Ex Vivo High-Throughput Flow Cytometry Screening Identifies Subsets of Responders to Differentiation Agents in Individual AML Patient Samples

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5206-5206 ◽  
Author(s):  
Sacha L. Prashad ◽  
Leylah Drusbosky ◽  
Hassan Sibai ◽  
Mark D. Minden ◽  
Stephen J. Western ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Ex Vivo ◽  
Surface Marker ◽  
Cell Surface Marker ◽  
Employment Equity ◽  
Surface Marker Expression ◽  
Cd163 Expression ◽  
Differentiating Agents ◽  
Equity Ownership

Abstract Background: Prognoses for acute promyelocytic leukemia (APL) patients improved drastically upon the introduction of differentiation therapy with all-trans-retinoic acid (ATRA) in combination with conventional chemotherapy. Unfortunately, this therapeutic approach has not translated to other genetic subtypes of acute myeloid leukemia (AML) where patients demonstrate marked heterogeneity to differentiating agents. To provide improved detection of drug-induced differentiation in AML patients, we have developed a high-throughput, flow cytometry-based personalized medicine platform. Methods: Total white blood cells were isolated from each patient sample by red cell lysis, plated in serum-free media in 384-well format and incubated with drugs for 3 days. Viable cells remaining after each drug treatment were identified and quantified using cell surface marker expression, cell membrane integrity, and morphology (FSC/SSC) to determine the compound's efficacy and specificity against the blast population. Changes in cell surface marker expression and shifts in morphology indicative of blast differentiation were also evaluated with each compound. As a control for ex vivo differentiation, two APL patient samples were treated ex vivo with ATRA and we observed the blasts gaining CD66b expression indicating granulocytic differentiation. Results: A refractory AML patient was identified whose leukemic blasts exhibited a strong differentiating response to dexamethasone treatment ex vivo. This resulted in loss of CD34 expression (a marker of immature blast cells), gain of CD163 expression (a marker of monocytic/macrophage maturation) and a significant change in cellular size and granularity. After being enrolled in a clinical trial (REB: 13-6962-C) the patient was treated based on the assay for 1 week (40 mg/day) with dexamethasone. Post-treatment samples from the peripheral blood and bone marrow of the patient exhibited the same morphological and cell surface marker changes predicted by the ex vivo assay. The CD163+ cells in the patient also gained additional markers of myeloid differentiation (CD11b, CD14, CD16). After additional cytarabine and fludarabine treatment, the patient remains in remission 4 months post-treatment. Conclusions: Following this initial study, we have continued to identify subgroups of both AML and Myelodysplastic Syndrome patients where blasts differentiate in response to dexamethasone, calcitriol, ATRA or other known differentiating agents using unique cell surface markers of monocytic and myeloid maturation. Flow cytometry expression changes correlated with changes in morphology as observed by May-Grunwald Giemsa staining. In the patient described above this included an increase in cytoplasm and vacuoles consistent with monocytic/macrophage differentiation, which positively correlates with CD163 expression. We aim to apply our assay towards the identification of subgroups of AML patients who respond to differentiation therapies and develop clinical trials to combine differentiating agents with chemotherapy. This approach has the potential to extend the clinical success of APL differentiation therapy to AML patients. Disclosures Prashad: Notable Labs: Employment, Equity Ownership. Western:Notable Labs: Consultancy. Biondi:Notable Labs: Employment. Shah:Notable Labs: Employment. Liu:Notable Labs: Employment, Equity Ownership. Nguyen:Notable Labs: Employment, Equity Ownership. Warnock:Notable Labs: Employment, Equity Ownership. Quinzio:Notable Labs: Employment, Equity Ownership. De Silva:Notable Labs: Employment, Equity Ownership. Schimmer:Novartis: Honoraria. Heiser:Notable Labs: Employment, Equity Ownership.

TT30, a Novel Human Protein Therapeutic, Selectively Modulates the Complement Alternative Pathway by Targeted Supplementation of Local Factor H Activity.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3021-3021 ◽  
Author(s):  
V. Michael Holers ◽  
Istvan Mazsaroff ◽  
Hillary Akana ◽  
Christopher G. Smith ◽  
J. Woodruff Emlen ◽  
...  
Keyword(s):  
Cell Surface ◽  
Potent Inhibitor ◽  
Ex Vivo ◽  
Alternative Pathway ◽  
Factor H ◽  
Employment Equity ◽  
Complement Alternative Pathway ◽  
Equity Ownership ◽  
The Complement System

