGFβ1 expression with patterns of overall survival points to a possible protective effect particularly for local recurrences. Hence, future studies focussing on potential heterogeneity within the CD66 subset along with the possible role of isoforms and intra-cellular roles would be essential.

Aloe vera extract functionalized zinc oxide nanoparticles as nanoantibiotics against multi-drug resistant clinical bacterial isolates

ZnO nanoparticles (ZnONPs) were synthesised through a simple and efficient biogenic synthesis approach, exploiting the reducing and capping potential of Aloe barbadensis Miller (A. vera) leaf extract (ALE). ALE-capped ZnO nanoparticles (ALE-ZnONPs) were characterized using UV–Vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM) analyses. XRD analysis provided the average size of ZnONPs as 15nm. FTIR spectral analysis suggested the role of phenolic compounds, terpenoids and proteins present in ALE, in nucleation and stability of ZnONPs. Flow cytometry and atomic absorption spectrophotometry (AAS) data analyses revealed the surface binding and internalization of ZnONPs in Gram +ve (Staphylococcus aureus) and Gram −ve (Escherichia coli) cells, respectively. Significant antibacterial activity of ALE-ZnONPs was observed against extended spectrum beta lactamases (ESBL) positive E. coli, Pseudomonas aeruginosa, and methicillin resistant S. aureus (MRSA) clinical isolates exhibiting the MIC and MBC values of 2200, 2400μg/ml and 2300, 2700μg/ml, respectively. Substantial inhibitory effects of ALE-ZnONPs on bacterial growth kinetics, exopolysaccharides and biofilm formation, unequivocally suggested the antibiotic and anti-biofilm potential. Overall, the results elucidated a rapid, environmentally benign, cost-effective, and convenient method for ALE-ZnONPs synthesis, for possible applications as nanoantibiotics or drug carriers.

Abstract B32: Microenvironmental distribution of trastuzumab in metastases and xenograft models is highly heterogeneous and decreases sharply when administered in combination with bevacizumab

Abstract Despite significant success, the response of Her2+ patients to trastuzumab (TzMAb, Herceptin ®) is varied, with many still experiencing tumor progression. We have used 3D tissue, tumor xenograft and metastatic models to examine the microregional distribution of TzMAb when administered alone or in combination with bevacizumab (BvMAb). Methods: Her2 positive (Her2+) SKOV-3 ovarian and MDA361, JIMT-1, BT474 mammary cancer cells were grown as 3D tissue discs, spheroids and as xenografts. Her2 expression was ranked as SKOV3 &amp;gt; BT474 &amp;gt; JIMT-1 &amp;gt; MDA361. Variable concentrations (25-100 µg/mL for in vitro; 2.5-10 mg/kg ip q3d for in vivo) of TzMAb, BvMAb or isotype IgG control antibodies were administered and tissues collected. Multiplexed immunohistochemistry generated maps of whole tissue sections for quantitative and qualitative analysis. In addition to direct visualization of fluorescent-tagged antibody therapeutics, features including Her2 expression (Her2), hypoxia (pimonidazole), blood vessels (CD31), vascular perfusion (carbocyanine fluorescent dye), pericytes (SMA, desmin), basal lamina (CIV) and tight junctions (ZO-1) were mapped relative to each other. Findings: In vitro: The rate of TzMAb distribution through 3D Her2+ tissue models was not significantly different to that of BvMAb or an isotype control (IgG). Inter-model variability was not correlated with the degree of Her2 expression. In vivo: All the xenograft models exhibited highly heterogeneous distribution of TzMAb, with variation at inter-vessel, inter-tumor and intra-tumor levels. There was no discernible pattern in the deposition of TzMAb; it was not limited to tumor margins or the central core, and was often bound up to 200 µm away from the nearest blood vessels. Other areas containing perfused vessels had little to no binding. Areas of limited TzMAb binding persisted after repeat dosing. SKOV-3 and BT474 metastatic lesions collected from liver and lung tissues had on average greater TzMAb staining intensity than was seen in subcutaneous tumors. However, there was a significant range in the ability of TzMAb to access and bind Her2+ve cells. Lesions ranged from less than 150 µm to several mm in diameter, with some metastases showing bound TzMAb on every cell and others showing no observable bound drug despite containing perfused vessels or being surrounded by highly perfused normal lung or liver tissue. For both subcutaneous tumors and metastatic lesions, no consistent, quantifiable difference or correlation was found between the amount of bound TzMAb and the presence of tight junctions, the density of nearest CD31 vessels, the location of hypoxic cells or the fraction of perfused, mature or immature vessels. Pretreatment with BvMAb for 24-72h resulted in significant reductions (up to 90% reduced) in tumor accumulation of TzMAb in all tumor models examined, including lung and liver metastases, despite the continued presence of perfused vessels. Summary: The extravascular distribution of TzMAb in Her2-overexpressing xenografts and metastases is heterogeneous and is not explained simply by the presence or absence of functioning vasculature. The relatively good distribution of TzMAb through 3D tissue models suggest that tight or irreversible binding to Her2 may not adequately explain the limited distribution of TzMAb seen in vivo. Pre-treatment with BvMAb dramatically decreases the access of TzMAb to the tumor microenvironment. Persistence of metastases and tissues with poor access to TzMAb suggests the possibility of inadequate drug exposure as a mechanism for resistance to TzMAb activity that could apply to additional targeted monoclonal antibody therapeutics. This resistance may be exacerbated when TzMAb or other antibodies are administered in combination with anti VEGF therapy. Citation Format: Jennifer H.E. Baker, Alastair H. Kyle, Jordan Cran, Haley Patrick, Maria-Jose Gandolfo, Andrew I. Minchinton. Microenvironmental distribution of trastuzumab in metastases and xenograft models is highly heterogeneous and decreases sharply when administered in combination with bevacizumab. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B32.

