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Leukemia-stimulated bone marrow endothelium promotes leukemia cell survival
Experimental Hematology 34 (2006) 610–621
Leukemia-stimulated bone marrow endothelium promotes leukemia cell survival J. Pedro Veigaa, Lara F. Costaa,b, Stephen E. Sallana, Lee M. Nadlera, and Angelo A. Cardosoa a
Departments of Medical Oncology and Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Mass., USA; b Unit of Tumor Biology, Institute of Molecular Medicine, University of Lisbon Medical School, Lisbon, Portugal (Received 5 December 2005; revised 19 January 2006; accepted 19 January 2006)
Extensive endothelial cell proliferation and marked neovascularization are the most pronounced microenvironmental changes consistently observed in the bone marrow (BM) of patients with acute lymphoblastic leukemia (ALL). It is not known whether ALL cells induce this phenotype and whether they receive critical signals from the tumor-associated BM endothelium. Here, we show that leukemia cells actively stimulate BM endothelium, promote de novo angiogenesis, and induce neovascularization in the leukemic BM. Soluble factors, present in the leukemic BM microenvironment, promote the proliferation, migration, and morphogenesis of BM endothelial cells, which are critical processes in tumor angiogenesis. We also show in vitro that leukemia cells display directional motion towards assembled BM endothelium and following adherence exhibit cell polarization, pseudopodia, and ultrastructural features that suggest the existence of leukemia-endothelium cross-talk. Finally, we show that BM endothelium promotes leukemia cell survival through a mechanism mediated through the anti-apoptotic molecule bcl-2. These studies indicate that ALL cells actively recruit BM endothelium and mediate the leukemia-associated neovascularization observed in ALL. Therefore, disruption of interactions between leukemia cells and BM endothelium may constitute a valid therapeutic strategy. Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc.
The expansion of malignant cells within the bone marrow (BM) of patients with acute lymphoblastic leukemia (ALL) is associated with the disruption of the normal marrow architecture and the development of marked neovascularization [1–3], a phenotype also observed in other malignancies evolving in the BM [2,4–10]. A consistent structural alteration of the BM microenvironment in ALL is the significant increase in the number of BM endothelial cells. A recent study showed that specialized endothelial microdomains serve as portals for the homing and maintenance of B-cell precursor (BCP) leukemia cells in the BM [11], suggesting a role for this endothelium in the formation of tumor-supportive tumor niches. It is presently unknown whether leukemia cells directly or indirectly induce the observed angiogenic phenotype and endothelial cell expansion. Most ALL patients have increased blood plasma and urine levels of basic fibroblast growth factor (bFGF; FGF-2) [1,2,12,13], suggesting a significant role for this Offprint requests to: Angelo A. Cardoso, M.D., Ph.D., Dana-Farber Cancer Institute, Room D-540B, 44 Binney Street, Boston, MA 02115; E-mail: [email protected]
pro-angiogenic cytokine in the leukemia-promoted endothelial phenotype observed. Other angiogenic factors seem to also contribute for this phenotype, as vascular endothelial factor (VEGF) levels are increased in some patients [2,13], and it has been shown that leukemia cells produce connective tissue growth factor (CTGF; CCN2) [14] and interleukin-8 (IL-8; CXCL8) [15,16]. Increasing evidence indicates that ALL cells receive signals from their microenvironment that impact on their growth and survival. Leukemia cells undergo spontaneous apoptosis ex vivo, but are responsive to stimuli from their microenvironment, as BM stroma can rescue them from apoptosis [17–19]. Similarly, IL-7, a cytokine produced by BM stromal cells and BM endothelium [20,21] that has been implicated in lymphoid tumorigenesis [22], promotes the survival and proliferation of ALL cells [23–26]. In light of the observed endothelial cell expansion in the leukemia BM, it is critical to determine how the BM endothelium contributes to the biology of ALL. In this study, we show that human leukemia cells transplanted into NOD/SCID mice develop in the animals’ BM with a morphological pattern that recapitulates the human
0301-472X/06 $–see front matter. Copyright Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 10.1016/j.exphem.2006.01.013
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disease. Moreover, plasma isolated from the BM of ALL patients induces neoangiogenesis and promotes proliferation, migration, and morphogenesis of primary BM endothelial cells. Importantly, cellular interactions between ALL cells and BM endothelium preserve the expression of anti-apoptotic proteins in leukemia cells, promoting their survival. Therefore, agents that disrupt the cross-talk between BM endothelium and leukemia cells may constitute valuable treatment strategies for ALL.
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Matrigel-plug assay C57BL/6NTac (C57BL/6) mice were purchased from Taconic Farms (Germantown, NY, USA) and maintained at the DFCI Animal Research Facility in accordance with protocols approved by the Animal Care and Use Committee. Plasmas from ALL patients (B-cell ALL) or normal donors (25% vol/vol) were mixed with Matrigel (BD Biosciences, San Jose, CA, USA) and 0.5 mL was injected into the subcutaneous space, in the abdominal paramedian region of 8- to 24-week-old C57BL/6 mice (2–3 mice/specimen) After 7 days, pellets were explanted retaining the peritoneal lining for support, photographed using a Leica MZFLIII stereomicroscope (Leica Systems, Switzerland), and processed for histology and immunohistochemistry.
Materials and methods Leukemia specimens Diagnostic BM and peripheral blood (PB) from ALL patients, or control specimens from age-matched healthy donors, were collected in heparinized tubes. Plasmas from leukemia or normal donors were separated by centrifugation, filtered (0.22 mm), and cryopreserved. After plasma collection, mononuclear cells were separated by density centrifugation and characterized by phenotypic analysis [27]. BM biopsies were obtained at diagnosis. Appropriate informed consent was obtained according to the Declaration of Helsinki, and the studies were approved by the Dana-Farber Cancer Institute’s Institutional Review Board. Bone marrow endothelial cells Bone marrow endothelial cells (BM-EC) were purified from BM aspirates of ALL patients and healthy donors, as described [28]. Briefly, BM-EC were purified using CD105 microbeads (Miltenyi Biotech, Auburn, CA, USA), and selected by culture in EGM-2 media (containing bFGF, VEGF, EGF, and IGF-1; Cambrex BioScience, Walkersville, MD, USA), which was also used for further expansion of these cells. Cultures were carried 37 C, 5% CO2. BM-EC between the second and the sixth passage were used for the experiments, and cell lineage was confirmed by phenotypic and functional analyses. These cells express CD31, CD106, Podocalyxin, Tie-2, VEGF, and FGF receptors, and incorporate acetylated LDL; they also respond to leukemic and recombinant pro-angiogenic stimuli by activating the PI3K/Akt and mTOR pathways [28]. NOD/SCID model of human ALL NOD.CB17-Prkdcscid/J (NOD/SCID) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and maintained at the DFCI Animal Resource Facility according to protocols approved by the DFCI Animal Care and Use Committee. Leukemia cells (18 B-cell, 2 T-cell ALL patients) were thawed and viable cells prepared using the Apoptotic Cell Removal Kit (Miltenyi). Five to 10 3 106 leukemia cells were injected intravenously in the tail vein of irradiated (250 cGy) NOD/SCID mice (3–5 animals/condition). Animals were monitored and sacrificed when moribund. Bone marrow, liver, and spleen samples were collected to determine engraftment levels by flow cytometry using antibodies specific for human CD45 and CD19, and mouse CD45 (BD Pharmingen, San Diego, CA, USA). Femurs, kidneys, liver, spleen, lungs, and abnormal masses were processed as described below for histology and immunohistochemistry.
