Endothelium-mediated survival of leukemic cells and angiogenesis-related factors are affected by lenalidomide treatment in chronic lymphocytic leukemia  Rossana Maffei, Stefania Fiorcari, Jenny Bulgarelli, Lara Rizzotto, Silvia Martinelli, Gian Matteo Rigolin, Giulia Debbia, Ilaria Castelli, Goretta Bonacorsi, Rita Santachiara, Francesco Forconi, Davide Rossi, Luca Laurenti, Giuseppe A. Palumbo, Daniele Vallisa, Antonio Cuneo, Gianluca Gaidano, Mario Luppi, Roberto Marasca  Experimental Hematology  Volume 42, Issue 2, Pages 126-136.e1 (February 2014) DOI: 10.1016/j.exphem.2013.10.007 Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 1 In vitro and in vivo effect of lenalidomide on CLL cells. (A) Purified CLL cells from nine patients were cultured in complete medium with the addition of lenalidomide (1, 0.1, 0.01 μmol/L). Viability was evaluated from 24 to 144 hours by annexin V-Propidium Iodide (PI) staining and flow cytometry and defined as percentage of Annexin V−/PI− cells. Graphics show three representative CLL samples. (B) WBC count (×109/L) and (C) ALC count (×109/L) for patients who completed at least 4 months of treatment (n = 16), including eight patients with a second time point from 5 to 13 months of treatment (end). Range bars represent standard error of the mean (p < 0.05, Wilcoxon test). Experimental Hematology 2014 42, 126-136.e1DOI: (10.1016/j.exphem.2013.10.007) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 2 Lenalidomide reduces EC-mediated CLL survival advantage and CLL-induced angiogenesis. (A) Purified CLL cells from nine patients were cultured in medium alone (CLL only) or in coculture on a monolayer of HUVECs (HC) in time course experiments from 24 to 168 hours. Lenalidomide (0.01 and 0.1 μmol/L) was added to CLL cocultures. Histograms represent percentage of viable cells (annexin V−/PI−) normalized on HC condition (100%). The survival advantage acquired by CLL cells in contact with endothelium was reduced by lenalidomide treatment (p < 0.01 compared with HC, Wilcoxon test). Data are presented as mean and SEM. (B) Absolute viability (%) measured by annexin V/PI stainings from 24 to 168 hours of a representative sample is shown. (C) HUVEC cells were plated onto Matrigel (ECMatrix) and incubated for 12 hours in medium alone (control) or in medium supplemented with supernatants (1:1 vol/vol) obtained from cultures of CLL cells alone (CM CLL only, n = 3) or CLL cells in contact with endothelial cells (CM CLL HC, n = 3). Lenalidomide at 100 μmol/L or vehicle (dimethyl sulfoxide) was added where indicated. The degree of tube formation was quantified by the pattern recognition method: 0 = individual cells, well separated; 1 = cells begin to migrate and align themselves; 2 = capillary tube visible, no sprouting; 3 = sprouting of new capillary tubes visible; 4 = closed polygons begin to form; 5 = complex meshlike structures develop. Histograms represent mean pattern value at 6 hours defined by two independent observers. Lenalidomide reduces the proangiogenic effect mediated by CLL-derived soluble factors in all experiments. (D) Pictures represent capillary tube formation at 6 hours for two patients. The supernatants of CLL cells cocultured on EC layers significantly increased the tube formation by HUVECs on the Matrigel matrix with closed polygons or complex meshlike structures just developed at 6 h. Conversely, isolated HUVECs or cells just starting to elongate and migrate to align themselves are visible in control samples. The inhibitory effect of lenalidomide is visible. Original magnification ×400. Experimental Hematology 2014 42, 126-136.e1DOI: (10.1016/j.exphem.2013.10.007) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 3 Modulation of angiogenesis-related factors in plasma samples of patients with CLL during treatment with lenalidomide. Plasmatic levels of bFGF, VEGF, THBS-1, Ang2, Ang1, and Ang in 25 patients with CLL treated with lenalidomide are shown. Pretreatment levels (n = 25) and measurements after 8 (n = 25) days and 4 months (n = 20) of treatment are represented as mean and SEM. A significant reduction of VEGF and THBS-1 protein was detected. Conversely, patients showed an increase in Ang2 plasma levels after 1 week of therapy with lenalidomide and in Ang plasma levels at month 4. p < 0.05 as indicated (Wilcoxon test). Experimental Hematology 2014 42, 126-136.e1DOI: (10.1016/j.exphem.2013.10.007) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 4 Different plasma levels of bFGF, VEGF, or Ang2/VEGF ratio characterized responders to therapy with lenalidomide and patients with TFR. (A) bFGF plasma levels decrease in patients (n = 10) who respond to lenalidomide just after 1 week of therapy, whereas they increase in nonresponders (n = 6). (B) Patients who respond to lenalidomide show a progressive reduction in VEGF plasma levels that are significantly lower at month 4 in responders compared with nonresponders. (C) Increase in Ang2/VEGF ratio is measured in patients who experience TFR. p < 0.05 as indicated, Mann–Whitney test for comparisons between groups and Wilcoxon test for pair-wise comparisons between time points. Experimental Hematology 2014 42, 126-136.e1DOI: (10.1016/j.exphem.2013.10.007) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 5 Modulation of circulating endothelial population in patients with CLL during therapy with lenalidomide. (A) Histograms represent the number of CECs, EPCs, and aCECs in 27 patients with CLL treated with lenalidomide. Flow cytometric evaluations were performed at baseline and after 8 days and 4 months of treatment. Note the decrement in absolute number of CECs, EPCs, and aCECs during treatment. (B) Flow cytometric plots of a representative CLL case displaying the increase of CECs undergoing apoptosis (APO-CEC, CD146+/AnnexinV+) after 4 months of lenalidomide treatment. The percentages of APO-CEC at baseline and after treatment are indicated above the plots. (C) Comparisons between groups reveal a significant reduction of CEC number only in responders at month 4 of therapy and (D) a higher percentage of activated CEC pretreatment in nonresponders. Data are reported as mean ± SEM. *p < 0.05 or indicated p value; Mann–Whitney test for comparisons between groups and Wilcoxon test for pair-wise comparisons between time points. Experimental Hematology 2014 42, 126-136.e1DOI: (10.1016/j.exphem.2013.10.007) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions