Expression of VEGF and Its Receptors by Myeloma Cells S Kumar1, TE Witzig1, M Timm1, J Haug1, L Wellik1, R Fonseca1, PR Greipp1 and SV Rajkumar1
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Leukemia (2003) 17, 2025–2031 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu Expression of VEGF and its receptors by myeloma cells S Kumar1, TE Witzig1, M Timm1, J Haug1, L Wellik1, R Fonseca1, PR Greipp1 and SV Rajkumar1 1Division of Hematology and Internal Medicine, Mayo Clinic, Rochester, MN, USA Angiogenesis or new vessel formation is an essential compo- and is being increasingly recognized.4 Increasing levels of tumor nent in the growth and progression of neoplasms and there is angiogenesis has been associated with poor prognosis for a growing evidence of its importance in hematological malig- nancies including multiple myeloma (MM). Vascular endothelial variety of hematological malignancies as well as solid tumors. growth factor (VEGF) is believed to play a role in tumor As in solid tumors, new vessel formation (occurring in the bone angiogenesis. We studied the expression of VEGF and its marrow) seems to play an important role in the pathophysiology receptors (VEGFR1 or Flt-1 and VEGFR2 or Flk-1/KDR) by of myeloma,5–8 leukemias9,10 and in myelofibrosis.11 Increased myeloma cell lines and plasma cells isolated from patients, bone marrow microvessel density in patients with myeloma using different methods. VEGF expression by the plasma cells appears to be a powerful prognostic indicator.7 was demonstrated by immunohistochemistry in 18 of 20 patients with MM. Enzyme-linked immunosorbent assay de- Various cytokines have been invoked as being responsible for monstrated VEGF secretion in all six different myeloma cell driving the process of neovascularization seen in the setting 12 lines studied. Five patient marrow samples and seven different of solid tumors and hematological malignancies. Vascular myeloma cell lines were then studied for VEGF mRNA expres- endothelial growth factor (VEGF) appears to have a particularly sion by reverse-transcriptase polymerase chain reaction (RT- important role in the biology of MM.13,14 Studies have shown PCR), which was positive in all. We further evaluated the that myeloma cells are capable of secreting VEGF contributing expression of both VEGFR1 and VEGFR2 in different myeloma cell lines and five sorted myeloma bone marrow samples by RT- to new vessel formation in the bone marrow in myeloma. In PCR. All the myeloma cell lines expressed VEGFR1 and three of turn, the microvascular endothelial cells as well as the marrow the cell lines expressed VEGFR2. VEGFR1 expression was stromal cells are stimulated by the VEGF resulting in increased detected in all and VEGFR2 in all but one of the sorted marrow secretion of interleukin (IL)-6, which has been shown to be a samples. Increased expression of VEGF by the myeloma cells potent growth factor for the malignant plasma cells.15,16 An taken in the context of the suspected prognostic value of autocrine pathway of myeloma cell stimulation by VEGF may marrow angiogenesis suggests a pathogenetic role for this cytokine and presence of its receptors on myeloma cells points also exist in addition to this paracrine pathway. We examined toward an autocrine mechanism. Demonstration of the pre- the expression of VEGF and their receptors by different methods sence of VEGFR2 in our study provides a potential biological on the plasma cells to examine this hypothesis further. explanation for the preclinical activity observed with VEGFR2 inhibitors. Leukemia (2003) 17, 2025–2031. doi:10.1038/sj.leu.2403084 Methods and materials Keywords: multiple myeloma; angiogenesis; vascular endothelial growth fector; VEGFR1; Flk-1 Isolation of patient plasma cells Introduction Bone marrow samples were obtained from patients with MM and plasma cells were isolated from the patient samples using Multiple myeloma (MM) is characterized by a clonal prolifera- MACS separation columns using antibodies against CD138 tion of abnormal plasma cells in the bone marrow resulting in a as described by the manufacturer (Miltenyi Biotec, Bergisch variety of different clinical manifestations like osteolytic bone Gladbach, Germany). These cells were further analyzed by lesions leading to pathologic fractures, anemia, life-threatening Wright–Giemsa stain and a purity of over 96% was confirmed. hypercalcemia and renal failure. It accounts for more than 10% of all hematological malignancies and nearly 1% of all malignancies. It is estimated that there will be 14 600 new Myeloma cell lines and cell culture patients with a diagnosis of myeloma in the United States alone during the year 2002 and there will be 10 800 estimated deaths Eight different myeloma cell lines were used for the study 17 18 19 18 due to myeloma in the same period.1 The median survival from (ARH9, OCI-My5, KAS 6/1, RPMI 8226, KP-6, ANBL- 20 21 diagnosis among patients treated with conventional chemother- 6, U266B1, OPM2). All cell lines were maintained at 371C apy is 3–4 years.2 Introduction of high-dose