Histology and immunohistochemistry Samples were fixed in 10% neutral buffered formalin (Sigma, St. Louis, MO, USA), embedded in paraffin, and 3-mm sections stained with hematoxylin/eosin (H&E). Additional sections were stained with rat anti-mouse CD31 (1:100; DB Pharmingen), rabbit anti-mouse Tie-2 (1:350; C-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or rabbit anti-human von Willebrand Factor (vWF; 1:500; Dako, Carpinteria, CA, USA) antibodies, as indicated. Antigen retrieval was performed with 0.25% trypsin (Sigma, St. Louis, MO, USA) for CD31 and vWF stainings, or citrate buffer, pH 6.0 (Biocare Medical, Walnut Creek, CA, USA) for Tie-2 staining. For CD31 staining, a rabbit anti-rat antibody was used (1:200; Dako). Slides were incubated with goat anti-rabbit horseradish peroxidase–conjugated antibody and developed with DAB chromogen (EnvisionD, Dako) per manufacturer’s instructions, and counterstained with hematoxylin. Measurement of angiogenic factors The levels of bFGF and VEGF in the BM plasma of ALL patients and normal donors were measured by competitive enzyme-linked immunosorbent assay (ELISA), using commercially available kits (Cytimune, Rockville, MD, USA), following the manufacturer’s instructions. BM-EC proliferation and migration BM-EC were plated on flat-bottom 96-well plates at 4 3 103 cells/ well, in EGM-2. After 12 hours, plates were washed with Hank’s balanced salt solution (HBSS; Sigma) and starved for 12 hours in cytokine-free EBM-2 media (Cambrex BioScience). Media were removed and BM-EC washed with HBSS. Test plasmas (patients or normal donors) were added at 25% vol/vol and incubated for 12 hours. BM-EC cultured in EBM-2 or EGM-2 were used as controls. Cells were pulsed with 0.5 mCi of [3H]-thymidine (PerkinElmer, Boston, MA, USA), incubated for an additional 24 hours, and harvested onto glass fiber filters. [3H]-thymidine incorporation was measured by liquid scintillation spectrophotometry (Betaplate; Amersham Biosciences, Piscataway, NJ, USA). Stimulation indexes (S.I.) were calculated for each individual experiment as: S.I. 5 (cpm experimental condition)/(cpm EBM-2 media). Endothelial cell migration was assessed by standard methodology [29,30] using 8-mm microporous Transwell inserts (Costar, Cambridge, MA, USA). Leukemia or normal plasmas were added to the lower compartment of cluster plates in serum-free media (AIM V; Invitrogen, Carlsbad, CA, USA). AIM V and EGM-2 media were used as controls. BM-EC (0.5–1 3 105) were placed on the Transwell inserts (upper compartment) and incubated for 6 to
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8 hours at 37 C, 5% CO2. Transwell inserts were then removed and the total number of migrated cells was quantified to include both the migrated cells that adhered to the plate (by microscopy; five random high-power fields; 3100) and those in suspension (by flow cytometry). Percentage of migrated cells was calculated by dividing by the number of input cells. BM-EC morphogenesis assay in Matrigel The assembly of endothelial cells into capillary-like or tube-like networks in Matrigel was assessed as previously described [31]. Briefly, BM or PB plasmas (25% vol/vol) were mixed with BMEC (1 3 105), plated onto 24-well plates coated with polymerized growth factor–reduced Matrigel, and incubated at 37 C, 5% CO2. Controls included plasmas from healthy donors and EGM-2. All assays were performed in duplicate or triplicate. Capillary-like structures are defined as tube-shape elongated networks of endothelial cells with multiple nodal points, and were scored at different time points using a Nikon Eclipse TE300 (Nikon Instruments, Melville, NY, USA) microscope and photographed. Kinetics of leukemia–BM-EC interactions Assembly of BM-EC into capillary-like structures in Matrigel was promoted using leukemia plasma (as indicated), adjusting the cell number and amount of Matrigel for the area of a 35-mm dish. After EC structures were formed (12–18 hours), leukemia cells were added at 4:1 ratio (ALL:BM-EC). Experiments were performed using a time-lapse video microscopy system consisting of a humidified chamber at 37 C, 5% CO2, an inverted microscope equipped with a digital camera, and image acquisition software (QED Imaging, Pittsburgh, PA, USA). Serial images were acquired and used to generate QuickTime movies (presented as supplemental data; available at http://www.research.dfci.harvard.edu/veiga). Transmission and scanning electron microscopy For transmission electron microscopy, BM-EC (3–5 3 104) were plated in sterile 7-mm circles of Aclar (Ted Pella, Redding, CA, USA) previously coated with diluted Matrigel (1:5 in EGM-2) or in a Transwell insert on top of a Matrigel layer (100 mL). After overnight incubation, leukemia cells (2 3 105) were added on top of BM-EC and incubated for 4 hours before processing. Cells were then fixed for 1 hour in FGP (1.25% formaldehyde, 2.5% glutaraldehyde, and 0.03% picric acid in 100 mM Cacodylate buffer) fixative and stained with a mixture of 1% OsO4 and 1.5% KFeCN6 for 1 hour, and with 1% uranyl acetate for 30 minutes, followed by conventional Epon embedding. Samples were then sectioned (80 nm) in a Reichert Ultracut-S microtome and analyzed in a JEOL 1200EX transmission electron microscope. For scanning electron microscopy, BM-EC (8 3 104) were plated in sterile 8- to 10-mm circles of Aclar previously coated with Matrigel (1:6 in EGM-2) or covered by a Matrigel layer (125 mL). BM-EC were allowed to form capillary-like structures for approximately 10 hours, after which leukemia cells (5 3 105) were added and incubated overnight prior to processing. Samples were fixed for 1 hour in 2.5% glutaraldehyde in 0.1 M phosphate buffer and dehydrated through graded series of ethanol followed by critical point dryer (CPD), Samdri-PVT-3B (Tousimis, Rockville, MD, USA), in a Touimis CPD machine and 5 minutes sputter coating in a Hummer V with a gold-palladium target. Samples were analyzed in a Leo 1450VP scanning electron microscope.
