Antiangiogenic treatment with endostatin inhibits progression of AML ...

Leukemia (2005) 19, 1312–1317 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu

Antiangiogenic treatment with endostatin inhibits progression of AML in vivo G Schuch1, L Oliveira-Ferrer2, S Loges1, E Laack1, C Bokemeyer1, DK Hossfeld1, W Fiedler1 and S Ergun2 1 2

Department of Medicine, Oncology and Hematology, University Hospital Hamburg-Eppendorf, Hamburg, Germany; and Institute of Anatomy, University Hospital Hamburg-Eppendorf, Hamburg, Germany

SPOTLIGHT

Increased vessel density in the bone marrow of patients with acute myeloid leukemia as well as elevated expression of proangiogenic factors by leukemic cells implies a central role of angiogenesis in hematological malignancies. Endostatin (ES), a fragment of collagen XVIII, is an endogenous inhibitor of angiogenesis that has shown therapeutic activity in solid tumors in various preclinical models. Using microencapsulation technology, we studied the therapeutic effect of ES in AML. While ES had no effect on proliferation of M1 murine leukemic cells in vitro, ES producing microbeads significantly inhibited growth of subcutaneous chloromas in SCID mice as compared to controls. In a leukemia model using M1 cells the concomitant treatment of mice with ES microbeads prolonged median survival significantly. Histological analysis revealed a decreased microvessel density and a reduced number of CD31positive single cells, putatively endothelial progenitor cells, in the bone marrow of treated animals. Taken together, ES has inhibitory effects on neo-angiogenesis in the bone marrow and on progression of leukemia in vivo. These experiments suggest a possible therapeutic role of antiangiogenic gene therapy with ES in AML. Leukemia (2005) 19, 1312–1317. doi:10.1038/sj.leu.2403824; published online 2 June 2005 Keywords: AML; angiogenesis; endostatin; encapsulation

Introduction Angiogenesis, the formation of a vascular network, has been established as an important prerequisite for tumor formation in solid malignancies.1,2 Therapies targeting this process are showing promising results in clinical trials.3 Recent developments focused on the role of angiogenesis in hematological malignancies. Comparable to solid tumors, leukemic cells express angiogenic growth factors, that is, vascular endothelial growth factor (VEGF) and thereby stimulate neovascularization in bone marrow.4,5 Expression of VEGF has been linked to adverse prognosis in acute myeloid leukemia (AML).6 Stimulation of angiogenesis results in an increased microvessel density in the bone marrow of patients with AML and other leukemias.7–9 Patients responding to chemotherapy have reduction of bone marrow vessel density to normal levels.10 In addition to the secretion of proangiogenic growth factors, leukemic cells also express several receptors for VEGF,5,11,12 angiopoietins, a different family of growth factors promoting vascular formation and their receptor Tie-2.13,14 Several studies demonstrated that besides a paracrine stimulation of endothelial cells, the angiogenic factors directly act on leukemic cells in an autocrine fashion through activation of VEGF receptors and Tie-2 leading to antiapoptotic and proliferative effects on myeloid blasts.15,16 In a previous study, we were able to show that VEGF promotes progression of leukemia in vivo while soluble Correspondence: Dr G Schuch, Department of Medicine, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany; Fax: þ 49 40 42803 2186; E-mail: [email protected] Received 12 January 2005; accepted 29 April 2005; published online 2 June 2005

neuropilin-1, an inhibitor of VEGF, significantly inhibited leukemic progression.17 Since M1 cells do not express VEGF receptor 2 and do not respond to VEGF, we could demonstrate inhibition of angiogenesis as an effective therapeutic principle in AML. Endostatin, a proteolytic fragment of basal membrane protein collagen XVIII, was identified as a potent endogenous inhibitor of angiogenesis and has shown activity in various solid tumor models.18–22 Endostatin induces apoptosis and inhibits proliferation of endothelial cells23 without affecting other cell types. The current work was performed to investigate whether a delivery system of microencapsulated cells producing constantly high levels of mouse endostatin could be used for antiangiogenic somatic gene therapy in AML.