CDK9 inhibitors selectively target estrogen receptor-positive breast cancer cells through combined inhibition of MYB and MCL-1 expression.

Our previous studies showed that MYB is required for proliferation of, and confers protection against apoptosis on, estrogen receptor-positive (ER+ve) breast cancer cells, which are almost invariably also MYB+ve. We have also shown that MYB expression in ER+ve breast cancer cells is regulated at the level of transcriptional elongation and as such, is suppressed by CDK9i. Here we examined the effects of CDK9i on breast cancer cells and the involvement of MYB in these effects. ER+ve breast cancer cell lines including MCF-7 were much more sensitive (> 10 times) to killing by CDK9i than ER−ve/MYB−ve cells. Moreover, surviving cells showed a block at the G2/M phase of the cell cycle. Importantly, ectopic MYB expression conferred resistance to apoptosis induction, cell killing and G2/M accumulation. Expression of relevant MYB target genes including BCL2 and CCNB1 was suppressed by CDK9 inhibition, and this too was reversed by ectopic MYB expression. Nevertheless, inhibition of BCL2 alone either by MYB knockdown or by ABT-199 treatment was insufficient for significant induction of apoptosis. Further studies implied that suppression of MCL-1, a well-documented target of CDK9 inhibition, was additionally required for apoptosis induction, while maximal levels of apoptosis induced by CDK9i are likely to also involve inhibition of BCL2L1 expression. Taken together these data suggest that MYB regulation of BCL2 underlies the heightened sensitivity of ER+ve compared to ER−ve breast cancer cells to CDK9 inhibition, and that these compounds represent a potential therapeutic for ER+ve breast cancers and possibly other MYB-dependent cancers.