Leukemia cell survival BM-EC were cultured in 6-well plates until confluent layers were formed. Viable leukemia cells (4–6 3 106) were added to the BMEC layers and the coculture was carried in B-cell media [27]. As control, ALL cells were cultured in the absence of BM-EC. After 3 to 5 days, cells were harvested, stained with annexin V and propidium iodide (PI) using the Apoptosis Detection Kit (R&D Systems), and analyzed by flow cytometry using a Coulter-XL or a Cytomics FC500 cytometer (Beckman-Coulter, Fullerton, CA, USA). In some experiments, soluble CD40L plus IL-4 was added to the cocultures (day 5) to stimulate the leukemia cells, as previously described [32]. Expression of anti-apoptotic molecules ALL cells were harvested from the BM-EC–ALL cocultures (day 3–5), counted, and stained with anti-CD19-PE antibodies (BD Pharmingen). Cells were then permeabilized with Cytoperm (BD Pharmingen) and intracellular staining was performed using FITC-conjugated anti-bcl-2 mAb (Dako) or purified anti-bcl-xL mAb (Santa Cruz Biotechnology) plus FITC-conjugated goat anti-rabbit (Southern Biotechnology Associates, Birmingham, AL, USA) as secondary antibody. Irrelevant isotype-matched antibodies were used as negative controls. Samples were analyzed by flow cytometry using a Coulter-XL or a Cytomics FC500 (Beckman-Coulter). Results were expressed as the ratio of mean fluorescence intensity (MFI) of the specific antibody stain over the MFI of the negative control antibody. Blockade of bcl-2 expression bcl-2 was abrogated using a specific antisense oligonucleotide (Biomol Research, Plymouth Meeting, PA, USA), which has been shown to effectively inhibit the expression of bcl-2 [33]. Briefly, viable ALL cells were plated at 2 3 106 cells/mL in 24-well plates and bcl-2 antisense or scrambled control oligonucleotides (200 nM) were added with Lipofectin (Life Technologies, Gaithersburg, MD, USA) and incubated for 4 hours at 37 C. Cells were harvested, washed in media, and plated on BM-EC monolayers (as described above), in the presence of the same concentration of the respective oligonucleotides. Additional controls included untreated leukemia cells plated on BM-EC layers or without BM-EC. ALL cell viability and bcl-2 protein expression was assessed after 5 days of culture. Statistical analysis The statistical significance was determined using the two-tailed Student’s t-test (with the Welch correction, when appropriate), the paired Student’s t-test, or the two-tailed nonparametric Mann-Whitney test. Differences were considered as statistically significant when a # 0.05.
Results Leukemia development in the bone marrow of NOD/ SCID mice recapitulates the angiogenic phenotype observed in the human disease The marked neovascularization observed in the BM of leukemia patients suggests that it plays a significant role in this malignancy, and indicates that ALL cells stimulate BM
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endothelial cells or their precursors to form new vessels. To investigate the pro-angiogenic potential of ALL cells, viable primary leukemia cells were transplanted into NOD/ SCID mice. Leukemia cells from ALL patients were used and animals were sacrificed when moribund. Engraftment was observed in 18 out of 20 ALL patients tested, with the mice developing a full-blown leukemia (terminal status at 34 to 276 days; mean, 154 days). Leukemia cells, identified as human CD45D cells, were present in the animal’s BM, spleen, liver, and kidneys. Histological analyses of femurs and tibias showed that the BM was largely replaced with human blasts (Fig. 1A). Importantly, these BMs
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showed evidence of active angiogenesis, as demonstrated by the presence of neovessels and endothelial sprouts (Fig. 1A,C,D). This angiogenic phenotype is in striking contrast with the BM morphology of age-matched NOD/ SCID mice (Fig. 1B), or of mice injected with normal human B-cell precursors (data not shown). Immunohistochemistry (IHC) analyses using an antibody specific for murine CD31 confirmed the endothelial origin of the cells forming these ‘‘angiogenic’’ structures (Fig. 1C,D). Importantly, the neovascularization induced by leukemia cells in the animals’ BM is comparable to the phenotype observed in the BM of ALL patients, as exemplified by
Figure 1. Development of human ALL cells in the BM of NOD/SCID mice recapitulates the neovascularization phenotype observed in the human disease. H&E stainings of the BM of NOD/SCID mice comparing animals transplanted with human leukemia cells (A) to age-matched control (B). Inset in (A) represents a 4003 magnification. Immunohistochemistry staining of the BM of transplanted NOD/SCID mice, using an antibody for murine CD31 (C,D) at magnifications of 1003 and 4003 respectively; area magnified in (D) is indicated in (C). Representative BM biopsy of an ALL patient, stained with anti-vWF antibody (E,F), at magnifications of 1003 and 6003 respectively. Arrows indicate blood vessels.