Materials and methods

Cell culture, transfection and microencapsulation of PAE cells The murine leukemic cell line M1 was purchased from DSMZ, Braunschweig, Germany, and cultured in RPMI with 10% FBS. Porcine aortic endothelial cells (PAE), a kind gift of Shay Soker, Boston, were grown in HAM/F-12 supplemented with 10% FBS. The coding sequence of murine ES (mES) provided by Judah Folkman, Boston, MA, USA, was transferred into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA) containing a CMV promoter and a neomycin resistance gene. PAE cells were transfected using the Lipofectamine PLUS reagent (Invitrogen). Stable clones were isolated after treatment with neomycin and expression of recombinant ES was monitored by Western blot analyses. Microencapsulation of PAE cells was performed as described previously.17 In brief, PAE cells were dissolved in 1.2% sodium alginate (MVP, Pronova, Oslo, Norway), the cell suspension was passed through a nozzle (Nisco Engineering, Zurich, Switzerland) and resulting capsules were collected in 1.5% mM CaCl2/ 13 mM HEPES buffer and subsequently coated with poly-L-lysine (Sigma).

Western blot for endostatin Conditioned media of transfected PAE cells were incubated with heparin agarose at 41C overnight, agarose was washed 3 times with PBS and gel loading buffer was added. After heating for 5 min at 951C, samples were loaded on 12.5% SDS-PAGE. Separated proteins were transferred to nitrocellulose membrane (Amersham Pharmacia, Freiburg, Germany) and blots were incubated with goat anti endostatin primary antibody (R&D, Wiesbaden, Germany). ES protein was visualized after incubation with secondary peroxidase labeled anti-rabbit antibody using ECL reagent (Amersham Pharmacia).

Endostatin inhibits progression of AML in vivo G Schuch et al

M1 cells (2 105 cells per well) were coincubated with microbeads containing mES producing or wild-type PAE cells in RPMI containing 10% FBS. Cell counts were determined after incubation for 24 and 48 h. Experiments were performed in triplicates.

Chloroma model M1 cells (1 106 cells per animal) were injected subcutaneously (s.c.) to SCID mice and encapsulated ES producing PAE cells or wild-type controls (1 105 cells per animal, 10 mice per group) were injected simultaneously s.c. nearby. After 4 weeks, chloromas were harvested, weight was determined and tissue was fixed for histological analyses. Systemic ES levels were measured by mouse ES specific ELISA (Cytimmune, College Park, MD, USA) from mouse serum obtained at day 30 post implantation.

Systemic leukemia model M1 cells (2 106 per animal) were injected intravenous (i.v.) to SCID mice (10 per group). Microbeads containing ES producing or wild-type cells were injected s.c. on day 3 and mice were followed for survival. In parallel, three mice were killed on day 35 and femoral bones were isolated for histological analyses.

ES has no effect on proliferation of leukemic cells in vitro Using ES producing microbeads in in vitro angiogenesis assays, we observed inhibition of endothelial cell function as determined by migration and permeability experiments (not shown). In order to test whether ES might act directly on leukemic cells since endothelial and hematopoetic cells share several features such as expression of VEGF receptors, the effect of ES on proliferation of M1 cells was tested in vitro. Murine leukemic M1 cells were coincubated with mES producing microbeads and proliferation of leukemic cells was determined by cell counting. As shown in Figure 2 there was no difference in the proliferation of ES treated and untreated cells. This observation confirms the lack of effect of endostatin on proliferation of nonendothelial cells as described by others.23

ES inhibits growth of chloromas in vivo In order to test the activity of endostatin produced by encapsulated cells in vivo, we performed experiments in a chloroma model using the M1 leukemic cell line. As published earlier, M1 cells develop solid tumors when injected s.c. into

Histological studies Tissue pieces were fixed in Bouin’s solution. For decalcification bones were incubated in 0.5 M Na-EDTA, pH 8.5 for 10 days. Tissue samples were embedded in paraffin. Paraffin sections were stained by H&E for histological evaluation. Furthermore, immunostaining using specific monoclonal antibodies for ES (R&D), laminin (Sigma, Saint Louis, MI, USA) or CD31 (DPC Biermann, Bad Nauheim, Germany) was performed and the staining was visualized by DAB using a Ni-enhanced glucose oxidase technique.24 All sections were counterstained using Calcium red after immunostaining. For determination of microvessel density in the bone marrow, CD31 positive vessel-like structures within marrow space were counted in 10 representative high power fields of each group in a blinded fashion. Numbers of single CD31 positive cells as putative endothelial progenitor cells, appearing larger in size and distinct from vessels, were determined separately.

