In vitro studies of endothelial progenitor cell (EPC) colony formation, growth, and capillary-like network formation before and after thalidomide. (A) Peripheral blood mononuclear cells were cultured in laminin-coated 6-well plates (i-iii) or in matrigel-coated 48-well plates (iv-vi). Colonies were quantitated 14 days after plating. Phase contrast micrographs of cultures from an MM patient were examined at the indicated time points: (i) an EPC colony characterized by a central cluster of rounded cells surrounded by radiating, thin, flat, elongated cells; (ii) smaller colonies at the indicated time point and a reciprocal increase in EC outgrowth; (iii) confluent expansion of endothelial cells; (iv) colony growth and tube formation on matrigel; (v) progression of tube formation; and (vi) EC network formation. Original magnification × 200. (B) Colony formation on laminin from patients with MM, stratified by change in M protein levels after treatment with thalidomide. P was determined by nonpaired analysis using Student t test. (C) Comparison of capillary-like network formation by EPCs from a patient before and after 3 months of treatment with thalidomide. The patient's serum M protein levels had decreased by 75% compared to pretreatment (not shown). PBMCs were grown on matrigel. Original magnification × 200. (D) Time course of thalidomide's effect on EC network formation. Bars represent mean ± SD of 3 patients' capillary-like tube formation scores which were determined sequentially at the indicated times during treatment with thalidomide. Tube formation score was assigned by phase contrast microscopy as described in “Patients, materials, and methods.” Mean of triplicate wells from each patient at indicated times was assigned as their score.