Phenotypic, Genotypic, and Functional Characterization Of AML-Derived Endothelial Cells
Authors: Pizzo Russell J , Coppage Myra , Rosell Karen , Morse Kimberly , Liesveld Jane L. 
Published: October 2019

Abstract

Background
In addition to participation in homing, egress, and transmigration of hematopoietic cells, marrow endothelium also contributes to regulation of hematopoiesis with effects on cell proliferation and survival. Characteristics of marrow—derived endothelial cells from normal subjects have been described (Blood 1994; 84: 10-19), but characterization of endothelial cells in leukemia states is incomplete. Angiogenesis is known to be increased in AML marrows, and circulating endothelial progenitors are increased and correlate with disease status and response to treatment. Furthermore, cytokines secreted by endothelial cells such as vascular endothelial growth factor (VEGF) have been found to serve as growth factors for leukemia, sometimes in a paracrine or autocrine fashion. Despite these findings, inhibition of VEGF with agents such as bevacizumab has not demonstrated clinical anti-leukemia activity. Since our group and others have shown that endothelial cells from multiple vascular beds (human umbilical vein endothelial cells—HUVECs), human microvascular endothelial cells derived from skin (HMEC-1 cell line), and normal subject—derived endothelial cells are able to prevent spontaneous or therapy-induced apoptosis in AML blasts, it is important to understand the phenotype and characteristics of endothelial cells isolated from AML patients to understand their functional roles and to see if they might have an angiogenic gene expression profile as has been described in multiple myeloma (Clin Cancer Res 2009 15:5369).

Methods
Endothelial cells were purified from marrow aspirates obtained with consent from normal subjects or from newly diagnosed AML patients. Cells were isolated using anti-CD105-PE (BD Bioscience) followed by anti-PE microbead selection (Miltenyi™) or after disruption of marrow spicules with subsequent selection for endothelial cells in endothelial cell selective medium (EGM-2, Lonza). Cells between 2nd and 4th passage were utilized for analysis. Protein expression was determined by flow cytometry, Western blotting, or RT-PCR. Matrigel™ tubule formation and acetyl-LDL expression were determined as per previously published methods, as were adhesion, CFU-L, and transmigration assays. RNASeq was performed by the Functional Genomics Core at the University of Rochester after extraction of polyadenylated RNA from purified total RNA. Conversion to cDNA occurred with the Illumina TruSeq™ preparation kit, and sequencing was accomplished with the Illumina Genome Analyzer IIx. CASAVA software was utilized for analysis.

Results
Marrow derived endothelial cells from normal and AML subjects express CD105 (endoglin), CD31(PECAM), CD106 (VCAM), CD146 (MCAM), CD54 (ICAM), and CD34. They do not express CD14 nor CD45, and they demonstrate low level expression of CD144 (VE-cadherin). By RT-PCR, they express Tie-2, VEGF, and eNOS (endothelial nitric oxide synthase). They express acetyl-LDL and form tubular structures in Matrigel™. Phosphorylated components of the mTOR and PI3K/Akt pathways were also expressed by Western blot analysis. Culture of AML cells with endothelial cells from both normal and AML subjects supported adhesion, transmigration, and CFU-L outgrowth, but no significant differences were noted in these functions between normal and AML—derived endothelial cells in vitro assays. RNASeq analysis revealed 130 genes significantly up—or down—regulated in AML derived endothelial cells as compared with those derived from normal marrow. Endothelial cells from both sources had a distinct signature from marrow—derived fibroblasts. The genes differentially expressed (p<0.001) were included in biological function categories involving cancer, cell development, cell growth and proliferation, cell signaling, inflammatory response, and cell death and survival. Further pathway analysis revealed upregulation of c-Fos, and this upregulation in AML vs. normal subject derived endothelial cells was confirmed by Western blot analysis. Genes involved in chemotaxis such as CXCL16 were also upregulated.

Conclusions
AML—derived endothelial cells exhibit similar phenotype and function as their normal marrow—derived counterparts, but genomic analysis suggests a differential signature with altered expression of genes which could play a role in leukemogenesis or leukemia cell maintenance in the marrow microenvironment.

Disclosures:
No relevant conflicts of interest to declare.