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a representative biopsy (Fig. 1E,F; endothelial cells stained with an anti-vWF antibody). These findings demonstrate that the growth of human leukemia cells in the BM of NOD/SCID mice recapitulates the morphology of the human leukemic BM, namely the leukemia-associated angiogenic phenotype in the BM microenvironment. Leukemic soluble microenvironment induces neovascularization Soluble factors play a critical role in the tumor-triggered ‘‘angiogenic switch’’ that stimulates endothelial cells to form new vessels [34,35]. To evaluate the pro-angiogenic properties of the leukemic soluble microenvironment, BM plasma from ALL patients was tested in vivo using a modification of the Matrigel-Plug assay. Leukemia BM plasma promoted neoangiogenesis as evidenced by the infiltration of the implanted Matrigel by vascular structures and endothelial sprouts (Fig. 2A), in the majority of ALL cases tested (12 out of 17 patients). Equivalent results were obtained using plasma collected from the peripheral blood of ALL patients (25 out of 42 patients). In contrast, plasma from normal controls did not promote vascularization or significant cellular infiltration of the implanted Matrigel (Fig. 2B). IHC analysis using a Tie-2-specific antibody confirmed that the cells form-
ing the vessels as well as most cells infiltrating the Matrigel were of endothelial origin (Fig. 2C,D). These vascular structures were functional, as shown by the presence of erythrocytes within their lumen (Fig. 2A,B) and the detection of hemoglobin in the Matrigel-Plug (data not shown). These findings demonstrate that the development of ALL in the human BM is associated with the production of soluble factors that promote neovascularization. We next screened the BM plasma from ALL patients for the presence of VEGF and bFGF, which are critical factors in the angiogenic phenotype associated with many hematological malignancies [36,37]. Different studies have shown that most ALL patients exhibit increased levels of circulating and urinary bFGF [1,2,12,13]. Using a competitive ELISA, we observed that the BM plasma from most ALL patients (58%) contained elevated levels of bFGF compared to BM plasma from normal donors (Fig. 3A; p ! 0.05). In contrast, the mean levels of VEGF did not significantly differ between leukemic and normal BM plasma (Fig. 3B; p O 0.1). These findings show that, in most cases, the leukemic BM contained increased levels of bFGF, but not of VEGF, extending previous observations that increased levels of bFGF are present in the peripheral blood and in the urine of most ALL patients [2,13].
Figure 2. Leukemia BM plasma promotes neoangiogenesis. Matrigel pellets containing BM ALL plasma (A,C,D) or normal plasma (B) were explanted from animals at day 7, and processed for histology (H&E; A,B) and immunohistochemistry. Arrows indicate blood vessels. Inset in (A) represents a 6003 magnification of a vessel, showing in detail the presence of intraluminal erythrocytes. Staining of ALL plasma pellets using an antibody specific for Tie-2 confirmed the endothelial origin of most cells infiltrating the Matrigel and vessel formation (C, 1003 magnification; D, 4003 magnification).
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Figure 3. Leukemia BM plasma contains elevated levels of bFGF but not VEGF. Competitive enzyme immunoassays were used to measure the levels of bFGF (A) and VEGF (VEGF165; VEGF121) (B) in the BM plasma of ALL patients or normal BM donors (p ! 0.05 for bFGF; p O 0.1 for VEGF). Measurements were performed in duplicate and as described in Materials and Methods. Mean (dotted lines) 6 2 S.E. (gray areas) are shown for each test group.
Leukemic soluble microenvironment promotes the proliferation, migration, and morphogenesis of human bone marrow endothelial cells We then evaluated the effects of leukemia BM plasma on the proliferation, migration, and morphogenesis of human BM endothelial cells, which are critical processes in neovascularization. BM plasma from the majority of ALL patients tested induced proliferation of BM-EC (Fig. 4A; S.I. 5 5.1 6 1.6), which was significantly higher (p ! 0.001) than that promoted by BM plasma from normal donors (Fig. 4A; S.I. 5 1.6 6 0.2). For chemotaxis studies, BM-EC were placed in Transwell inserts and tested for their responsiveness to plasma from ALL patients or normal donors. As shown in Figure 4B, leukemia BM plasma induced significant migration of BM-EC (2.2% 6 0.7%; n 5 15), which was significantly higher (p ! 0.001) than that promoted by control plasmas (0.5% 6 0.1%; n 5 5). These observations demonstrate that the leukemic microenvironment contains soluble factors capable of stimulating the proliferation and migration of human BM endothelial cells.
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Figure 4. Leukemia BM plasma stimulates BM endothelial cell proliferation (A) and migration (B). Mean (dotted lines) 6 2 S.E. (gray areas) are shown for each test group. For proliferation assays, S.I. was calculated as described, and the basal media used was EBM-2. For migration experiments, percentage of migrating cells was calculated as described. Leukemia BM plasma was significantly more effective than normal BM plasma in promoting BM-EC proliferation (p ! 0.001) and chemotaxis (p ! 0.001).
To determine the effect of the leukemic soluble microenvironment on BM endothelial cell morphogenesis, we assessed the ability of ALL plasma to promote the differentiation of this endothelium into capillary-like structures in Matrigel. BM endothelial cells from leukemia patients or normal donors were cultured in Matrigel in the presence of either BM plasma, a combination of angiogenic cytokines, or control media. In all cases tested, leukemia plasma (n 5 18; 17 B-cell and 1 T-cell ALL) promoted the formation of capillary-like BM endothelial structures (Fig. 5A), with an efficacy comparable to that observed using a cocktail of recombinant pro-angiogenic cytokines (Fig. 5B). No structures were formed in the absence of cytokines or plasma (Fig. 5C). In 2 out of 10 cases tested, BM plasma from normal donors also induced the formation of endothelial networks in Matrigel, but these were considerably less complex than those promoted by leukemia plasma (reduced branching and fewer nodal points; data not shown).
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Figure 5. Leukemia soluble milieu stimulates BM endothelium to assemble as capillary-like structures. Experiments were performed in growth factor– reduced Matrigel, and BM-EC were rested in growth factor–free media for 12 hours prior to use. Images show assembly of BM endothelium at 12 hours in response to BM leukemia plasma (A), a cocktail of pro-angiogenic cytokines (bFGF, VEGF, EGF, and IGF-1) (B), and control media (C). At later time points (18–24 hours) structures promoted by recombinant cytokines evolve to form more complex networks.