Embigin Regulates HSPC Homing and Quiescence and Acts As a Cell Surface Marker for a Niche Factor-Enriched Subset of Osteolineage Cells

Background. Discovery of niche-derived HSPC regulators is critical for further development of novel therapeutic approaches to promote HSPC regeneration. We have previously reported a proximity-based approach to the study of the bone marrow niche, which is based on the transcriptional comparison of osteolineage cells (OLCs) located close (proximal OLCs) or further away from transplanted HSPC (Blood 2014;124: 773), which lead to identification of IL18 as a quiescence regulator of early progenitors. We now report the results of functional validation for another molecule - Embigin - identified through this strategy as a hematopoietic regulator.Results. Embigin is a cell adhesion molecule with poorly characterized function. Our analysis showed that Embigin expression in proximal OLCs was significantly higher. Since the genetic tools for this molecule do not exist, we used a neutralizing antibody against Embigin to investigate the effect of Embigin blockade on primitive hematopoietic cells. We found that injection of anti-Embigin resulted in mobilization of myeloid progenitors and colony-forming cells (CFC) into the blood. In contrast, Embigin blockade was associated with a homing defect when LKS cells [known to express Embigin] were either pre-incubated with anti-Embigin antibody or injected into anti-Embigin pretreated animals, overall suggesting that Embigin regulates retention and localization of primitive hematopoietic cells in the bone marrow. Moreover, animals treated with anti-Embigin antibody had a higher frequency and proliferative activity of primitive hematopoietic cells, as demonstrated by cell cycle and BrdU incorporation studies and an increased CFC, consistent with Embigin-mediated localization also affecting HSPC cell cycling. Bone marrow from anti-Embigin treated mice reconstituted poorly when competitively transplanted with untreated animal marrow into irradiated recipients, likely due to the impaired homing and increased cell cycling. Finally, pre-treating irradiated recipients with anti-Embigin resulted in increased proliferation of transplanted WT LKS cells. Collectively, these data are consistent with Embigin serving as a regulator of HSPC localization and quiescence.Given a functional role of Embigin in the bone marrow niche and an overlapping pattern of expression with VCAM1, a known niche-derived HSPC regulator, in proximal OLCs we explored the use of Embigin in conjunction with VCAM1 as cell surface markers for prospective isolation of niche factor-enriched OLC subset by flow cytometry. Using this strategy, we were able to purify a rare OLC subset of CD45- Ter119-V CAM-1+E mbigin+ cells (termed VE cells), which were enriched for niche factor expression as compared to their non-VE counterparts. VE cells were transcriptionally distinct from other, previously defined niche subsets such as nestin-GFPdim mesenchymal stem cells, nestin-GFPbright pericytes and N-cadherin-positive osteoblastic cells. In particular, VE cells expressed higher levels of most niche factors than nestin-GFPbright cells - a cell population recently characterized as regulating HSPC quiescence. Interestingly, the expression profile of VE cells from animals transplanted with LT-HSCs and those which were irradiated but injected with saline alone demonstrated upregulation of cell adhesion molecules in the LT-HSC-injected group, suggesting that VE cells are involved in bidirectional communication within the niche.To investigate a functional role of VE cells, we performed co-culture experiments in which we compared the effect of VE and non-VE cells on HSPC proliferation and engraftment. We found that in the presence of VE cells, HSPCs proliferated at slower rate and generated a lower hematopoietic colony number, consistent with quiescence-inducing effect. Upon transplantation, VE-cultured HSPCs generated a higher level of chimerism compared to those cultured on non-VE layer, indicating a superior ability of VE cells to support engraftment and reconstitution properties of HSPC during the in vitro culture.Conclusion. Our work defines Embgin as a previously unrecognized hematopoietic regulator and a cell surface marker for a niche factor-enriched subset of the osteolineage cells which regulates HSPC quiescence. Pharmacological blockade of Embigin signaling may serve as a potential therapeutic tool to enhance hematopoietic regeneration. DisclosuresNo relevant conflicts of interest to declare.