Abstract Abstract 3021 Poster Board II-997 The complement system is activated through three pathways: classical, lectin/mannose and alternative. Polymorphisms and mutations that promote Complement Alternative Pathway (CAP) activity are associated with human diseases including atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration (AMD). The complement system is also centrally involved in many hemolytic disorders, including paroxysmal nocturnal hemoglobinuria (PNH) where the CAP initiates complement activation resulting in intravascular hemolysis (IVH) after engagement of C5 and formation of the membrane attack complex (MAC). Systemic neutralization of C5 with the anti-C5 monoclonal antibody, eculizumab, abrogates IVH when plasma concentrations are maintained above the minimal efficacious concentration (Cmin = 35 μg/mL). However, because eculizumab does not inhibit CAP activity prior to C5, C3 fragments (C3frag) continue to covalently bind to and accumulate on PNH red blood cells (RBCs). Clearance by the reticuloendothelial system of PNH RBCs that are C3frag-coated is a putative cause of extravascular hemolysis (EVH) in eculizumab-treated patients. In order to selectively modulate CAP activity, we developed TT30, a novel therapeutic 65kD fusion protein linking the first four short consensus repeat (SCR) domains of human complement receptor type 2 (CR2/CD21) with the first five SCR of human factor H (fH). CR2 SCR1-4 encompasses the antigen-fixed C3frag (iC3b, C3dg and C3d) binding domain. Factor H is the primary soluble phase, negative regulator of CAP activity functioning via the SCR1-5 domains. The unique mechanism of TT30 utilizes CR2 SCR1-4 to recognize and bind to C3frag on cells in which complement activation is occurring, thus delivering cell surface-targeted inhibition of CAP activity via fH SCR 1-5. TT30 both prevents CAP-dependent hemolysis of rabbit RBCs in human serum and blocks accumulation of C3frag on the RBC surface. By design, TT30 should also be a potent inhibitor of the CAP, but with minimal inhibition of the complement classical (CCP) and mannose (lectin; CMP) pathways. To test this hypothesis, we utilized sensitive pharmacodynamic assays that allow in vitro or ex vivo assessment in an ELISA format of individual complement pathway activity present in human serum. In this format, TT30 is a potent and selective inhibitor of CAP activity in normal human complement-preserved serum, with EC50 and EC100 values of ∼0.1 and 1 μg/mL serum. As predicted by the use of fH in its construction, TT30 is a much less potent inhibitor of the CCP and CMP, with EC100 values of ∼65 μg/mL. By contrast, in these assays a monoclonal and polyclonal anti-C5 antibody each demonstrate non-selective inhibition of CAP and CCP activity at all effective concentrations. TT30 activity is dependent upon CR2 binding to C3frag, as an anti-CR2 monoclonal antibody reverses the surface inhibition of CAP activity. This surface-targeting approach to delivering fH SCR1-5 results in a molecule with a 10-fold potency gain in CAP inhibition relative to added purified fH and an ∼30-fold potency gain relative to the total fH present in the serum used in the assay. TT30 administered as a single IV injection at 20 mg/kg to rats, rabbits and monkeys results in Cmax values of ∼400, 500 and 300 μg/mL and concentration-dependent inhibition of CAP activity. At serum concentrations of TT30 that induced maximal (100%) inhibition of systemic CAP activity for up to 12 hours, CCP activity is modestly (∼35-60%) inhibited for only 2 hours. CAP activity returns to baseline levels in a predictable fashion. Pharmacokinetic analysis indicates no gender-related differences and the expected scaling of parameters across species. TT30 is pharmacologically active in monkeys, rabbits and mice. TT30 administered as a single subcutaneous injection at 20 mg/kg to monkeys results in Cmax values of ∼25 μg/mL, and EC100 values identical to those observed with IV administration, but with a 3-fold prolongation of the maximal pharmacodynamic effect. The novel therapeutic TT30 has been shown in vitro and ex vivo to deliver cell surface-targeted control of CAP activation with minimal CCP and CMP inhibition and effective blockade of C3frag accumulation and MAC formation. As a result, TT30 has potential utility for the treatment of complement-mediated diseases such as PNH, AMD and aHUS, in which cell surface-targeted control of CAP activation may be clinically beneficial. Disclosures Holers: Taligen Therapeutics: Employment, Equity Ownership, Patents & Royalties, Research Funding. Mazsaroff:Taligen Therapeutics: Employment. Akana:Taligen Therapeutics: Employment. Smith:Taligen Therapeutics: Employment. Emlen:Taligen Therapeutics: Employment, Equity Ownership. Marians:Taligen Therapeutics: Employment. Horvath:Taligen Therapeutics: Employment.