BM endothelial networks induce directional motion of primary ALL cells To evaluate potential cross-talk between ALL cells and BM endothelium, leukemia cells were plated in Matrigel containing assembled capillary-like networks of human BM endothelium (induced by leukemia plasma, as in Fig. 5A). The movement and kinetics of leukemia cells in Matrigel were studied at sequential time points using either light microscopy or time-lapse video microscopy (TLVM). Leukemia cells actively moved through the Matrigel and, after 4 to 8 hours, most tumor cells surrounded the BM-EC structures (Fig. 6A). As shown in this figure, small, round leukemia cells trafficked and adhered to the outline of the BM endothelial structures. TLVM studies showed that the leukemia cell movement towards the BM endothelial structures is directional, suggesting the involvement of chemotaxis (see Film 1A, Supplemental Data available at http://www.research.dfci.harvard.edu/veiga). In the absence of BM endothelium, leukemia cells cultured in the presence of leukemia BM plasma did not cluster, remaining dispersed in the gel (Fig. 6B), and displayed random motion (Film 1B, Supplemental Data). Interestingly, the directional movement is not observed or is markedly reduced when leukemia cells are added to Matrigel containing organized clusters or structures of BM mesenchymal stem cells or BM stromal myofibroblasts (data not shown). Scanning electron microscopy showed that leukemia cells in direct contact with assembled BM-EC exhibited pseudopodia and were often polarized (Fig. 6C). This is in agreement with the continuous movement of leukemia cells in contact with the assembled BM endothelium observed in TLVM. Ultrastructural analysis using transmission electron microscopy showed endothelial cellular processes and abluminal differentiation with deposition of matrix elements. Importantly, it revealed the existence
of junctional complexes between the plasma membrane of the leukemia cells and the BM endothelial cells (Fig. 6D), suggesting that active cross-talk between these cells may occur. BM endothelium promotes leukemia cell survival To determine whether BM endothelial cells impact on leukemia cell survival, primary ALL cells (n 5 6) were cultured in monolayers of BM endothelial cells and assessed for viability using an annexin V/PI assay. Coculture with BM-EC significantly (p ! 0.05) reduced leukemia cell apoptosis. In contrast, leukemia cells cultured in control conditions underwent spontaneous cell death (Fig. 7A). The mean viability of leukemia cells in contact with BM endothelium was 68%, in comparison to 47% in control conditions (n 5 6 patients). This anti-apoptotic effect was observed in both B-cell and T-cell leukemia specimens. To evaluate whether the leukemia cells rescued by BM endothelium maintain their functional properties, we examined their capacity to respond to physiologic signals. We have previously shown that, in most patients, stimulation of B-cell precursor ALL by CD40L/CD154DIL-4 results in cell clustering, proliferation, and induction/ upregulation of MHC and B7 family molecules [32,38]. Leukemia cells cocultured with BM-EC responded to sCD40L/IL-4 by forming homotypic clusters of leukemia cells (Fig. 7B) and upregulating MHC class I and class II molecules as well as CD80/B7-1 and CD86/B7-2 (data not shown). In contrast, leukemia cells cultured for identical periods without BM-EC did not demonstrate either cluster formation (Fig. 7B) or upregulation of MHC or B7 family molecules (data not shown). These results show that BM endothelium provides a survival signal to ALL cells that preserves their capacity to respond to physiological stimuli.
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Figure 6. Assembled BM endothelium promotes leukemia cell directional motion. Leukemia cells were added to Matrigel containing assembled tube-like endothelial structures promoted by ALL plasma (A) or, as control, to Matrigel containing ALL plasma (B). Cell motion was tracked using time-lapse video microscopy. Snapshots in (A) document the leukemia cell movement towards endothelium. The movies of the experiments depicted in (A) and (B) are included as Supplemental Data. Leukemia cells were plated on preassembled BM endothelium in Matrigel (C; for scanning electron microscopy) or a BM endothelial cell layer (D; for transmission electron microscopy). Images in (C) show ALL cells adhering to multicellular, branching endothelial structures, with cell polarization and pseudopodia (image on the right shows magnification of the indicated area). Images in (D) show the contact interface between BM endothelial cells (EC) and leukemia cells (LC), indicating the existence of junctional complexes.
Human BM endothelium promotes leukemia cell survival through modulation of bcl-2 To examine the molecular mechanisms involved in the BM-EC–mediated leukemia cell survival, we evaluated the expression in ALL cells of the anti-apoptotic molecules bcl-2 and bcl-xL, which have been implicated in the regulation of leukemia cell viability. Coculture with BM-EC promoted the maintenance of high levels of both bcl-2 and bcl-xL in ALL cells, supporting their potential involvement in the BM-EC–promoted ALL survival (Fig. 7C; 4 B-ALL patients). In contrast, ALL cells cultured in control conditions showed downregulation of bcl-2 and bcl-xL (Fig. 7C). To confirm that BM-EC–induced ALL survival was mediated through an anti-apoptotic signal, experiments were performed using anti-bcl-2–specific antisense oligonucleotides, which have been shown to effectively and specifically abrogate bcl-2 expression [24,33]. As shown in Figure 7D, bcl-2 anti-sense oligonucleotides significantly inhibited the BM-EC–promoted leukemia cell survival, in contrast to control missense oligonucleotides (p ! 0.001). These results demonstrate that the viability effect
promoted by BM-EC involves the maintenance of antiapoptotic genes and is mediated, at least in part, through modulation of bcl-2.
Discussion The BM of most patients with ALL exhibit marked neovascularization and increased numbers of endothelial cells [1–3]. Little is known on how ALL cells induce the angiogenic phenotype observed in the BM, and whether the BM endothelial cells provide signals that are advantageous to the leukemia cells. Our data using in vivo and in vitro models provide compelling evidence that ALL cells are responsible for the development of the observed BM ‘‘angiogenic’’ microenvironment. Reciprocally, BM endothelial cells provide signals to ALL cells that promote their survival through the modulation of pro-apoptotic molecules. Taken together, these observations support the hypothesis that cross-talk between ALL cells and BM endothelium is critical to the development of ALL.
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Figure 7. BM endothelium promotes leukemia cell survival, which is abrogated by blockade of bcl-2 expression. (A): Cell viability and apoptosis were determined using the annexin V/PI assay; dead ALL cells were identified as strongly binding annexin V. Representative case from 6 different ALL patients (4 B-cell and 2 T-cell), and 5 independent experiments. (B): Responsiveness to stimulation by CD40LDIL-4 was assessed by the formation of homotypic clusters of ALL cells. ALL cells were incubated with BM endothelium for up to 5 days prior to addition of CD40L/IL-4. One representative case is shown from 3 individual experiments using cells from 5 ALL patients. (C): Expression of bcl-2 and bcl-xL was measured by flow cytometry. Black symbols indicate ALL cells cultured in BM-EC and open symbols indicate cells cultured in control conditions; solid and dotted lines indicate bcl-2 and bcl-xL respectively. Representative case from 4 B-cell ALL patients. (D): Leukemia cells were cultured with anti-sense bcl-2 oligonucleotides or control missense oligonucleotides prior to their addition to BM endothelial cells. Cell viability and apoptosis were determined using the annexin V/PI assay. The effects of anti-sense bcl2 oligonucleotides (black bars; coculture with BM-EC) and control media (gray bars; no BM-EC) were calculated relative to the maximal survival effect of BM-EC in the presence of missense oligonucleotides (white bars; normalized to 100% survival). n.d., not determined.