Single Cell Whole Transcriptome Analysis Reveals Distinct Molecular Signatures of Therapy-Resistant Chronic Myeloid Leukemia Stem Cells

Molecularly targeted therapy frequently induces remissions in cancer, but rarely achieves complete disease eradication, with resulting risk of disease relapse/progression. Chronic myeloid leukemia (CML) is a good example, with rare, propagating stem cells (SCs) that are incompletely eradicated by BCR-ABL directed tyrosine kinase inhibitor (TKI) therapy in most cases. Consequently, CML relapse usually occurs following treatment discontinuation. Further, some patients fail to achieve a satisfactory response to TKI therapy and are at risk of disease progression. Ultimately, the depth of response to TKIs is dictated by CML-SCs, however, it has proven challenging to characterise this crucial population of cells as they reside in the same compartment as their normal hematopoietic stem cell (HSC) counterparts from which they cannot be reliably distinguished throughout the disease course.Advances in single cell transcriptomics are opening up unprecedented opportunities to unravel heterogeneity in cell populations. However, to date, this technology has primarily been used to analyse normal tissues, partly reflecting the lack of sensitivity for detection of somatic mutations using current techniques. Herein, we developed a novel protocol allowing targeted amplification of BCR-ABL during single cell cDNA library generation using a modification of a published protocol (PMID:24385147). We then applied this method to analyse 1082 bone marrow SCs (defined as Lin-CD34+CD38-) from a cohort of 10 patients with CML and 3 normal controls.We first validated our method using the BCR-ABL +ve K562 cell line. Surprisingly, using standard methods, BCR-ABL was only detected in a small minority of cells. With our modified technique, we demonstrate robust detection of BCR-ABL in 100% of single K562 cells with parallel whole transcriptome amplification from the same cell. Further, using a plasmid Òspike-inÓ, we demonstrate sensitivity to detect a single molecule of BCR-ABL per cell, without false +ve BCR-ABL amplification in 142 cells tested. We then analysed 40 SCs from a patient with CML who was in a cytogenetic remission (CyR) following 3 months of TKI therapy. BCR-ABL was detected in 16 cells and was absent in 24 cells. With an average of 3.3 million mapped reads per single cell, we detected over 4600 genes per cell with good correlation of single cell ensemble data with bulk analysis. Using this approach we were uniquely able to compare BCR-ABL +ve and -ve cells and identify a large number of differentially expressed genes, which would not have been detected through bulk analysis. We selected a number of genes of biologic interest for validation by Q-PCR and 80% of them were confirmed as differentially expressed, a number of these are highlighted in the heatmap (see Figure).We then analysed 717 single SCs from 8 CML diagnostic samples. This analysis identified marked heterogeneity of BCR-ABL expression with frequencies comparable to the published literature (9% to 92%). Unsupervised clustering analysis revealed many genes that were differentially expressed between BCR-ABL +ve and -ve SCs e.g. CML-SCs showed upregulation of cell cycle signatures and decreased expression of HSC-affiliated genes. We next analysed 223 single SCs from a series of 5 patients in CyR following TKI initiation. BCR-ABL positive SCs could still be detected, albeit at a reduced frequency (0.5% to 45%). In contrast to the diagnostic samples, CML-SCs showed quiescence and HSC signatures that were comparable to normal HSCs. Furthermore, through analysis of serial samples from diagnosis to remission from the same patient, this single-cell approach uniquely allowed analysis of gene expression specifically of the CML-SCs which evade TKI therapy, facilitating the identification of a number of candidate gene-sets including cell surface markers and potentially ÒdruggableÓ targets. Finally, through analysis of a patient with early blast crisis transformation of CML during TKI therapy, we illustrate how single cell RNA sequencing might be applied to predict such early disease progression events.We herein describe a new approach for single cell RNA sequencing of CML-SCs that might be applied to fate-map persistent CML-SCs during and following treatment for discovery research and also to refine precision medicine in CML. This approach could be applied across a range of clonal disorders with potential broader relevance for cancer research. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Combination of Anti-CD123 and Anti-CD19 Chimeric Antigen Receptor T Cells for the Treatment and Prevention of Antigen-Loss Relapses Occurring after CD19-Targeted Immunotherapies

