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Continuous Infusion of Endostatin Inhibits Differentiation, Mobilization, and Clonogenic Potential of Endothelial Cell Progenitors¹

Division of Hematology-Oncology, Department of Medicine [P. M., C. D., G. M., F. B.], Department of Experimental Oncology-IFOM/Fondazione Italiana Ricerca sul Cancro Institute of Molecular Oncology [M. C., A. G., S. M.], and Department of Pathology-Laboratory Medicine [G. P.], European Institute of Oncology, 20141 Milan, Italy, and The Jackson Laboratory, Bar Harbor, Maine 04609 [L. S.]

	ABSTRACT

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Purpose: We investigated the effect of endostatin on differentiation, mobilization, and clonogenic potential of circulating endothelial cell (EC) progenitors, and whether the effect of endostatin was improved by continuous infusion (CI) versus bolus administration.

Experimental Design: Four-color flow cytometry and clonogenic EC cultures were used to study EC progenitors in tumor-free mice, tumor-bearing immunodeficient mice, and immunodeficient mice xenotransplanted with human bone marrow (BM) cells.

Results: Endostatin significantly reduced the number of circulating EC progenitors in tumor-free BALB/c mice. The effect of endostatin on EC progenitors was enhanced significantly in mice treated with CI drug treatment. When immunodeficient mice xenotransplanted with human BM cells were treated with CI of endostatin we observed a significant decrease in the engraftment and differentiation of human BM-derived EC progenitors. Numbers of circulating EC progenitors increased 7-fold in immunodeficient mice bearing human lymphoma. In this preclinical model, treatment with CI of endostatin inhibited host murine EC progenitor mobilization and human tumor growth. Furthermore, the clonogenic potential of EC progenitors was impaired severely.

Conclusions: Endostatin is a potent inhibitor of the mobilization and clonogenic potential of human and murine EC progenitors, and its preclinical activity is increased significantly in CI compared with bolus administration. These observations might be useful in the design of future clinical trials.

	INTRODUCTION

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Tumor growth and metastasis requires the generation of new blood vessels (1) . Recent in vivo studies have indicated that early angiogenesis in the tumor microenvironment is characterized by corecruitment and proliferation of BM-derived³ EC and hematopoietic progenitors (2) . Endostatin, a fragment of collagen XVIII, is able to inhibit angiogenesis and tumor growth in an EC-specific manner (3 , 4) . Endostatin induces EC apoptosis in vivo (5) , and inhibits EC proliferation and migration (6) . In the current study we have investigated effects of bolus and CI of endostatin on differentiation, mobilization, and clonogenic potential of BM-derived EC and hematopoietic progenitors. Furthermore, we evaluated two animal models of human lymphoma to compare the preclinical activity of CI versus bolus endostatin.

	MATERIALS AND METHODS

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Animal Models.
As described previously (4) , 6–8-week-old NOD/SCID mice were injected i.p. with 10 x 10⁶ Namalwa or Granta 519 cells (American Type Culture Collection, Manassas, VA). Namalwa cells (phenotype CD3-, CD10+, CD13-, CD19+, CD20+, and GlyA-) were derived from an EBV+ Burkitt’s lymphoma and cultured in RPMI 1640–8% fetal bovine serum (HyClone, Logan, UT). Granta 519 cells (phenotype CD3-, CD10-, CD13(+), CD19+, CD20+, and CD30+) were derived from PB at relapse of a high-grade B-cell mantle cell lymphoma in leukemic transformation and cultured in DMEM-10% fetal bovine serum. Tumor growth was evaluated every other day, tumors were measured by calipers, and the formula (width² x length x 0.52) was applied for approximating the volume of a spheroid (3 , 4) . In separate xenograft studies, 6–8-week-old NOD/RAG (7) mice sublethally irradiated at 500 rads were injected i.v. with 6 x 10⁶ human BM cells from healthy subjects.

Tumor-bearing NOD/SCID mice (n = 6/study group, treatment beginning day 2 after tumor inoculation), tumor-free BALB/c mice (n = 6/study group), and chimeric NOD/RAG mice (n = 5/study group, treatment beginning day 15 after transplant of human BM) were treated with bolus endostatin (150 µg/mouse/day i.p. or s.c. in a site remote from the inoculated tumor), CI of endostatin [150 µg/mouse/day for 21–28 days, delivered by an osmotic pump releasing 1 µl/hour for 1 week or 0.5 µl/hour for 2 weeks produced by Alzet (Cupertino, CA) and implanted s.c.] or CI of PBS as a control. Clinical grade, recombinant human endostatin (expressed in Pichia pastoris) was kindly provided by EntreMed (Rockville, MD).

