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Tumor Biology |
Division of Pediatric Oncology, Department of Pediatrics [E. S. J. M. d. B., F. S., T. M., W. A. K.] and Division of Hematology, Department of Internal Medicine [E.V.], University Hospital Groningen, 9700RB Groningen, the Netherlands, and Department of Cell Biology, Section Histology and Immunology, University of Groningen, 9713 AV Groningen, the Netherlands [J. E. J. G., N. A. B.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Asahara et al. (10) and Takahashi et al. (11) reported that CD34+ endothelial progenitors have the potential to differentiate in vitro to endothelial cells and can be incorporated in vivo into sites of neoangiogenesis. Even stronger evidence was provided by Shi et al. (12) , who demonstrated that bone marrow-derived CD34+ endothelial progenitors had the capacity to line an implanted vascular prosthesis. More recently, emerging data show that a subset of human CD34+ cells coexpressing VEGFR-2 and AC133 have phenotypic features of endothelial cell progenitors playing a role in postnatal angiogenesis (13 , 14) .
The process of new vessel formation plays a critical role in solid tumor outgrowth, invasion, and metastasis (15 , 16) . Recently, in hematological malignancies, neovascularization has also been observed, especially in lymphoma, multiple myeloma, acute lymphoblastic leukemia and acute myeloid leukemia (17, 18, 19, 20, 21, 22) .
The process of neovascularization has long been considered as angiogenesis (vessel sprouting from preexisting vessels; Ref. 16 ). However, more recent studies have indicated that postnatal vasculogenesis implying in situ vessel formation from CD34+ hematopoietic stem cells might occur due to the expansion and differentiation of a specific subset of hematopoietic VEGFR-2-expressing stem cells called hemangioblasts to (precursor) endothelial cells that are also able to express ANG-2 (10 , 23) . Evidence indicates that ANG-2 plays a critical role in vessel outgrowth, remodeling, and maturation (24 , 25) .
Based on these results, we hypothesized that the infused mobilized CD34+ cells might facilitate the outgrowth of tumor or residual tumor. We demonstrated that (a) i.v. infused human CD34+ mobilized cells enhance tumor growth in a xenotransplantation mouse NOD/SCID model of human non-Hodgkin’s lymphoma and (b) mobilized human CD34+ cells are incorporated in the tumor and differentiate along the endothelial lineage.
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MATERIALS AND METHODS |
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After obtaining informed consent, peripheral blood MNCs were obtained from leukapheresis samples of multiple myeloma patients (n = 3) undergoing autologous peripheral blood stem cell mobilization. Cells were collected by apheresis during the regeneration phase after high-dose cyclophosphamide (6 g/m2) in the presence of G-CSF (5 µg/kg). CD34+ cells were isolated by positive selection using Isolex-300 (Baxter, Deerfield, IL) according to the manufacturer’s instructions. At the end of the procedure, CD34+ cell purity was reanalyzed by flow cytometry using anti-HPCA-2 and was >90%. No CD138+ (BB4) cells were detected in stem cell collections.
Animal Study.
NOD/SCID mice (kindly provided by Dr. L. D. Shultz, The Jackson Laboratory, Bar Harbor, ME) were bred and maintained at the Central Animal Facility, University of Groningen. Animals were kept under laminar flow conditions during the experiment. Our model was generated by s.c. injecting 10 x 106 Daudi cells into the neck region of sublethally irradiated (2 Gy) 6–8-week-old NOD/SCID mice. The mice were evaluated for tumor growth. At day 11 after tumor inoculation, human CD34+ cells were injected in the tail vein, and control mice received PBS. To control for outgrowth of myeloma, mice were injected with mobilized human CD34+ cells (3 x 106 cells) after 3 Gy of irradiation. Tumor-bearing mice were killed by carbon dioxide asphyxiation, and tumor engraftment was confirmed by histological and immunohistochemical studies. All procedures involving animals were done in accordance with local ethical animal laws and policies.
Phenotypic Analysis.
Antihuman CD34, CD38, CD10, CD33, CD56, CD7, and anti-GPA phycoerythrin-1- or FITC-conjugated antibodies were purchased from Becton Dickinson (Mountain View, CA), and antihuman CD138 was obtained from Immunoquality Products (Groningen, the Netherlands). Biotinylated rat antimouse CD18 (PharMingen, San Diego, CA) developed with streptavidin-allophycocyanin was used to differentiate between mouse and human cells. Cells (105) were labeled with FITC- and/or phycoerythrin-1-conjugated antibodies or isomatched controls for 30 min on ice. Subsequently, the cells were washed in PBS containing 1% BSA and 0.1% NaN3. Cells were analyzed on a FACScan flow cytometer using CellQuest software from Becton Dickinson.
