High-speed flow cytometric analysis of nanoparticle targeting to rare leukemic stem cells in peripheral human blood: preliminary in-vitro studies

Leukemic cancer stem cells are both stem-like and leukemic-like. This complicates their detection as rare circulating tumor cells in peripheral blood of leukemia patients. The leukemic stem cells are also highly resistant to standard chemotherapeutic regimens so new therapeutic strategies need to be designed to kill the leukemic stem cells without killing normal stem cells. In these initial studies we have designed an antibody-targeted and fluorescent (Cy5.5) nanoparticle for targeting these leukemic stem cells and then introducing new strategies for killing them. Multicolor flow cytometric analyses were performed on a BD FACS Aria III. Human leukemic stem cell-like cell line RS4;11 (with putative immunophenotype CD123+/CD24+/CD38-/CD10-/Flt-3-) was used as a model human leukemic stem cell systems and were spiked into normal human peripheral blood cells containing normal blood stem-progenitor cells (immunophenotype CD123-/CD34+/CD38-) and Cy5.5-labeled nanoparticles with targeting molecule anti-CD123 antibody. An irrelevant antibody (CD71) which should not bind to any live leukemic stem cell or normal stem cell (binds erythrocytes) was used as a way of distinguishing between true-positive live and false-positive damaged/dead cells, the latter occurring at much higher frequencies than the very rare (e.g. 0.001 to 0.0001 percent frequency true leukemic stem cells). These studies are designed to measure the targeting sensitivity and specificity of the fluorescent nanoparticles to the putative rare leukemic stem cells with the eventual design to use the nanoparticles to direct killing therapeutic doses to the leukemic stem cells but not to the normal stem-progenitor cells.

[1]  T. Barbui,et al.  Improved risk classification for risk-specific therapy based on the molecular study of minimal residual disease (MRD) in adult acute lymphoblastic leukemia (ALL). , 2009, Blood.

[2]  B. Wood,et al.  9-color and 10-color flow cytometry in the clinical laboratory. , 2006, Archives of pathology & laboratory medicine.

[3]  J. Hanley,et al.  The meaning and use of the area under a receiver operating characteristic (ROC) curve. , 1982, Radiology.

[4]  J. V. van Dongen,et al.  Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of the AIEOP-BFM ALL 2000 study. , 2010, Blood.

[5]  A J van der Sluijs-Gelling,et al.  Improved flow cytometric detection of minimal residual disease in childhood acute lymphoblastic leukemia , 2013, Leukemia.

[6]  John A. Swets,et al.  Evaluation of diagnostic systems : methods from signal detection theory , 1982 .

[7]  E. Thiel,et al.  Adult patients with acute lymphoblastic leukemia and molecular failure display a poor prognosis and are candidates for stem cell transplantation and targeted therapies. , 2012, Blood.

[8]  Dario Campana,et al.  Minimal residual disease in acute lymphoblastic leukemia. , 2010, Hematology. American Society of Hematology. Education Program.

[9]  James F. Leary,et al.  High-speed cell classification systems for real-time data classification and cell sorting , 1997, Photonics West - Biomedical Optics.

[10]  V. V. D. van der Velden,et al.  Defining consensus leukemia-associated immunophenotypes for detection of minimal residual disease in acute myeloid leukemia in a multicenter setting , 2013, Blood Cancer Journal.

[11]  D. Hoelzer,et al.  Modern therapy of acute lymphoblastic leukemia. , 2011, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[12]  D. Campana,et al.  Immunologic minimal residual disease detection in acute lymphoblastic leukemia: a comparative approach to molecular testing. , 2010, Best practice & research. Clinical haematology.

[13]  Peter G. Smith,et al.  Drug Efflux Transporters and Multidrug Resistance in Acute Leukemia: Therapeutic Impact and Novel Approaches to Mediation , 2012, Molecular Pharmacology.

[14]  K. Anderson,et al.  Distinct patterns of hematopoietic stem cell involvement in acute lymphoblastic leukemia , 2005, Nature Medicine.

[15]  Sean C. Bendall,et al.  Extracting a Cellular Hierarchy from High-dimensional Cytometry Data with SPADE , 2011, Nature Biotechnology.

[16]  Robert Gentleman,et al.  flowCore: a Bioconductor package for high throughput flow cytometry , 2009, BMC Bioinformatics.

[17]  J F Leary,et al.  Rare-event analysis methods for detection of fetal red blood cells in maternal blood. , 1984, Cytometry.

[18]  J. Hanley.  Receiver operating characteristic (ROC) methodology: the state of the art. , 1989, Critical reviews in diagnostic imaging.

[19]  Bernard,et al.  Flow cytometric monitoring of red blood cell chimerism after bone marrow transplantation , 1999, Transfusion medicine.

[20]  Leslie L Robison,et al.  Acute lymphoblastic leukaemia , 2018, Radiopaedia.org.

[21]  R. Stam.  The ongoing conundrum of MLL-AF4 driven leukemogenesis. , 2013, Blood.

[22]  J R Beck,et al.  The use of relative operating characteristic (ROC) curves in test performance evaluation. , 1986, Archives of pathology & laboratory medicine.