Human ALL cells transplanted into NOD/SCID mice promote a significant expansion of the murine BM endothelium with neovascularization that recapitulates the ALL microenvironment. These studies demonstrate that leukemia cells can, either directly or indirectly, stimulate BM endothelial cells. Since the BM contains multipotent stem cells and endothelial cell precursors [39–41], it is likely that leukemia cell transformation and expansion create local conditions leading to the recruitment of these BM precursors. Interestingly, myeloma and myeloid leukemia cells, developing in the BM of immunodeficient mice, also induced increased neovascularization [42–45], suggesting that the ability to stimulate BM endothelial cells is a common trait of malignant cells of different lineages and distinct genetic programs. In support of the biologic relevance of these observations to human ALL is the demonstration that comparable BM phenotype has been observed in patients with other hematological malignancies [2,6,46–49]. This is further reinforced by the recent observation that patients with breast cancer BM micrometastases have increased microvessel density [50]. Moreover, it has been recently reported that endothelial cells purified from the BM of
myeloma patients exhibit an activated, ‘‘angiogenic’’ phenotype [51]. To define the mechanisms by which ALL cells stimulate BM endothelium, we elected to use plasma isolated from the BM of leukemia patients since it more accurately represents the complexity of the soluble microenvironment of the leukemic BM, in contrast to the use of single recombinant pro-angiogenic factors. The observation that leukemia BM plasma is angiogenic and stimulates BM endothelial cells to proliferate, migrate, and assemble into capillarylike structures suggests that ALL cells secrete one or more soluble factors that induce the observed neovascularization. In support of the concept that multiple cytokines are responsible for this phenotype is our preliminary evidence that neutralization of individual cytokines (bFGF, VEGF, aFGF/FGF-1, TGF-a) failed to abrogate or significantly inhibit the effects of the leukemic BM plasma (Veiga JP and Cardoso AA; unpublished observations). Although others have not formally examined the angiogenic properties of BM soluble microenvironment in other hematological malignancies, studies have shown that conditioned media from malignant leukocytes possess pro-angiogenic activity
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in the CAM assay or have the ability to stimulate vascular endothelial cells in vitro [9,46,52]. The observation that bFGF is increased in the urine and peripheral blood of the majority of ALL patients suggests that bFGF is a key player in the leukemia-promoted stimulation of BM endothelium. We observed that bFGF is elevated in the BM plasma of ALL patients, at a frequency concordant with that reported in the blood and urine of these patients [2,12,13]. Whether bFGF is sufficient to promote the angiogenic phenotype observed in ALL remains to be determined. Relevant to this issue is the discrepancy between the frequency of ALL patients with BM neovascularization and the comparatively lower occurrence of patients who have elevated levels of bFGF and/or VEGF. Other studies have shown that serum VEGF, in contrast to other hematological cancers [2], is elevated in only a small fraction of ALL patients [13]. Interestingly, it has been reported that ALL patients with increased levels of VEGF at diagnosis have a poor prognosis[13] and that patients with higher numbers of VEGF transcripts have lower relapse-free intervals and overall survival [53]. The observation that only a fraction of ALL patients have elevated VEGF does not exclude, however, a role for VEGF in leukemia-associated neovascularization. It is possible that VEGF, produced by leukemia cells and the BM endothelium (Veiga and Cardoso; unpublished observations), is mostly bound to the extracellular matrix [54] and may act as a juxtacrine factor for the BM-EC. A second possibility is that although the levels of VEGF produced in ALL are lower than observed in other BM malignancies, they may be sufficient to stimulate the endothelium in the leukemic BM. Our findings also indicate that other factors may be involved in the leukemia-promoted stimulation of BM endothelium, and that a more comprehensive analysis of pro-angiogenic mediators and putative endogenous inhibitors in the ALL microenvironment is necessary. Likely candidates include CXCL8/IL-8 and CTGF/CCN2, which seem to be produced by ALL cells or the leukemia microenvironment [14–16]. The fact that BM endothelial cells, which are markedly increased in the leukemic BM, provide survival signals to leukemic cells supports the concept that cell nonautonomous signals play an important role in leukemia development. Many studies have shown that bcl-2 exerts an important role in lymphoid leukemogenesis through its cell-autonomous cooperation with oncogenic events [55– 57]. Here, we show that the leukemia microenvironment also modulates bcl-2 expression, thus promoting leukemia cell survival. Similarly, we and others have previously shown that IL-7, which is produced by BM endothelium and therefore is present in the leukemic microenvironment, mediates survival of leukemia cells through bcl-2 [23,24,58]. Additional evidence for the role of the microenvironment in leukemia cell survival is derived from the observation that BM-derived stroma can rescue ALL cells
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from apoptosis and protect them from the cytotoxic effects of conventional drugs [17,57,59,60]. Our in vitro observation that ALL cells move towards and interact with assembled BM endothelium in Matrigel provides additional support for the importance of leukemia-endothelium cross-talk. Although it remains to be formally demonstrated that BM endothelium plays an essential role in ALL pathogenesis and maintenance, studies support the biological relevance of tumor-promoted neovascularization in BM malignancies. Studies in animal models of myeloid leukemia showed that specific inhibition of the tumor-promoted angiogenesis resulted in significant suppression of tumor growth [43,61]. Since the therapeutic agents employed (neutralization of leptin-R; endostatin; PI-88) did not target the tumor cells, these studies formally demonstrated that abrogation of the endothelial component of the disease affected leukemia development [43,61]. These studies and our present findings suggest that targeting the BM-EC and tumor-promoted BM neovascularization will likely impact the outcome of leukemia therapy and may offer a rationale for therapeutic intervention in ALL.
Acknowledgments We thank Drs. W. Nicholas Haining, Nadia Carlesso, Ana Limo´n, and Gordon Freeman for critical review of the manuscript. We are indebted to Maria Ericsson and Rebecca Stearn for their assistance in the studies of scanning and transmission electron microscopy, and to Drs. Ravi Salgia and Patrick Ma for their help in the studies of time-lapse video microscopy. We thank Dr. Roderick Bronson for his kind assistance in interpreting the murine pathology slides. This work was supported by grants from the National Institutes of Health (P01-CA68484; to SES, LMN, AAC), the Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT-Portugal; SAU/13240; to AAC), and the National Leukemia Research Association (to AAC). JPV and LFC were supported by scholarships from Programa PRAXIS XXI, FCT-Portugal.