Introduction: Anti-CD19 chimeric antigen receptor T cells (CART19) and bi-specific anti-CD19/CD3 antibodies (blinatumomab) generate unprecedented complete response rates of 45-90% in relapsing/refractory acute lymphoblastic leukemia (r/r B-ALL). However, a subset of patients treated with these targeted approaches will relapse and a significant portion of these relapses are characterized by the loss of detectable CD19 (about 30% of relapses after blinatumomab and up to 50% after CART19), a clear manifestation of immunoediting. Hence, novel effective strategies are needed in order to be able to treat those patients and ideally prevent antigen-loss. CD123, the interleukin-3 receptor alpha, is involved in hematopoiesis and has been shown to be expressed in several hematologic neoplasms, including acute myeloid leukemia (AML) and more recently also B-ALL. Targeting CD123 with chimeric antigen receptor T cells (CART123) was shown to lead to deep and long-term responses in human primary AML xenografts. The goal of this study was to pre-clinically evaluate the impact of targeting CD123 and CD19 with chimeric antigen receptor T cells for the treatment and prevention of CD19-negative leukemia relapses occurring after CD19-directed therapies.Results: We analyzed the expression of CD123 in 42 r/r B-ALL samples and found that CD123 is highly and homogeneously expressed on the surface of most B-ALL blasts (81.75%, range: 5.1-99.6), making it a promising candidate for targeted therapy in B-ALL. Moreover, CD123 was also found to be expressed in the putative leukemia stem cells (LSC), identified as CD34-pos CD38-neg. Notably, in some Ph+ B-ALL samples we found CD19-ve CD123+ve cells with a BCR-ABL translocation by FISH, suggesting that these cells too are malignant. The expression of CD123 was detected in all (n=6) CD19-negative B-ALL blasts analyzed after relapse from CART19 treatment (Figure 1). These findings indicate that CD123 represents an ideal marker to target CD19-neg ALL blasts occurring after CART19 or blinatumomab. Therefore, we generated anti-CD123 chimeric antigen receptor T cells costimulated with 4-1-BB using a lentiviral vector (CART123). We then evaluated the CART123 anti-B-ALL efficacy both in vitro and in vivo against primary B-ALL and cell line (NALM-6). CART123 showed intense anti-B-ALL ex vivo activity, as defined by specific CD107a degranulation, cytokine production, cytotoxicity and proliferation, not statistically different from that of CART19. In order to test the role of CART123 to target CD19-negative relapses we developed a novel in vivo model, engrafting immunodeficient NSG mice with blasts obtained from a patient relapsing with CD19-ve disease after CART19 treatment. At day 14 mice were randomized to receive CART19, CART123 or control T cells (untransduced, UTD). CART19 and control T cell treated mice had no anti-tumor activity, while CART123 led to complete eradication of the disease and long-term survival. We next developed a murine model to test the hypothesis that a combined approach simultaneously targeting CD123 and CD19 could treat and prevent CD19-negative relapses. NSG mice were injected with a mixture of primary CD19-neg and CD19-pos blasts from the same patient that were labeled with different click beetle luciferases (red or green) in order to be able to track the respective clones in vivo. Mice were then randomized to receive UTD, CART19 or the combination of CART19 and CART123 (same total dose of T cells). As shown in Figure 2, mice treated with UTD had progression of both leukemic clones (CD19-pos and CD19-neg) while CART19 showed rapid progression mostly of the CD19-neg disease (red luciferase); on the contrary only mice treated with the combination of CART123 and CART19 showed rapid clearance of the disease, with improved overall survival (64 days for CART19, not reached for CART19+CART123). As a final strategy, we expressed both CAR19 and CAR123 in the same T cells and showed potent anti-leukemia activity (CD107a degranulation 81.7%).Conclusions: Here we demonstrate that CD123 is expressed in CD19-negative B-ALL relapses occurring after CD19-directed therapies, and that combining CART123 cells with CART19 cells is an effective therapy for the treatment and prevention of antigen-loss relapses in B-ALL murine xenografts. [Display omitted] [Display omitted] DisclosuresRuella:Novartis: Patents & Royalties, Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding. Scholler:Novartis: Patents & Royalties. Lacey:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding. Hunter:Surface Oncology - SAB: Membership on an entity's Board of Directors or advisory committees, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding. Grupp:Novartis: Consultancy, Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.