chemotherapy with in RPMI 1640 media supplemented with 10% heat-inactivated stem cell rescue has led to improved survival, but these patients fetal bovine serum (FBS), penicillin, streptomycin and eventually relapse from their disease and it remains an incurable L-glutamine. Three of these cell lines, KP6, ANBL-6 and KAS disease with the currently available therapeutic modalities.3 6/1, were also supplemented with IL-6 at 5 ng/ml. Angiogenesis or new blood vessel formation from existing blood vessels (in contrast to vasculogenesis or de novo formation of blood vessels) occurs physiologically during Enzyme-linked immunosorbent assay normal growth, tissue healing and regeneration. The role of abnormal, increased angiogenesis in the development and Intracellular and secreted levels of VEGF were estimated by spread of tumors had been suspected for nearly two decades enzyme-linked immunosorbent assay (ELISA) using Quanti- kines human VEGF assay (R and D systems, Minneapolis, 6 Correspondence: Dr SV Rajkumar, Mayo Clinic, 200 First Street MN, USA). For intracellular levels of VEGF, 5 Â 10 cells from SW, Rochester, MN 55905, USA; Fax: þ 1 507 266 4972 each cell line were washed in RPMI Â 2, snap frozen in liquid Received 30 December 2002; accepted 16 June 2003 nitrogen, and then thawed at 371C. The snap freezing and Vascular endothelial growth factor in myeloma S Kumar et al 2026 thawing was repeated two more times. Cellular debris was then bone marrow was performed using a labeled streptavidin–biotin spun down and supernatant saved for analysis. Secreted levels peroxidase method, on a Ventana ES automated immuno- were estimated using cell culture supernatants taken on day 7 histochemistry stainer (Ventana Medical Systems, Tucson, AZ, from each cell line. Briefly, 200 ml of cell culture supernatant, USA) using buffers and detection reagents supplied by the cell lysate or standard (recombinant human VEGF165) was added manufacturer. After deparaffinization, the primary antibody to the microplate wells with the recommended diluent and (VEGF mouse monoclonal antibody, Santacruz Biotechnology, incubated at room temperature for 2 h. The wells were washed Santa Cruz, CA, USA, diluted 1:500) was incubated with tissue and 200 ml of VEGF conjugate (polyclonal antibody against sections for 24 min. The AEC (amino ethyl carbazole) detection VEGF conjugated to horseradish peroxidase) was added and kit (Ventana Medical Systems) was used for antigen visualiza- further incubation carried out at room temperature for 2 h. The tion; sections were counterstained with a light hematoxylin and wells were washed and 200 ml of hydrogen peroxide and then cover slipped with Kaiseri’s glycerol jelly (Mayo Medical tetramethylbenzidine was added and incubated for 20 min at Laboratories, Rochester, MN, USA). VEGF expression was RT. After stopping the reaction with 2 N sulfuric acid, the optical graded as follows: À, no staining; þ , weak staining (0–30% density was read at 450 nm using a microtiter plate reader. plasma cells positive); þþ, weak to moderate staining (30–60% plasma cells positive); þþþ, strong staining (460% plasma cells positive). Reverse-transcriptase polymerase chain reaction All patients had given written informed consent for research use of bone marrow and serum specimens. Approval for the Total RNA was isolated from cell lines and patient samples using s study was obtained from the Mayo Institutional Review Board in an RNeasy Kit as described by the manufacturer (Qiagen, Inc., accordance with federal regulations and the Declaration of Hilden, Germany). To generate cDNA and for amplification, Helsinki. 100 ng of RNA was used in a one-step reverse-transcriptase polymerase chain reaction (RT-PCR) kit as described by the manufacturer (Qiagen, Inc., Hilden, Germany). Nested PCR was Functional studies performed to detect VEGFR1 and VEGFR2, and one round of PCR to detect VEGF. We chose an RT-nested-PCR approach to Ability of VEGF to activate intracellular downstream kinases was improve the sensitivity for detection of the VEGF receptors, since examined in three different myeloma cell lines. Phosphorylation previous studies using RT single-step PCR have failed to of the mitogen-activated protein kinase (MAPK) and members of demonstrate its presence consistently. Amplification of the the signal transducers and activators of transcription (Stat) family housekeeping gene b-actin was used to verify mRNA isolation members Stat1, Stat3 and Stat5 was evaluated after stimulation and RT-PCR techniques. Primer sequences were as follows: with differing concentrations of VEGF. Human MM cell lines VEGF 50-GAA GTG GTG AAG TTC ATG GAT GTC-30 (forward) JJN3, 8226 and OPM2 were maintained in RPMI 1640 contain- and 50-CGA TCG TTC TGT ATC AGT CTT TCC-30 (reverse); ing 10% FBS, 2 mML-glutamine, 100 U/ml penicillin and 100 mg/ VEGFR1 (outer) 50-GAG AAT TCA CTA TGG AAG ATC TGA TTT ml streptomycin. Cells were starved 16 h in RMPI 1640 and then CTT ACA GT-30 (fwd) and 50-GAG CAT GCG GTA AAA TAC stimulated with 10, 20 or 50 ng/ml VEGF and VEGF .