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Leukemia-stimulated bone marrow endothelium promotes leukemia cell survival J. Pedro Veigaa, Lara F. Costaa,b, Stephen E. Sallana, Lee M. Nadlera, and Angelo A. Cardosoa a
Departments of Medical Oncology and Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Mass., USA; b Unit of Tumor Biology, Institute of Molecular Medicine, University of Lisbon Medical School, Lisbon, Portugal (Received 5 December 2005; revised 19 January 2006; accepted 19 January 2006)
Extensive endothelial cell proliferation and marked neovascularization are the most pronounced microenvironmental changes consistently observed in the bone marrow (BM) of patients with acute lymphoblastic leukemia (ALL). It is not known whether ALL cells induce this phenotype and whether they receive critical signals from the tumor-associated BM endothelium. Here, we show that leukemia cells actively stimulate BM endothelium, promote de novo angiogenesis, and induce neovascularization in the leukemic BM. Soluble factors, present in the leukemic BM microenvironment, promote the proliferation, migration, and morphogenesis of BM endothelial cells, which are critical processes in tumor angiogenesis. We also show in vitro that leukemia cells display directional motion towards assembled BM endothelium and following adherence exhibit cell polarization, pseudopodia, and ultrastructural features that suggest the existence of leukemia-endothelium cross-talk. Finally, we show that BM endothelium promotes leukemia cell survival through a mechanism mediated through the anti-apoptotic molecule bcl-2. These studies indicate that ALL cells actively recruit BM endothelium and mediate the leukemia-associated neovascularization observed in ALL. Therefore, disruption of interactions between leukemia cells and BM endothelium may constitute a valid therapeutic strategy. Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc.
The expansion of malignant cells within the bone marrow (BM) of patients with acute lymphoblastic leukemia (ALL) is associated with the disruption of the normal marrow architecture and the development of marked neovascularization [1–3], a phenotype also observed in other malignancies evolving in the BM [2,4–10]. A consistent structural alteration of the BM microenvironment in ALL is the significant increase in the number of BM endothelial cells. A recent study showed that specialized endothelial microdomains serve as portals for the homing and maintenance of B-cell precursor (BCP) leukemia cells in the BM [11], suggesting a role for this endothelium in the formation of tumor-supportive tumor niches. It is presently unknown whether leukemia cells directly or indirectly induce the observed angiogenic phenotype and endothelial cell expansion. Most ALL patients have increased blood plasma and urine levels of basic fibroblast growth factor (bFGF; FGF-2) [1,2,12,13], suggesting a significant role for this Offprint requests to: Angelo A. Cardoso, M.D., Ph.D., Dana-Farber Cancer Institute, Room D-540B, 44 Binney Street, Boston, MA 02115; E-mail: [email protected]
pro-angiogenic cytokine in the leukemia-promoted endothelial phenotype observed. Other angiogenic factors seem to also contribute for this phenotype, as vascular endothelial factor (VEGF) levels are increased in some patients [2,13], and it has been shown that leukemia cells produce connective tissue growth factor (CTGF; CCN2) [14] and interleukin-8 (IL-8; CXCL8) [15,16]. Increasing evidence indicates that ALL cells receive signals from their microenvironment that impact on their growth and survival. Leukemia cells undergo spontaneous apoptosis ex vivo, but are responsive to stimuli from their microenvironment, as BM stroma can rescue them from apoptosis [17–19]. Similarly, IL-7, a cytokine produced by BM stromal cells and BM endothelium [20,21] that has been implicated in lymphoid tumorigenesis [22], promotes the survival and proliferation of ALL cells [23–26]. In light of the observed endothelial cell expansion in the leukemia BM, it is critical to determine how the BM endothelium contributes to the biology of ALL. In this study, we show that human leukemia cells transplanted into NOD/SCID mice develop in the animals’ BM with a morphological pattern that recapitulates the human
0301-472X/06 $–see front matter. Copyright Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 10.1016/j.exphem.2006.01.013
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disease. Moreover, plasma isolated from the BM of ALL patients induces neoangiogenesis and promotes proliferation, migration, and morphogenesis of primary BM endothelial cells. Importantly, cellular interactions between ALL cells and BM endothelium preserve the expression of anti-apoptotic proteins in leukemia cells, promoting their survival. Therefore, agents that disrupt the cross-talk between BM endothelium and leukemia cells may constitute valuable treatment strategies for ALL.
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Matrigel-plug assay C57BL/6NTac (C57BL/6) mice were purchased from Taconic Farms (Germantown, NY, USA) and maintained at the DFCI Animal Research Facility in accordance with protocols approved by the Animal Care and Use Committee. Plasmas from ALL patients (B-cell ALL) or normal donors (25% vol/vol) were mixed with Matrigel (BD Biosciences, San Jose, CA, USA) and 0.5 mL was injected into the subcutaneous space, in the abdominal paramedian region of 8- to 24-week-old C57BL/6 mice (2–3 mice/specimen) After 7 days, pellets were explanted retaining the peritoneal lining for support, photographed using a Leica MZFLIII stereomicroscope (Leica Systems, Switzerland), and processed for histology and immunohistochemistry.
Materials and methods Leukemia specimens Diagnostic BM and peripheral blood (PB) from ALL patients, or control specimens from age-matched healthy donors, were collected in heparinized tubes. Plasmas from leukemia or normal donors were separated by centrifugation, filtered (0.22 mm), and cryopreserved. After plasma collection, mononuclear cells were separated by density centrifugation and characterized by phenotypic analysis [27]. BM biopsies were obtained at diagnosis. Appropriate informed consent was obtained according to the Declaration of Helsinki, and the studies were approved by the Dana-Farber Cancer Institute’s Institutional Review Board. Bone marrow endothelial cells Bone marrow endothelial cells (BM-EC) were purified from BM aspirates of ALL patients and healthy donors, as described [28]. Briefly, BM-EC were purified using CD105 microbeads (Miltenyi Biotech, Auburn, CA, USA), and selected by culture in EGM-2 media (containing bFGF, VEGF, EGF, and IGF-1; Cambrex BioScience, Walkersville, MD, USA), which was also used for further expansion of these cells. Cultures were carried 37 C, 5% CO2. BM-EC between the second and the sixth passage were used for the experiments, and cell lineage was confirmed by phenotypic and functional analyses. These cells express CD31, CD106, Podocalyxin, Tie-2, VEGF, and FGF receptors, and incorporate acetylated LDL; they also respond to leukemic and recombinant pro-angiogenic stimuli by activating the PI3K/Akt and mTOR pathways [28]. NOD/SCID model of human ALL NOD.CB17-Prkdcscid/J (NOD/SCID) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and maintained at the DFCI Animal Resource Facility according to protocols approved by the DFCI Animal Care and Use Committee. Leukemia cells (18 B-cell, 2 T-cell ALL patients) were thawed and viable cells prepared using the Apoptotic Cell Removal Kit (Miltenyi). Five to 10 3 106 leukemia cells were injected intravenously in the tail vein of irradiated (250 cGy) NOD/SCID mice (3–5 animals/condition). Animals were monitored and sacrificed when moribund. Bone marrow, liver, and spleen samples were collected to determine engraftment levels by flow cytometry using antibodies specific for human CD45 and CD19, and mouse CD45 (BD Pharmingen, San Diego, CA, USA). Femurs, kidneys, liver, spleen, lungs, and abnormal masses were processed as described below for histology and immunohistochemistry.
