Figure 1.
Molecular characterization of OSU-CLL and OSU-NB cell lines.
Analysis is performed in patient sample from which the cell lines were derived as well as cultured cells from both lines (approximate timing: early = 3 months, intermediate = 6 months, late = 9 months). A. Flow cytometric analysis for CLL related surface molecules. Flow cytometric analysis for selected B-cell markers, shown as the percent of cells positive. B. Fluorescence in situ hybridization. Interphase FISH analysis for a panel of cytogenetic abnormalities associated with CLL: trisomy 12 (centromere), del(13q14) (D13S319), del(11q22.3) (ATM), del(17p13.1) (TP53), t(11;14) (CCND1-IGH fusion), +8 (MYC), +3 (BCL6), and del(6q22.3) (c-MYB). C. Karyotype analysis. Metaphase karyotype analysis to determine any additional chromosomal abnormalities not identified by FISH analysis.
Figure 2.
Mutational status in OSU-CLL and OSU-NB cell lines.
Analysis is performed in patient sample from which the cell lines were derived as well as cultured cells from both lines (approximate timing: early = 3 months, intermediate = 6 months, late = 9 months). A. IGHV mutational status. Gene mutational status (relative to the reference genome), and immunoglobulin gene usage determined by sequence analysis. B. Somatic gene mutation status. Existence of mutations for common CLL variants was explored by sequence analysis.
Figure 3.
OSU-CLL and OSU-NB exhibit differential in vitro migration properties towards chemokine.
Cells were suspended (5 x 106 cells/mL) and placed in the upper well of 24-well transwell plates. The bottom wells contained either media alone, or media with recombinant CXCL12 (200 ng/mL) or CXCL13 (1000 ng/mL). Cells in the lower chamber were collected after 3 hours; percent migration is calculated relative to the input.
Figure 4.
Viability OSU-CLL in response to CLL therapeutic agents.
A. Viability of OSU-CLL either untreated (media), treated with vehicle control (DMSO), or increasing doses of chlorambucil (A), fludarabine (B) and dexamethasone (C) for 48 hours determined by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. D. Viability at 48 hours in response to the indicated therapeutic antibodies was determined by AnnexinV/ propidium iodide (Ann/PI) staining. Antibodies were used as a concentration of 10 µg/mL in the presence of 50 µg/mL anti-Fc crosslinking antibody. Abbreviations: Trastuzumab (Tras, HER2), Rituximab and Ofatumumab (Ritux and Ofa, CD20) and Alemtuzumab, (Alem, CD52). All results shown are representative of 4 independent experiments.
Figure 5.
OSU-CLL engrafts into immunodeficient mice.
A. Survival curve for NOG mice engrafted intravenously with OSU-CLL (1 x 107 cells; N = 10). B. White blood cell count (WBC) at 7, 14 and 21 days post-engraftment, determined by Giemsa staining of peripheral blood smears. C. Spleens collected at the time of sacrifice from a non-engrafted control animal (left) and two animals engrafted with OSU-CLL (middle, right). D. Flow cytometric analysis of surface human CD19 and human CD5 in a non-engrafted control animal and two representative animals engrafted with OSU-CLL. Leukemic cells were detected in both the peripheral blood and spleen cells of the engrafted animals.