Comparative Histological Study on the Therapeutic Effect of Green Tea and Stem Cells in Alzheimer's Disease Complicating Experimentally Induced Diabetes.

Background and ObjectivesAlzheimer’s disease (AD) is a devastating neurodegenerative disorder. Increasing evidence implicates diabetes mellitus (DM) as a risk factor for AD. Green tea (GT) has several beneficial effects attributed to its anti-oxidant phenolic compounds. Adipose tissue is a rich source of adipose-derived mesenchymal stem cells (ADSCs). This study was designed to evaluate and compare the possible therapeutic effect of green tea extract (GTE) and ADSCs on AD complicating induced DM in male rat.Methods31 adult male albino rats were divided into 5 groups. Group I (Control), Group II received GTE, 50 mg/kg daily orally for 4 weeks, Group III received a single intraperitoneal injection of Streptozotocin (STZ), 50 mg/kg, Group IV: received STZ followed by GTE and Group V: received STZ followed by human ADSCs (hADSCs) intravenously.ResultsMultiple acidophilic masses, deformed neurons, Congo red +ve masses and Caspase 3 +ve neurons were seen in group III, became few in group IV and occasional in group V. Multiple Prussian blue +ve cells were detected in group V. Some CD44 +ve cells were noticed in group III, became multiple in groups IV and V. The mean area of neurons exhibiting acidophilic cytoplasm, mean area of amyloid plaques and mean area % of Caspase 3 +ve cells indicated a significant increase in group III. The mean area % of CD44 +ve cells recorded a significant increase in group IV.ConclusionshADSCs exerted a more marked therapeutic effect on the neurodegenerative changes complicating DM and corresponding to AD.

Fibroblast Growth Factor-9 Activates c-Kit Progenitor Cells and Enhances Angiogenesis in the Infarcted Diabetic Heart.

We hypothesized that fibroblast growth factor-9 (FGF-9) would enhance angiogenesis via activating c-kit positive stem cells in the infarcted nondiabetic and diabetic heart. In brief, animals were divided into three groups: Sham, MI, and MI+FGF-9. Two weeks following MI or sham surgery, our data suggest that treatment with FGF-9 significantly diminished vascular apoptosis compared to the MI group in both C57BL/6 and db/db mice (p < 0.05). Additionally, the number of c-kit+ve/SM α-actin+ve cells and c-kit+ve/CD31+ve cells were greatly enhanced in the MI+FGF-9 groups relative to the MI suggesting FGF-9 enhances c-Kit cell activation and their differentiation into vascular smooth muscle cells and endothelial cells, respectively (p < 0.05). Histology shows that the total number of vessels were quantified for all groups and our data suggest that the FGF-9 treated groups had significantly more vessels than their MI counterparts (p < 0.05). Finally, echocardiographic data suggests a significant improvement in left ventricular output, as indicated by fractional shortening and ejection fraction in both nondiabetic and diabetic animals treated with FGF-9 (p < 0.05). Overall, our data suggests FGF-9 has the potential to attenuate vascular cell apoptosis, activate c-Kit progenitor cells, and enhance angiogenesis and neovascularization in C57BL/6 and db/db mice leading to improved cardiac function.

Oestrogen receptor positive breast cancer metastasis to bone: inhibition by targeting the bone microenvironment in vivo.