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Figure 1 (a) Western blot analysis for endostatin secreted by engineered PAE cells. Different clones of PAE cells obtained after transfection and selection using G418 showed stable expression of mES detected as a single band at 20 kDa. (b) Light microscopy of microbeads. PAE cells expressing high levels of mES were encapsulated with sodium alginate and poly-L-lysine. Cells are visible at 10 and 40 magnification within microbeads.

SPOTLIGHT

In vitro proliferation assay

Results

Preparation of microencapsulated ES-producing cells PAE cells were engineered for secretion of mouse ES by lipofection using a CMV-driven expression plasmid. Expression of mES was monitored by Western blot analyses from conditioned media (Figure 1a). Stable clones were utilized for microencapsulation. Alginate microcapsules were manufactured for transplantation of engineered cells as described earlier.17 The configuration of PAE cells within the microbeads is demonstrated in Figure 1b. Stable production of mES from microbeads was observed over a period of more than 4 weeks (data not shown).

Figure 2 Effect of ES on proliferation of M1 cells in vitro. Effect on proliferation of M1 cells was determined by coincubation of M1 cells with microbeads containing mES producing cells. Leukemic cells were counted after 24 and 48 h. Leukemia


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SCID mice.17 These chloromas contain vessels as seen in solid tumors and antiangiogenic treatment has been shown to inhibit growth of chloromas.17 In the present experiment, M1 cells were injected s.c. On the same day, microbeads containing either wild-type or ES producing cells were placed nearby. While all

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control animals developed chloromas, in half of the ES-treated animals no tumor could be detected. The size of the chloromas in the treatment group was significantly lower than in controls (Po0.01, Figure 3a). As tested by a mouse ES specific ELISA, levels of circulating ES in the blood were increased significantly in the treatment group compared to controls (Po0.02, Figure 3b). These results demonstrate that our delivery system enables therapy with ES in vivo and that the protein produced by transfected cells has antiangiogenic properties. Tissue sections were analyzed by immunohistochemistry for ES using an ES-specific antibody not crossreacting with collagen XVIII.25 In untreated chloromas a faint staining was seen in vessels at the outer growth zone of tumors (Figure 4a). In contrast, ES staining was more abundant in vessels of ES-treated chloromas (Figure 4b) and around ES producing microcapsules (Figure 4c). The staining pattern suggests that endogenous ES is involved in neo-angiogenesis in tumor growth. Delivery of excess amounts of ES might inhibit tumor growth by keeping vessels in a stabilized form. Normal vessels, on the other hand, are already stable and not activated and may lack the expression of ES binding partners.

Effect of ES on AML in vivo

Figure 3 ES released from encapsulated NMuMG cells inhibits growth of M1 chloromas in SCID mice. Leukemic M1 cells (2 106 per animal) and microbeads containing ES producing cells were injected simultaneously s.c. to SCID mice. After 4 weeks, chloromas were harvested and weight was determined (a). Systemic levels of mES in serum of mice were measured by ELISA (b).