All of the procedures involving animals were done in accordance with national and international laws and policies.

Measurement of EC and Hematopoietic Progenitors by FC.
Murine PB EC progenitors were enumerated by four-color FC using a panel of monoclonal antibodies reacting with murine CD45 (to exclude hematopoietic cells; Ref. 8 ) and endothelial murine markers VEGF receptor 2 FLK, CD34, and CD117 (PharMingen BD, San Diego, CA). On some occasions, nuclear staining (Procount; BD, San Jose, CA) was used to ascertain whether platelets or cell debris hampered the accuracy of EC progenitor enumeration. After red cell lysis, cell suspensions were evaluated by a FACScalibur (BD) using analysis gates designed to exclude dead cells, platelets, and debris. After acquisition of at least 100,000 cells/sample, analyses were considered as informative when adequate numbers of events (i.e., >50, typically 100–200) were collected in the EC progenitor enumeration gates. Percentages of stained cells were determined and compared with appropriate negative controls. Positive staining was defined as being greater than nonspecific background staining. Annexin V and 7AAD were used to depict and exclude apoptotic and dead cells (4 , 8) .

The presence of BM-derived human EC progenitors and mature EC cells was evaluated in chimeric NOD/RAG mice by FC as described previously (9) with few modifications. Monoclonal antibodies reacting with human CD45 were used to exclude human hematopoietic cells, and monoclonal antibodies reacting with human (but not murine) endothelial markers CD31, CD34 (PharMingen BD), and CD133 (Miltenyi Biotec, Auburn, CA) were used to depict human EC and EC progenitors. After red cell lysis, cell suspensions were evaluated by a FACScalibur as described above.

Evaluation of EC Progenitor Clonogenic Potential.
PB cells from tumor-bearing NOD/SCID and tumor-free BALB/c mice were evaluated for the presence of circulating clonogenic EC progenitors by seeding 0.5 (NOD/SCID) or 1 x 10⁶ (BALB/c) nucleated cells in Petri dishes coated previously with fibronectin in the presence of collagen gel, EC medium, 12.5% FCS, and 12.5% horse serum supplemented by VEGF (100 ng/ml) and b-FGF (5 ng/ml). Cells were cultured at 37°C. On day 14 of culture, colonies with endothelial morphology (slightly elongated, sprouting or spindle, sometimes multinucleated cells) were enumerated, and subclones established by picking colonies and resuspending the cells in 24-well plates in the presence of VEGF and b-FGF. Fresh medium and cytokines were added weekly. After 4-week culture, when seeded cells showed EC characteristics (patterned, tubular networks, sometimes multinucleate cells), the EC phenotype of cultured cells was evaluated and confirmed by FC.

Statistical Analysis.
Statistical comparisons were performed using the t test, ANOVA and linear regression when data were normally distributed, and the nonparametric analyses of Spearman and Mann-Whitney when data were not normally distributed. Values of P < 0.05 were considered as statistically significant.