Clinical trials have shown that adjuvant Zoledronic acid (ZOL) reduces the development of bone metastases irrespective of ER status. However, post-menopausal patients show anti-tumour benefit with ZOL whereas pre-menopausal patients do not. Here we have developed in vivo models of spontaneous ER+ve breast cancer metastasis to bone and investigated the effects of ZOL and oestrogen on tumour cell dissemination and growth. ER+ve (MCF7, T47D) or ER-ve (MDA-MB-231) cells were administered by inter-mammary or inter-cardiac injection into female nude mice ± estradiol. Mice were administered saline or 100 μg/kg ZOL weekly. Tumour growth, dissemination of tumour cells in blood, bone and bone turnover were monitored by luciferase imaging, histology, flow cytometry, two-photon microscopy, micro-CT and TRAP/P1NP ELISA. Estradiol induced metastasis of ER+ve cells to bone in 80-100 % of animals whereas bone metastases from ER-ve cells were unaffected. Administration of ZOL had no effect on tumour growth in the fat pad but significantly inhibited dissemination of ER+ve tumour cells to bone and frequency of bone metastasis. Estradiol and ZOL increased bone volume via different mechanisms: Estradiol increased activity of bone forming osteoblasts whereas administration of ZOL to estradiol supplemented mice decreased osteoclast activity and returned osteoblast activity to levels comparable to that of saline treated mice. ER-ve cells require increased osteoclast activity to grow in bone whereas ER+ve cells do not. Zol does not affect ER+ve tumour growth in soft tissue, however, inhibition of bone turnover by ZOL reduced dissemination and growth of ER+ve breast cancer cells in bone.

Abstract 13831: Atrial and Ventricular Cardiac Stem Cells From the Same Heart Exhibit Differential Functional Characteristics for Cardiac Regeneration but Are Superior to the Endothelial Progenitor Cells From the Same Patient

Introduction: Deciding the best cell type for cardiac regeneration remains a challenge, however, no studies have directly compared the functional efficacy of cardiac stem cells (CSCs) with extra- cardiac SCs isolated from the same patient. Objective: To compare the functional characteristics of endothelial progenitor cells (EPCs), right atrial (RAA) CSCs and left ventricular (LV) CSCs isolated from the same patients. Methods and Results: Flow-cytometry analysis of CSCs (n=12 patients) revealed higher expression of CD90 (involved in cell adhesion and migration) in RAA CSCs (CD90 &amp; CD105 ++ve cells: 71±15% in RAA vs. 47±27% in LV, P&lt;0.05). However, scratch assay showed superior wound closure in LV CSCs (80±10% in LV vs. 60±5% in RAA, P&lt;0.05) suggesting that LV CSCs have a better migration potential. Moreover, the expansion properties of RAA CSCs were better than LV CSCs in terms of higher cumulative population doublings in culture. To simulate in vivo ischemic environment, cells were subjected to hypoxia (1% O2) and serum starvation for 3 days. RAA and LV CSCs exhibited similar pattern of resistance to apoptotic cell death (evaluated as percentage of annexin +ve cells, n=5) and proliferation under hypoxia. Interestingly, EPCs exhibited highest resistance to apoptotic cell death, but they also showed the lowest proliferation under hypoxia (P&lt;0.05). Secretion of paracrine and pro-angiogenic factors have been thought to be most important mechanisms behind cardiac regeneration. Therefore, to compare the paracrine angiogenic and wound healing potential, HUVECs were cultured in culture supernatant (CS) from all the 3 cell types. RAA-CS showed the highest increase in tube formation capacity (P&lt;0.05, n=6), while the scratch assay showed comparable wound closure with both LV and RAA CS (67.56 ± 7.36% and 70.02 ± 5.70%, n=5), but was lowest in EPC CS (54±7%). The secretion of the angiogenic growth factor VEGF-A (n=5) was lowest in EPCs (0.36± 0.20 ng/mL) and comparable between RAA (3.44±1.94 ng/mL) and LV CSCs (4.33± 1.57 ng/mL). Conclusion: Stem cells from different regions exhibit differential functional characteristics within the same patient. The selection of the best cell type should therefore be influenced by the nature of the injury and the type of effect desired.