scholarly journals Shared Cell Surface Marker Expression in Mesenchymal Stem Cells and Adult Sarcomas

2012 ◽  
Vol 2 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Stefan Wirths ◽  
Elke Malenke ◽  
Torsten Kluba ◽  
Simone Rieger ◽  
Martin R. Müller ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Surface ◽  
Surface Marker ◽  
Cell Surface Marker ◽  
Surface Marker Expression ◽  
Marker Expression ◽  
Cell Surface Marker Expression

scholarly journals Transcriptional, Protein-Level and Functional Profiling of Human Fetal Liver-Derived Hematopoietic Stem Cells at Single Cell Resolution

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1187-1187
Author(s):  
Kim Vanuytsel ◽  
Carlos Villacorta-Martin ◽  
Wilfredo Garcia Beltran ◽  
Taylor Matte ◽  
Alejandro Balazs ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Surface ◽  
Single Cell ◽  
Protein Level ◽  
Fetal Liver ◽  
Surface Marker ◽  
Cell Surface Marker ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Surface Marker Expression

Intro: In the mouse, hematopoietic stem cells (HSCs) can be isolated and characterized at single cell resolution using a well-defined panel of markers. While it is possible to enrich for human HSCs using a panel of associated markers, similar resolution has not been attained. By profiling HSCs residing in the human fetal liver (FL) using a novel technique called CITE-Seq that combines single cell RNA sequencing (scRNAseq) and cell surface marker interrogation using oligo-tagged antibodies, we aimed to establish an accurate molecular signature of engraftable human HSCs shortly after they arise in development. As HSCs are defined functionally, we have coupled this transcriptomic and protein-level characterization with transplantation assays in immunocompromised NOD scid gamma (NSG) mice to connect expression profiles of cell subsets with functional engraftment. Methods: CITE-Seq was performed on human FL cells (week 19) that showed robust engraftment capability in NSG mice. CD34+ and CD34- cells were magnetically separated and stained with a panel of 19 oligo-tagged antibodies that were deemed relevant to characterize HSCs, including classical HSC markers but also novel targets that were identified in a previous pilot scRNAseq experiment conducted on CD34+ FL cells. From the CD34+ fraction, we sorted live-gated cells (CD34+bulk) as well as a population of cells that was further enriched based on the expression of GPI-80, a marker tightly linked to engraftment potential (CD34+GPI-80+, ~3%). CD34-GlycophorinA(GYPA)- cells were also sorted to assay for the presence of CD34- HSCs. These fractions were then loaded onto the 10x Genomics platform for capture of single cells and subsequent reverse transcription and amplification of both mRNAs and antibody-derived tags (ADTs). Results: Both mRNA and ADT libraries were successfully sequenced, yielding 29-43,000 reads/cell for the mRNA portion and >1,500 reads/cell for the ADT fraction. After quality control and filtering, this effort resulted in 8,775 CD34+bulk cells, 7,279 CD34+GPI-80+ cells, and 6,937 CD34-GYPA- cells available for further analysis. Simultaneous transplantation experiments of the fractions assayed by CITE-seq revealed superior engraftment potential of the CD34+GPI-80+ fraction, confirming enrichment for bona fide HSCs at the functional level. This was also reflected in the scRNAseq data where we found enrichment for known HSC markers such as VNN2 (GPI-80), PROM1 (CD133), PROCR (EPCR), THY1 (CD90), ITGA6 (CD49f), HMGA2, CLEC9A and HLF in the CD34+GPI-80+ fraction compared to CD34+bulk cells. As our pilot studies revealed considerable differences in transcriptional expression (via scRNAseq) as compared to protein-level expression (via cell surface marker expression), integration of the transcriptomic and cell surface marker expression data will further refine the signature of engraftable HSCs. Both layers of information at single cell resolution will allow for the identification of novel markers or unique combinations of markers that are directly correlated with engraftment potential. Conclusion: By isolating the GPI-80+ population within the CD34+ fraction in human FL, we have achieved unprecedented resolution of the signature of engraftable HSCs as confirmed by transplantation experiments. The in-depth characterization of this compartment as well as the surrounding CD34+ and CD34- cells within the FL is expected to yield valuable insights with respect to several biological questions. This data can be directly harnessed in improving the purification and expansion of engraftable HSCs as well as in guiding the in vitro generation of HSCs from pluripotent stem cells. Disclosures No relevant conflicts of interest to declare.

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