INTERACTION BETWEEN ENDOTHELIUM AND CHRONIC LYMPHOCYTIC LEUKEMIA B-CELLS RESCUES FROM APOPTOSIS AND MODULATES GENE EXPRESSION PROFILE OF LEUKEMIC CELLS

Background. Despite an apparent long life in vivo, CLL cells die rap- idly in vitro. This observation suggests that the apoptotic resistance is not intrinsic to leukemia B cells but extrinsic factors are necessary for CLL prolonged survival. Aims. we investigated the interactions be- tween endothelial cells and CLL cells, highlighting molecular net- works involved in this cellular crosstalk. Methods. we co-cultured CLL cells on HUVEC endothelial monolayer (HC) or in medium alone (CLL only). Then, we detected CLL viability by flow cytometry and we performed whole-genome high density microarrays. Results. we found that endothelial cells protected CLL from spontaneous apop- tosis. After 48h, increased number of alive CLL cells was present in HC condition (59.7 ± 4.2%) compared to CLL alone (22.9 ± 5.1%) (p<0.0001). Moreover, we found that spontaneous in vitro apoptosis was higher in unmutated IGHV CLL (UM-CLL) compared to mutated ones (M-CLL). In HC condition, similar survival was detected be- tween M-CLL and UM-CLL, implying a 2.2-fold increase in relative viability in M-CLL and a 6.1-fold increase in UM-CLL. Moreover, the endothelial cell layer decreased the in vitro sensitivity of CLL cells to Fludarabine-induced apoptotic cell death. The mean viability of CLL cells treated with 10 µM Fludarabine was 19.8% (±4.4%) after 48 hours and 3.8% (±1.3%) after 72 hours. In HC with Fludarabine ad- dition, the mean viability of CLL cells was 37.8% (±9.1%) after 48 hours and 14.3% (±3.2%) after 72 hours. Then, we compared gene expression profiles (GEP) between CLL cultured in contact with EC layer and CLL at baseline to unravel the transcriptional modifications induced by EC cells. Overall 1944 genes were found to be modulated (FC≥2, p<0.05). CLL cells in HC condition showed a 22.6-fold in- crease of CCL2, able to recruit tumor-activated monocytes (p=0.0032) and a 6.5-fold increase of PDGFC, chemoattractant for mesenchymal stromal cells (p=0.0051). Other soluble factors up-reg- ulated by EC/CLL contact were VEGFC (FC=9.4, p=0.0061), ANGTL4 (FC=8.6, p=0.015), EDN1 (FC=9.2, p=0.0061), AMOTL2 (FC=4.3, p=0.019) and THBS1 (FC=45.1, p=0.0004) as well as the metalloproteases MMP2 (FC=8.3, p=0.02) and MMP4 (FC=3.0, p=0.039). The GEP data were confirmed by evaluating the secreted levels of soluble factors in conditioned medium collected after 48h- HC culture. In addition, CLL cells on endothelial layer maintained or increased the expression levels of anti-apoptotic factors Bcl-2, Bcl2A1, BIRC3/c-IAP2 and BIRC5/Survivin compared to CLL cells at baseline. Of interest, the Ang2 tyrosine kinase receptor Tie2 mRNA was found to be increased in CLL cells in co-culture (FC=10.7, p=0.017). We confirmed GEP data by flow cytometry finding a 2-fold and a 4.3-fold increase of percentage of Tie2+CLL cells at 48h and 72h in HC. Conclusion. our results demonstrate a role of endothelial cells in CLL survival advantage and Fludarabine-resistance. The inti- mate contacts with EC seem to determine a microenvironmental- driven angiogenic switch of CLL phenotype, improve the secretion of cytokines involved in regulation of microenvironmental elements such as stromal cells and macrophages and increase the expression of anti-apoptotic molecules.