Abstract 10198: Endothelin B Receptor Agonist, IRL-1620 is an Effective Neurovascular Remodeling Agent in Diabetic Rats With Cerebral Ischemia

Endothelin B (ETB) receptor agonist, IRL-1620 administered at 2, 4, 6 hr post-cerebral ischemia, has been shown to significantly aid in neuroprotection and neurovascular remodeling in normal rats. The incidence of type 2 diabetes mellitus (diabetes) is on the rise and it can be expected that there will be an upsurge of associated diseases, such as ischemic stroke in patients with diabetes. The present study was conducted to determine the efficacy and neurovascular remodeling effect of IRL-1620 in normal (non-diabetic) and diabetic rats following cerebral ischemia induced by right middle cerebral artery (MCAO). The induction of diabetes was achieved through a high-fat diet and single low dose (45 mg/kg) of streptozotocin. IRL-1620 (5 μg/kg) was administered intravenously at 4, 6, and 8 hr after MCAO on day 0, 3 and 6 (total of 15 μg/kg in three divided doses on day 0, 3 and 6). A mortality of 46% was observed by day 7 in normal rats with MCAO when treated with vehicle, while it was 17% when treated with IRL-1620. In diabetic rats 90% mortality was observed by day 7 post-MCAO when treated with vehicle, while it was 45% when treated with IRL-1620. Functional outcome for the IRL-1620 group was significantly better in both non-diabetic and diabetic rats compared to the vehicle group. In both non-diabetic (80.81%; p=0.02) and diabetic (69.58%; p=0.04) rats a significant reduction in infarct volume was seen in the IRL-1620 group compared to the vehicle group. Significantly more blood vessels/30μm brain slice were observed in the IRL-1620 versus vehicle group. Immunofluorescence staining revealed a significant increase in ETB receptor expression for rats treated with IRL-1620, compared to vehicle. Co-localization of VEGF+ve and endothelial cells in diabetic rats was higher (p=0.0009) in the IRL-1620 group (15.5±1.1%) compared to the vehicle group (10.2±0.81%). In diabetic rats, the IRL-1620 group had significantly (p=0.0012) more neurons in the cortex compared to the vehicle group as well as the highest levels of neuronal replication. Administration of BQ-788, a specific ETB receptor antagonist, attenuated the effects of IRL-1620. It is concluded that IRL-1620 is an effective neurovascular remodeling agent and may be useful in the treatment of cerebral ischemia.

Isolation and Characterization of Human Adult Epithelial Stem Cells from the Periodontal Ligament

We report a novel method for the isolation of adult human epithelial stem cells (hEpiSCs) from the epithelial component of the periodontal ligament—the human epithelial cell rests of Malassez (hERM). hEpiSC-rich integrin-α6+ve hERM cells derived by fluorometry can be clonally expanded, can grow organoids, and express the markers of pluripotency (OCT4, NANOG, SOX2), polycomb protein RING1B, and the hEpiSC supermarker LGR5. They maintain the growth profile of their originating hERM in vitro. Subcutaneous cotransplantation with mesenchymal stem cells from the dental pulp on poly-l-lactic acid scaffolds in nude mice gave rise to perfect heterotopic ossicles in vivo with ultrastructure of dentin, enamel, cementum, and bone. These remarkable fully mineralized ossicles underscore the importance of epithelial-mesenchymal crosstalk in tissue regeneration using human progenitor stem cells, which may have already committed to lineage despite maintaining hallmarks of pluripotency. In addition, we report the clonal expansion and isolation of human LGR5+ve cells from the hERM in xeno-free culture conditions. The genetic profile of LGR5+ve cells includes both markers of pluripotency and genes important for secretory epithelial and dental epithelial cell differentiation, giving us a first insight into periodontal ligament–derived hEpiSCs.