Since leukemia is a systemic disease and only rarely grows as chloromas, we tested the effect of ES in an established leukemic model using M1 cells in SCID mice. In this model, the leukemic cells automatically settle in the bone marrow after i.v. application leading to death by leukemia after approximately 5 weeks.17 In the current experiment, ES producing microcapsules were injected s.c. 3 days after inoculation of leukemic cells. Animals were followed for survival. The median survival for control animals was 34 days. Median survival of ES-treated animals was

Figure 4 Immunohistochemical analyses of M1 chloromas treated with ES. Chloromas were fixed in Bouin’s solution and sections were stained using antibodies specific for ES visualized by DAB. In controls a faint staining for ES was seen in vessels demarcating the growth zone of chloromas (a). In ES-treated chloromas the staining was more pronounced (b); (c) shows staining next to microbeads with ES producing cells. ES was restricted to malignant tissue while surrounding normal tissue was free of staining (d). Leukemia


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Figure 5 Effect of mES on survival of SCID mice with leukemia. Leukemic M1 cells (2 106 per animal) were injected i.v. to SCID mice (10 per group). Microbeads containing ES producing or wild-type cells were injected s.c. on day 3 and mice were followed for survival. Survival in ES treated animals was significantly prolonged (Po0.003).

number of CD31 positive endothelial progenitor cells as compared to wild-type beads. Taken together these results demonstrate that ES has antileukemic activity in an in vivo model through its antiangiogenic action. In fully established leukemia its antiangiogenic activity alone might not be sufficiently effective enough to prevent leukemia progression.

Discussion The present results demonstrate that antiangiogenic therapy with endostatin inhibits the progression of AML by suppression of neo-angiogenesis in the bone marrow without affecting proliferation of leukemic cells. Somatic gene therapy using microencapsulation of endostatin releasing cells allows systemic therapy resulting in elevated systemic ES levels, inhibition of bone marrow microvessel density and prolonged survival of treated animals. ES was initially isolated as an endogenous protein inhibiting tumor growth by its antiangiogenic properties.18 ES inhibits proliferation, migration and tube formation of endothelial cells in vitro.26–28 A discrete modulation of leukemic cell proliferation by endostatin was seen in a small subset of primary AML only in the presence of additional growth factors.29 In vivo, ES has antitumor activity in various models of solid neoplasms20 and lymphoma.30,31 Previously, we were able to demonstrate a causal role of VEGF in AML progression in an animal model.17 In the current study we investigated whether the antiangiogenic protein ES would exhibit inhibitory activity in leukemia. The therapeutic protein was delivered using a system of ex vivo modified cells encapsulated in alginate microbeads and

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increased to 39 days resulting in a significant survival benefit (Po0.003, Figure 5). Histological analyses of bone marrow sections by staining for the endothelial-specific antigen CD31 revealed a decrease of microvessel density in ES treated mice by one third as compared to control animals (Figure 6). This finding was confirmed by staining for the basal membrane component laminin. Additionally, single CD31-positive cells not incorporated into vascular structures were less frequent in the bone marrow of ES-treated mice. Since ES has been shown to decrease the amount of endothelial progenitor cells,34 CD31positive cells aside from capillaries may represent endothelial precursor cells. Treatment with ES microbeads decreased the

Figure 6 Immunostaining of bone marrow sections from leukemic mice treated with ES producing microbeads. Staining for endothelialspecific CD31 revealed a decrease in microvessel density and number of putative endothelial progenitor cells in the bone marrow of treated mice (a) as compared to untreated mice (b). Immunostaining for laminin reveals a similar pattern and confirms the vascular character of stained structures (c). In the negative control, there is no specific staining (d). All sections were counterstained with Calcium Red. Leukemia