	RESULTS

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CI of Endostatin Inhibits EC Progenitor Mobilization.
Fig. 1 shows murine and human EC and EC progenitor evaluation by four-color FC. According to Rafii (10) , human EC progenitors were enumerated as CD45-, CD31+, and CD34+ CD133+, and human differentiated EC as CD45-, CD31+, CD34+, and CD133-. Because the CD133 antigen has not been characterized in mice, according to Kocher et al. (11) , murine EC progenitors were enumerated as CD45-, FLK+, CD34+, and CD117+. The detection limit of EC progenitor detection by FC was 0.1 cell/µl, specificity being >90% (8 , 9) . Nuclear staining indicated that platelets did not interact with EC enumeration.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Representative murine (top) and human (bottom) EC progenitor evaluation by FC. A, analysis gate used to exclude platelets, dead cells, and debris. B, gate used to exclude CD45+ hematopoietic cells. C, gate for enumeration of CD45-, FLK+, CD34+ EC. D, enumeration of CD117+ EC progenitors. E, gate for the enumeration of human CD45-, CD31+ EC. F, enumeration of human CD34+ CD133+ EC progenitors.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. A, circulating murine EC progenitors in tumor-free BALB/c mice treated with CI of PBS as a control (n = 6), bolus IP endostatin (n = 6), and CI of endostatin (n = 6, * = P < 0.01 versus bolus). B, human EC progenitors (CD45-, CD31+, CD34+, CD133+, ) and differentiated EC (CD45-, CD31+,CD34+, CD133-, ) in the BM of tumor-free (i.e., uninjected) NOD/RAG mice xenotransplanted with human BM and treated with CI of endostatin (n = 5), and with CI of PBS as a control (n = 5, * = P < 0.001). C, circulating hematopoietic progenitors (CD45+, CD34+, CD117+) in tumor-free BALB/c mice (n = 6/study arm), xenotransplanted, tumor-free NOD/RAG mice (n = 5/study arm) and tumor-bearing NOD/SCID mice (n = 6/study arm) treated with CI of PBS as a control (), bolus IP endostatin (), or CI of endostatin (, * = P < 0.05 versus control). D, circulating EC progenitors in tumor-bearing NOD/SCID mice treated with CI of PBS as a control (n = 6), bolus IP endostatin (n = 6), and CI of endostatin (n = 6, * = P < 0.0001 versus bolus); bars, ±SD.

CI of Endostatin Abrogates Lymphoma Growth in Vivo.
Two weeks after tumor injection, the total number of circulating EC progenitors (CD45-, CD34+, FLK+, and CD117+) was increased 7-fold in the PB of lymphoma-bearing NOD/SCID mice (P = 0.012 versus tumor-free NOD/SCID mice evaluated as control). Fig. 3 shows Namalawa and Granta 519 tumor growth in NOD/SCID mice treated with endostatin or PBS as a control. In both models, bolus daily endostatin (either s.c. or i.p.) significantly inhibited tumor growth, and CI of endostatin abrogated tumor growth. In bolus endostatin-treated mice, minced Namalwa and Granta tumors had decreased murine viable EC (FLK + 7AAD-) and increased apoptotic EC (FLK+ 7AAD+; P < 0.01; Fig. 3B ).

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Namalwa (A) and Granta 519 (C) tumor growth in NOD/SCID mice treated for 21 (Namalwa) or 28 days (Granta 519) with CI of endostatin (n = 6), bolus IP, or Sc endostatin (n = 6), and CI of PBS as a control (n = 6). B, frequency (expressed as percentage of nucleated cells in minced tumor suspensions) of murine viable (FLK +7AAD-, ) and apoptotic EC (FLK +7AAD+, ) in Namalwa tumors from mice treated with bolus IP endostatin versus control (* = P < 0.01, the FC procedure for EC evaluation has been described in detail in Ref. 4 ). Namalwa tumors did not grow in mice treated with CI of endostatin; bars, ±SD.

CI of Endostatin Inhibits the Clonogenic Potential of EC Progenitors.
Fig. 4 shows morphology and phenotype characteristics of colonies and subclones generated by circulating EC progenitors in tumor-bearing NOD/SCID mice. A median of 11 and 3 EC-like colonies was found in cultures of tumor-bearing NOD/SCID mice and tumor-free BALB/c mice, respectively. EC progenitors were considered as clonogenic when adherent cell cultures derived from single colonies displayed a predominant EC phenotype (i.e., >70% CD45-, CD31+, P1H12+ cells at FC; Fig. 4, E–H ). This phenotype was observed in 20–70% of secondary cultures (median 60%). Endostatin effect on EC clonogenic potential was higher in mice treated with CI versus mice treated with bolus drug (Fig. 4I ; P < 0.01).

View larger version (96K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Clockwise, from top left. A, representative EC colonies evaluated after 14-day culture in the presence of collagen gel, VEGF, and b-FGF. Colonies including EC-like spindle cells (B) were picked and cells resuspended in 24-well plates. After 4-week culture, when seeded cells showed EC characteristics (patterned, tubular networks of adherent, sometimes multinucleate cells, see arrows in C and D), the EC phenotype of cultured cells was evaluated and confirmed by FC. E, forward and side scatter evaluation used to generate the analysis gate; F, negative controls; G and H, frequency of viable (7AAD-) cells expressing EC markers P1H12 (G), and CD31 and lacking the hematopoietic CD45 marker (H). I, inhibition of the clonogenic potential of EC progenitors in tumor bearing NOD/SCID and tumor-free BALB/c mice treated with CI (, n = 6) or bolus IP endostatin (, n = 6; * = P < 0.01). Inhibition was evaluated versus mice treated with CI PBS as a control (n = 6); bars, ±SD.