Immunohistochemical Stainings.
Tumor tissue and other used tissues from mice were collected and snap-frozen until analysis. Tumor specimens were cut in 4-µm sections and fixed in acetone. Slides were treated with different antibodies diluted in PBS/1% BSA. An appropriate peroxidase-labeled second antibody was used, followed by the addition of substrate for peroxidase. The following antibodies (obtained from PharMingen) were used: (a) rabbit antihuman FVIII (dilution, 1:3200; DAKO AS, Glostrup, Denmark); (b) rabbit antihuman IgM (dilution, 1:4000); (c) mouse antihuman CD20 and CD22 (dilution, 1:1000); and (d) rat antimouse CD31 (0.5 mg/ml). As secondary antibodies, we used peroxidase-labeled swine antirat IgG (dilution, 1:40) and biotin-labeled swine antirabbit (Fab')2 (dilution, 1:300; both from Southern Biotechnology Associates, Birmingham, AL). Streptavidin peroxidase-labeled second step reagent (1:300) was obtained from DAKO.
RT-PCR Analysis for Several Angiogenic Factors.
Total RNA from tumor tissues was extracted by the Trizol method, following the manufacturer’s instructions (Life Technologies, Inc., Grand Island, NY). cDNAs were prepared by reverse transcription at 37°C for at least 1 h in a 20-µl reaction mixture containing 2 µg of total RNA, random hexamers (Pharmacia), 5x first strand buffer, RNasin, and 1 µl of reverse transcriptase (Life Technologies, Inc.). cDNA was amplified in the presence of primers, 10x buffer, 1.5 mM MgCl2, deoxynucleotide triphosphates, and Taq (Life Technologies, Inc.). The mixture was amplified in a Perkin-Elmer apparatus with PCR cycle conditions specific for the PCRs tested. PCR product was analyzed by electrophoresis in a 1.5% agarose gel. Gels were stained with ethidium bromide and photographed. Specific primers for ß2-microglobulin were CCA-GCA-GAG-AAT-GGA-AAG-TC (sense) and GAT-GCT-GCT-TAC-ATG-TCT-CG [antisense; PCR product, 268 bp; 22 cycles; Tann (annealing temperature) 55°C]. Specific primers for IL-8 were TTG-GCA-GCC-TTC-CTG-ATT (sense) and AAC-TTC-TCC-ACA-ACC-CTC-TG (antisense; PCR product, 247 bp; 28 cycles; Tann 55°C; 1.25 mM MgCl2 and 5% DMSO). Specific primers for VEGF were GAG-TGT-GTG-CCC-ACT-GAG-GAG-TCC-AAC (sense) and CTC-CTG-CCC-GGC-TCA-CCG-CCT-CGG-CTT (antisense; PCR products, 177, 312, and 384 bp; 30 cycles; Tann 60°C). The primers for VEGF span the splice junctions, allowing the amplified product of each splice variant to be separated electrophorically. Specific primers for bFGF were [GCC-TTC-CCG-CCC-GGC-CAC-TTC-AAG-G (sense) and GCA-CAC-ACT-CCT-TTG-ATA-GAC-ACA-A (antisense; PCR product, 180 bp; 34 cycles; Tann 56°C], and for restriction analysis of the PCR product, AflIII (mouse specific; 60 and 115 bp) and AluI (human specific; 54 and 124 bp) were used. For ANG-2 [PCR product, 234 bp; 34 cycles; Tann 55°C (R&D Systems)], ScaI (mouse specific; 100 and 130 bp) and FokI (human specific; 35 and 200 bp) were used for restriction analysis of the PCR product. For MMP-2, specific primers GGC-CCT-GTC-ACT-CCT-GAG-AT (sense) and GGC-ATC-CAG-GTT-ATC-GGG-GA (antisense; PCR product, 473 bp; 30 cycles; Tann 55°C) were used, and for restriction analysis of the PCR product, ScaI (mouse specific; 235 and 240 bp) and AccI (human specific; 184 and 290 bp) were used. For MMP-9, specific primers CAA-CAT-CAC-CTA-TTG-GAT-CC (sense) and CGG-GTG-TAG-AGT-CTC-TCG-CT (antisense; PCR product, 479 bp; 30 cycles; Tann 55°C) were used, and for restriction analysis of the PCR product, AluI (mouse specific; 420 bp) and EcoI (human specific; 200 and 280 bp) were used. All restriction enzymes were obtained from Promega.
