Microvascular endothelial cells increase proliferation and inhibit apoptosis of native human acute myelogenous leukemia blasts Kimberley Hatfield 1 , Anita Ryningen 1 , Matthias Corbascio 2 and Øystein Bruserud 1,3 * 1 Section for Hematology, Institute of Medicine, The University of Bergen, Bergen, Norway 2 Cardiothoracic Surgery, Department of Heart Disease, Haukeland University Hospital, Bergen, Norway 3 Department of Internal Medicine, Section for Hematology, Haukeland University Hospital, Bergen, Norway Interactions between acute myelogenous leukemia (AML) blasts and neighbouring endothelial cells in the bone marrow seem impor- tant both for disease development and susceptibility to chemother- apy. We investigated the effects of soluble mediators released by microvascular endothelial cells on native human AML cells. AML cells derived from 33 patients were cocultured with microvascular endothelial cells, separated by a semipermeable membrane. We investigated the effect of coculture on AML cell proliferation, via- bility/apoptosis and cytokine release. Coculture increased AML cell proliferation, and this growth enhancement included the clonogenic leukemia cell subset. Increased release of several soluble mediators was also detected (interleukin 3, interleukin 6, granulocyte-macro- phage and granulocyte colony-stimulating factors) in cocultures. Our cytokine neutralization experiments suggest that an intercellu- lar crosstalk involving several soluble mediators contribute to the increased leukemia cell proliferation. The presence of endothelial cells had an additional antiapoptotic effect on the AML cells. The endothelial cells did not have any growth-enhancing effect on native human acute lymphoblastic leukemia cells. Our in vitro results sug- gest that the release of soluble mediators by microvascular endothe- lial cells supports leukemic hematopoiesis through paracrine mech- anisms by direct enhancement of AML blast proliferation and by inhibition of leukemic cell apoptosis. ' 2006 Wiley-Liss, Inc. Key words: acute myelogenous leukemia; angiogenesis; endothelial cells; apoptosis Acute myelogenous leukemia (AML) is an aggressive disorder characterized by accumulation of immature malignant cells in the bone marrow. 1 Leukemia relapse is an important cause of death in these patients, but the relapse risk differs considerably between patients and depends both on the genetic abnormalities as well as the in vivo susceptibility of AML blasts to chemotherapy. 2–6 The observation of increased microvessel density in the bone marrow of AML patients suggests that angiogenesis is involved in leuke- mogenesis, 7 and recent clinical studies suggest that regulation of angiogenesis is important also for the risk of leukemia relapse af- ter intensive chemotherapy. 8,9 Furthermore, microvascular endo- thelial cells represent the border between the intravascular and extravascular compartments, and these cells are thereby involved in a wide range of biological processes including modulation of local cytokine networks. 10–12 They are separated from the extrac- ellular bone marrow compartment by the basal membrane, and their direct influence on hematopoiesis must be mediated through their release of soluble mediators. 13 Previous in vitro studies sug- gest that endothelial cells may participate in paracrine growth reg- ulation in AML, 13 and data from experimental animal models fur- ther support the hypothesis that endothelial cells are involved in regulation of malignant cell proliferation in the bone marrow. 14 In the present study we investigated a large group of consecutive patients and describe a growth-enhancing and antiapoptotic effect of microvascular endothelial cells on native human AML cells. Material and methods Cell preparation Acute leukemia cells. The study was approved by the local Ethic’s Committee and samples collected after informed consent. AML blasts were derived from 33 consecutive patients with high peripheral blood blast counts (Table I). We investigated 15 females and 18 males (median age 61 years, range 29–80 years). One patient had leukemia relapse (Table I, patient 29) and a minority of the other patients had AML secondary to primary myelodysplastic syndrome (Table I, patients 2, 8, 20, 22, 26), chronic myeloid leukemia (patient 12) or pre- vious chemotherapy (patient 21). All patients had at least 90% AML blasts among peripheral blood leukocytes (median blast count 73.4 3 10 9 /l, range 5.6–342). Acute lymphoblastic leukemia (ALL) blasts were derived from 9 consecutive patients with B-cell disease. 15 Leukemic peripheral blood mononuclear cells (PBMC) were isolated by density gradient separation (Lymphoprep; Axis-Shield, Oslo, Norway; specific density 1.077) from peripheral blood. The percentage of blasts among leukemic PBMC exceeded 95% judged by light microscopy or flow cytometry. 16,17 The cells were stored frozen in liquid nitrogen, 18 and the thawed cells showed a viability exceeding 70%. 17,18 Endothelial cells. Human microvascular endothelial cells were obtained as frozen vials (Cambrex BioScience, Walkersville, MD) and stored in liquid nitrogen. The cells were then thawed and used directly in coculture assays. Lung microvascular endothelial cells were derived from a healthy 16 years old male Caucasian (product code CC-2527, lot no. 3F1056), whereas dermal cells were derived from a 56 years old female African (product code CC- 2543, lot no. 2F0188). The cells showed a doubling time in culture of 18 and 21 hr, respectively, and stained positive for acetylated LDL uptake stain, factor VIII related antigen and PECAM. Cells stained negative for a-actin. The cells tested negative for myco- plasma, human immunodeficiency virus 1, hepatitis B and hepati- tis C (polymerase chain reactions) (distributor’s information). Endothelial cell-enriched bone marrow stromal cells. Collec- tion of normal bone marrow was approved by the local Ethic’s Committee. After informed consent bone marrow was collected from the sternum during thoracotomy for 2 patients without hemato- logical disorders. Mononuclear bone marrow cells were isolated by gradient separation (see earlier) and subsequently dissolved in the EGM-2MV endothelial cell medium (see later) at 1 3 10 6 cells/ml. These cells were incubated for 18 hr in Transwell plates (1 3 10 6 cells in 1 ml) before nonadherent cells were removed and the adher- ent cells further cultured for additional 4/6 days. Transwell cocul- tures with AML cells were then prepared, and the cocultures were incubated for 7 days as described later. When the cocultures were ended the stromal cells had been in culture for a total of 11/13 days, and then the adherent cells included a subset that stained positive for CD31 (8 and 10%, respectively) and factor VIII (5 and 10%, respec- tively) judged by light microscopy after immunoperoxidase staining using the DAKO Envision Kit (DAKO, Copenhagen, Denmark). Grant sponsor: Norwegian Cancer Society. *Correspondence to: Medical Department, Haukeland University Hos- pital, N-5021 Bergen, Norway. Fax: 155-97-29-50. E-mail: oystein.bruserud@haukeland.no Received 11 October 2005; Revised 12 May 2006; Accepted 2 June 2006 DOI 10.1002/ijc.22180 Published online 18 August 2006 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 119, 2313–2321 (2006) ' 2006 Wiley-Liss, Inc. Publication of the International Union Against Cancer