	DISCUSSION

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Endostatin, the COOH-terminal M_r 20,000 fragment of collagen XVIII, is known to inhibit tumor growth by interfering with angiogenesis in an EC-specific fashion. The mechanism of endostatin action, still poorly understood, may rely on interference with integrins (15) or interaction with tropomyosin-containing microfilaments (16) that lead to disruption of microfilament function and induction of EC apoptosis. In the current study we have observed that endostatin significantly inhibits EC progenitor mobilization in both tumor-free and tumor-bearing mice. In immunodeficient mice xenotransplanted with human BM cells, endostatin administration was associated with decreased differentiation of human BM cells to an EC progenitor phenotype (CD133+) and to mature (CD133-) EC. Along this line, we observed another novel effect of endostatin on the clonogenic potential of EC progenitors. In fact, endostatin impaired their ability to generate colonies and subclones. Our studies also confirm previous observations from Kisker et al. (17) indicating that CI of endostatin administration by osmotic pump improves its efficacy. This is in line with other observations indicating that effective delivery of other antiangiogenic drugs such as angiostatin and IFN requires continuous rather than bolus administration for prolonged periods of time (18 , 19) .

Considering that early angiogenesis seems to involve mobilization of EC and hematopoietic progenitors, we also studied the impact of endostatin on the latter cells. BM homing of hematopoietic progenitors is known to rely on complex (and still poorly understood) molecular pathways where integrins play a dominant role (20) . Thus, the interaction of endostatin with 5 and v integrins (15) might explain, at least in part, the trend toward increased mobilization of hematopoietic progenitors that we observed in mice treated with endostatin. However, our data collected in tumor-bearing mice treated with endostatin indicate that mobilization of hematopoietic progenitors is not sufficient to support angiogenesis in the absence of an adequate mobilization of EC progenitors.

Taken together, our data support two major findings. First, endostatin acts as a potent inhibitor of the mobilization and clonogenic potential of human and murine EC progenitors. Second, endostatin effect on EC progenitors and its preclinical activity are increased significantly in CI compared with bolus administration. These observations also confirm our previous findings (4) on the therapeutic potential of endostatin in preclinical models of human lymphoma. In recent reports of Phase I clinical studies of i.v. bolus or CI endostatin (21 , 22) , circulating EC were found to decrease >10-fold in patients with either a minor response or stable disease, whereas circulating EC were not changing or increasing in patients with progressive disease. Thus, the prospective evaluation of circulating EC seems promising as a surrogate marker of endostatin clinical activity, and results of the present study might be useful in the design of clinical trials in lymphoma and other neoplastic diseases.

	ACKNOWLEDGMENTS

 
We thank Aron Goldhirsch for critical reading of the manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by Fondazione Italiana Ricerca sul Cancro, Associazione Italiana Ricerca sul Cancro, and NIH Grants AI30389 and DK57199. F. B. is a scholar of the United States National Blood Foundation.

² To whom requests for reprints should be addressed, at Division of Hematology-Oncology, European Institute of Oncology, via Ripamonti 435, 20141 Milan, Italy. Phone: 39-02-57489535; Fax: 39-02-57489537; E-mail: francesco.bertolini{at}ieo.it

³ The abbreviations used are: BM, bone marrow; EC, endothelial cell; CI, continuous infusion; FC, flow cytometry; NOD/RAG, nonobese diabetes/recombination activating gene (NOD-Rag1^null); NOD/SCID, nonobese diabetes/severe combined immunodeficiency (NOD/-prkdc^scid); PB, peripheral blood; VEGF, vascular endothelial growth factor; FLK, vascular endothelial growth factor receptor 2 fetal liver kinase 1; 7AAD, 7-aminoactinomycin D; b-FGF, basic fibroblast growth factor.

⁴ F. Bertolini, S. Paul, and P. Mancuso, unpublished observations.

Received 4/22/02; revised 7/ 9/02; accepted 7/15/02.

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