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SPOTLIGHT Leukemia

transplanted in mice carrying leukemia. Microbeads can easily be used for experiments in vitro or transplanted to animals for in vivo studies. This system not only provides immune protection in immune competent individuals but also protects the transplanted cells upon injection. We and others have shown the feasibility of this system in various models including the M1 AML in mice.32–34 Since NMuMG cells used for encapsulation previously17 appeared to lose expression of transfected plasmids over time, we have changed to PAE cells in the actual study. These cells are easy to transfect and maintain expression without decline over time. Although isolated as endothelial cells, PAE cells have lost endothelial properties and are not affected by ES or other angiogenic inhibitors. Pilot studies showed no interference of PAE cells with tumor growth in vivo. Release of ES by encapsulated PAE cells resulted in significant reduction of chloroma growth in mice. Immunohistochemistry for ES performed on chloromas showed staining in vessel walls in the marginal tumor zone of untreated chloromas where tumor growth takes place and angiogenesis is active. This was more pronounced in ES treated animals while no staining was noted in normal adjacent tissue. These data show that ES accumulates preferentially in angiogenic endothelia and not in stable vessels like in normal tissue and in the tumor center suggesting that only activated endothelial cells are targeted by ES probably through binding to proteins selectively expressed in angiogenic vessels. This question has been addressed in various studies. Specific RGD motifs have been identified as potential binding partners for ES and similar antiangiogenic proteins.35 Experiments performed in order to test effects of ES delivered by Alzet pumps on endothelial permeability in vivo showed inhibition of VEGF-induced permeability only after 3 days of treatment (unpublished data), supporting that ES has to saturate all binding targets before a systemic effect can be observed. Since systemic concentration of ES reached by microbeads is only slightly elevated over normal levels, we speculate that selective accumulation in sensitive endothelia achieved by continuous delivery might be sufficient for the effect observed. It was already shown that continuous delivery of ES is superior to intermittent bolus applications.36 Higher doses may even lead to inverse effects as reported by Abdollahi.37 Treatment of systemic AML with mES by s.c. transplantation of microbeads resulted in inhibition of leukemia progression as determined by prolonged median survival. When treatment was delayed by injecting ES microbeads 3 weeks after inoculation of leukemic cells, there was no difference in survival between both groups, although analyzed blood showed elevated levels of mES in treated animals. On the other hand, pretreatment of mice with ES for 14 days before injection of leukemic cells prolonged median survival to 52 days (data not shown). ES treatment decreased the microvessel density in the bone marrow as a surrogate marker of inhibition of leukemia-induced neo-angiogenesis. Interestingly, we observed a lower count of CD31-positive cells in the bone marrow not incorporated into vessels. One might speculate whether these cells represent endothelial progenitor cells that have been shown to be affected by ES.34 However, no additional analyses were performed in the present study. The molecular basis of ES-induced inhibition of endothelial cells is not fully understood, yet. Given the selective accumulation of ES in angiogenic vessels, where VEGFR-2 and integrins are upregulated, one might speculate whether these proteins are primary targets of ES leading to consecutive signaling events. It was already shown that ES inhibits VEGF-induced phosphorylation of VEGFR-2.38 Wickstrom et al 39 demonstrated the

association of ES with integrins and the effects on src-mediated regulation of the cytoskeleton. So far, little information on antiangiogenic therapy with ES in leukemia has been published. In acute lymphocytic leukemia, ES delivered by retrovirally transduced hematopoetic stem cells failed to show therapeutic activity.40 In contrast, other studies demonstrated therapeutic benefit of ES in leukemia. ES reduced the amount of leukemic cells in the bone marrow of rats with juvenile myelomonocytic leukemia or AML.41 In a recent study, it was reported that ES inhibited the progression of a myeloproliferative disorder in bcr/abl transgenic mice.42 Retroviral gene therapy with combination of angiostatin and endostatin achieved responses in leukemic mice.43 In clinical trials, antiangiogenic approaches targeting VEGF and VEGF receptors in AML have shown promising results as published recently.44,45 Taken together, these results demonstrate that ES inhibits leukemia-induced neo-angiogenesis in the bone marrow. Progression of AML can be delayed significantly by somatic gene therapy using ES-producing cells in vivo. When leukemic cells have already expanded and induction of angiogenesis with increase of microvessel density has taken place ES does not affect leukemic progression. Given the mechanism of ES promoting stability of activated vessels it becomes understandable that ES alone has limited activity in late stage disease either in solid tumors or in leukemia. In the context of AML therapy, treatment with ES would fit into the maintenance setting when the microvessel density in the bone marrow is low. Combination with conventional chemotherapy or different angiogenic inhibitors should be investigated in future studies.

Acknowledgements This work was in part supported by the Erich-Roggenbuck-Stiftung (GS, SE) and a BMBF grant (SL, WF). The authors are grateful to Kirsten Miethe and Katrin Kluge for their excellent technical assistance.

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