Nested RT-PCR for VEGFR-2 was performed as follows: (a) first PCR was performed using CTT-CAA-CCA-GTC-TGG-GAG-TGA-GA (sense) and CTC-TCC-TGC-TCA-GTG-GGC-TGC-ATG-T (antisense; PCR product, 860 bp; 25 cycles; 1 mM MgCl2 and 5% DMSO; Tann 60°C); and (b) nested PCR was performed using TGG-AAG-TGG-CAT-GGA-ATC-TC (sense) and TTG-CCG-CTT-GGA-TAA-CAA-GG (antisense; PCR product, 571 bp; 25 cycles; Tann 53°C) with 4 µl of the first PCR product.
Nested RT-PCR for ANG-2 was performed as follows: (a) first PCR was performed using TGA-GCA-AAC-GCG-GAA-GT (sense) and TTC-TTC-TTT-AGC-AAC-AGT-GGG (antisense; PCR product, 423 bp; 30 cycles; 1 mM MgCl2; Tann 62°C); and (b) nested PCR was performed using CTT-GGA-ACA-CTC-CCT-CTC-G (sense) and GTC-CTT-AGC-TGA-GTT-TGA-TGT-GG (antisense; PCR product, 330 bp; 30 cycles; 1 mM MgCl2; Tann 60°C) with 4 µl of the first PCR product. Restriction analysis of this PCR product was performed using NheI (human specific; 106 bp) and BstOI (mouse specific; 169 bp).
To control for the addition of cDNA in the first PCR reaction, ß2-microglobulin PCR was performed from the same first PCR product as used for the second VEGFR-2 PCR (nested PCR). The first product was split; 4 µl were used for the ß2-microglobulin PCR to control for the cDNA addition in the first PCR.
Statistical Analysis.
Nonparametric tests such as the Mann-Whitney U test and Spearman analyses were used for the abnormally distributed data. P < 0.05 was considered to represent significance.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Tumors grew as solid masses in the neck region. Tumors were composed of CD20+/CD22+ cells, with membrane IgM expression typical for Daudi cells (Fig. 1B)
. No large areas of necrosis were observed (data not shown). When left untreated, the tumors reached a weight of approximately 600 mg at day 24. In this s.c. model, no metastases were found ever during the therapeutic window.
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. No differences in vessel number could be observed between the center and peripheral areas of the tumors (data not shown). Accordingly, we determined the expression of several angiogenic factors including ANG-2, bFGF, MMP-2, MMP-9, and VEGF by RT-PCR. In addition, we assessed whether these factors were of mouse or human origin. In vitro cultures of Daudi cells demonstrated the expression of human VEGF and MMP-9 mRNA, but not transcripts for ANG-2, bFGF, and/or MMP-2 (Fig. 2A)
. However, in the tumor sections, not only were VEGF and MMP-9 found (as expected), but mouse-specific ANG-2, MMP-2, and bFGF were found as well, as determined by restriction enzyme analysis (Fig. 2B)
. No differences in expression of the investigated angiogenic factors were observed between the center of the tumors and the peripheral areas (data not shown).
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shows that the tumor weight in the xenotransplanted mice infused with human CD34+ cells is nearly 2-fold greater than that seen in xenotransplanted mice infused with control PBS [median CD34+ group (1086 mg; range, 411-1287 mg; n = 12) versus PBS group (586 mg; range, 270–976 mg; n = 11), P = 0.004]. When using the CD34- fraction of MNCs from leukapheresis samples, the tumor growth was not enhanced compared with that seen in PBS controls [median CD34- fraction (749 mg; range, 685–858 mg; n = 3) versus median PBS controls (586 mg; range, 270–976 mg; n = 11). In addition to 0.5 x 106 CD34+ cells, 0.1 x 106 CD34+ cells were injected to show a relation between the number of injected CD34+ cells and the increased tumor growth. A 5-fold lower amount of CD34+ cells resulted in a tumor of 443 mgr (range, 424–462 mgr; n = 2). This suggests that a certain number of injected CD34+ cells are needed to increase the size of the tumors. In Fig. 3B,
the difference in tumor growth is visualized for two representative animals. No differences could be found with regard to morphology, IgM, CD20/CD22, CD31, and FVIII stainings between tumors from mice infused with CD34+ cells and tumors from mice injected with PBS. No differences were found in the number of vessels/high-power field; however, the total tumor mass was increased by 2-fold.
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Human CD34+ Hematopoietic Stem Cells Can Be Found in the Tumors.
To demonstrate that the injected human CD34+ cells have been homed to the tumors and differentiated along the endothelial lineage to (precursor) endothelial cells, RT-PCR studies were performed with oligonucleotides specific for human VEGFR-2, which is expressed on CD34+ cells. As depicted in Fig. 4A,
VEGFR-2 mRNA is expressed by the tumors from mice injected with CD34+ cells, whereas in the control mice, no transcript was shown. In all samples, ß2-microglobulin PCR was performed with the same first PCR product to confirm the equivalent addition of cDNA.
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shows the results; ANG-2 mRNA (human specific) is expressed by tumors from mice injected i.v. with CD34+ cells, whereas in control mice, no ANG-2 transcript was found. In addition, CD34+ cells and Daudi cells in culture are ANG-2-. With enzyme restriction analysis (Fig. 4C)
, it was demonstrated that the ANG-2 PCR bands were of human origin and not of murine origin. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This xenotransplantation model was chosen because it has the advantage of 100% engraftment, and spontaneous regression of tumors is rarely seen (27) . As shown by Hudson et al. (27) , optimal growth of Daudi cells is achieved using NOD/SCID mice in conjunction with sublethal radiation. The disadvantage of this model is the ectopic localization of the tumor outside its normal environment. However, the actual biology or related immune function was not the aim of this study.
Tumor-induced blood vessel formation is the result of a strong interaction between tumor cells and surrounding normal cells, such as endothelial cells or their precursors. In the present study, this is reflected by the production of VEGF and MMP-9 of human origin and MMP-2, bFGF, and ANG-2 of murine origin; it is likely that all these factors contribute to tumor growth because murine MMP-2 and bFGF affect human cells.
Recent data have indicated that a circulating common precursor for hematopoietic and endothelial cells might exist that is phenotypically characterized by the expression of VEGFR-2 (12, 13, 14, 15) . The existence of a bipotential precursor (hemangioblast) for hematopoietic and endothelial cells was suggested by the finding that embryos lacking VEGFR-2 demonstrated defects in both hematopoietic and endothelial lineages (28 , 29) . In normal homeostasis, a low amount of mature endothelial cells (0.05%) can be detected in the peripheral circulation (30, 31, 32) . It is believed that these circulating endothelial cells are mainly the result of turnover from vessel wall endothelium and can be expanded slowly in in vitro cultures (33) . In contrast, bone marrow-derived endothelial cells demonstrate a 50-fold faster expansion but are even 20-fold more sparse in the peripheral blood. However, during the mobilization of bone marrow hematopoietic precursors, the number of circulating progenitors in the peripheral blood is increased from 0.05% or less up to 1–5%. This increase is comparable to the effect of i.v. infusion of human CD34+ cells in this mice tumor model. It is not known whether these mobilized circulating endothelial cells are involved in enhanced outgrowth of tumors in humans. However, in human chronic myeloid leukemia, it was recently shown that the BCR/ABL fusion transcript was expressed in a variable proportion of endothelial cells (34) .
Endothelial precursors originating from hematopoietic stem cells remain VEGFR-2+ and might become ANG-2+ during their differentiation, whereas hemangioblasts committed to the hematopoietic lineage showed no Ang-2 transcripts and lose their VEGFR-2 expression. Our results strongly suggest that injected CD34+ human cells from peripheral blood stem cell sources can be found in the tumor as human VEGFR-2+ and ANG-2+ cells. The possibility that outgrowth of circulating endothelial cells from the peripheral blood enhance tumor growth by new vessel formation cannot be excluded. However, the number and proliferating potential of circulating endothelial cells are far less then those of bone marrow-derived CD34+ cells, as discussed above. Dedifferentiation of Daudi cells to precursor endothelial cells cannot be excluded in this model. However, Daudi cells are CD34-, VEGFR-2-, and ANG-2-, and dedifferentiation of Daudi cells has not been described until now.
These results show for the first time that malignant tumors are capable of incorporating mobilized CD34+ hematopoietic cells, resulting in enhanced tumor growth. The study suggests that leukapheresis and resubmission of mobilized stem cells in patients with residual tumor might lead to enhanced tumor growth and must be done with caution. Further investigations are warranted to ensure that leukapheresis and/or stem cell reinfusion in the setting of autologous stem cell transplantation in patients with residual disease are a safe method of treatment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Research Grant SKOG 96-002 from the Foundation for Pediatric Oncology Research Groningen. 
2 To whom requests for reprints should be addressed, at Department of Pediatrics, Division of Pediatric Oncology/Hematology, University Hospital Groningen, P. O. Box 30.001, 9700RB Groningen, the Netherlands. Phone: 31-50-361-4213; Fax: 31-50-361-1671; E-mail: e.de.bont{at}bkk.azg.nl
3 The abbreviations used are: NON/SCID, nonobese diabetic/severe combined immunodeficient; MNC, mononuclear cell; VEGFR, vascular endothelial growth factor receptor; VEGF, vascular endothelial growth factor; ANG-2, angiopoietin-2; RT-PCR, reverse transcription-PCR; G-CSF, granulocyte colony-stimulating factor; IL, interleukin; bFGF, basic fibroblast growth factor; MMP, matrix metalloproteinase.
Received 2/14/01. Accepted 8/13/01.
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