Histology and immunohistochemistry Samples were fixed in 10% neutral buffered formalin (Sigma, St. Louis, MO, USA), embedded in paraffin, and 3-mm sections stained with hematoxylin/eosin (H&E). Additional sections were stained with rat anti-mouse CD31 (1:100; DB Pharmingen), rabbit anti-mouse Tie-2 (1:350; C-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or rabbit anti-human von Willebrand Factor (vWF; 1:500; Dako, Carpinteria, CA, USA) antibodies, as indicated. Antigen retrieval was performed with 0.25% trypsin (Sigma, St. Louis, MO, USA) for CD31 and vWF stainings, or citrate buffer, pH 6.0 (Biocare Medical, Walnut Creek, CA, USA) for Tie-2 staining. For CD31 staining, a rabbit anti-rat antibody was used (1:200; Dako). Slides were incubated with goat anti-rabbit horseradish peroxidase–conjugated antibody and developed with DAB chromogen (EnvisionD, Dako) per manufacturer’s instructions, and counterstained with hematoxylin. Measurement of angiogenic factors The levels of bFGF and VEGF in the BM plasma of ALL patients and normal donors were measured by competitive enzyme-linked immunosorbent assay (ELISA), using commercially available kits (Cytimune, Rockville, MD, USA), following the manufacturer’s instructions. BM-EC proliferation and migration BM-EC were plated on flat-bottom 96-well plates at 4 3 103 cells/ well, in EGM-2. After 12 hours, plates were washed with Hank’s balanced salt solution (HBSS; Sigma) and starved for 12 hours in cytokine-free EBM-2 media (Cambrex BioScience). Media were removed and BM-EC washed with HBSS. Test plasmas (patients or normal donors) were added at 25% vol/vol and incubated for 12 hours. BM-EC cultured in EBM-2 or EGM-2 were used as controls. Cells were pulsed with 0.5 mCi of [3H]-thymidine (PerkinElmer, Boston, MA, USA), incubated for an additional 24 hours, and harvested onto glass fiber filters. [3H]-thymidine incorporation was measured by liquid scintillation spectrophotometry (Betaplate; Amersham Biosciences, Piscataway, NJ, USA). Stimulation indexes (S.I.) were calculated for each individual experiment as: S.I. 5 (cpm experimental condition)/(cpm EBM-2 media). Endothelial cell migration was assessed by standard methodology [29,30] using 8-mm microporous Transwell inserts (Costar, Cambridge, MA, USA). Leukemia or normal plasmas were added to the lower compartment of cluster plates in serum-free media (AIM V; Invitrogen, Carlsbad, CA, USA). AIM V and EGM-2 media were used as controls. BM-EC (0.5–1 3 105) were placed on the Transwell inserts (upper compartment) and incubated for 6 to
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8 hours at 37 C, 5% CO2. Transwell inserts were then removed and the total number of migrated cells was quantified to include both the migrated cells that adhered to the plate (by microscopy; five random high-power fields; 3100) and those in suspension (by flow cytometry). Percentage of migrated cells was calculated by dividing by the number of input cells. BM-EC morphogenesis assay in Matrigel The assembly of endothelial cells into capillary-like or tube-like networks in Matrigel was assessed as previously described [31]. Briefly, BM or PB plasmas (25% vol/vol) were mixed with BMEC (1 3 105), plated onto 24-well plates coated with polymerized growth factor–reduced Matrigel, and incubated at 37 C, 5% CO2. Controls included plasmas from healthy donors and EGM-2. All assays were performed in duplicate or triplicate. Capillary-like structures are defined as tube-shape elongated networks of endothelial cells with multiple nodal points, and were scored at different time points using a Nikon Eclipse TE300 (Nikon Instruments, Melville, NY, USA) microscope and photographed. Kinetics of leukemia–BM-EC interactions Assembly of BM-EC into capillary-like structures in Matrigel was promoted using leukemia plasma (as indicated), adjusting the cell number and amount of Matrigel for the area of a 35-mm dish. After EC structures were formed (12–18 hours), leukemia cells were added at 4:1 ratio (ALL:BM-EC). Experiments were performed using a time-lapse video microscopy system consisting of a humidified chamber at 37 C, 5% CO2, an inverted microscope equipped with a digital camera, and image acquisition software (QED Imaging, Pittsburgh, PA, USA). Serial images were acquired and used to generate QuickTime movies (presented as supplemental data; available at http://www.research.dfci.harvard.edu/veiga). Transmission and scanning electron microscopy For transmission electron microscopy, BM-EC (3–5 3 104) were plated in sterile 7-mm circles of Aclar (Ted Pella, Redding, CA, USA) previously coated with diluted Matrigel (1:5 in EGM-2) or in a Transwell insert on top of a Matrigel layer (100 mL). After overnight incubation, leukemia cells (2 3 105) were added on top of BM-EC and incubated for 4 hours before processing. Cells were then fixed for 1 hour in FGP (1.25% formaldehyde, 2.5% glutaraldehyde, and 0.03% picric acid in 100 mM Cacodylate buffer) fixative and stained with a mixture of 1% OsO4 and 1.5% KFeCN6 for 1 hour, and with 1% uranyl acetate for 30 minutes, followed by conventional Epon embedding. Samples were then sectioned (80 nm) in a Reichert Ultracut-S microtome and analyzed in a JEOL 1200EX transmission electron microscope. For scanning electron microscopy, BM-EC (8 3 104) were plated in sterile 8- to 10-mm circles of Aclar previously coated with Matrigel (1:6 in EGM-2) or covered by a Matrigel layer (125 mL). BM-EC were allowed to form capillary-like structures for approximately 10 hours, after which leukemia cells (5 3 105) were added and incubated overnight prior to processing. Samples were fixed for 1 hour in 2.5% glutaraldehyde in 0.1 M phosphate buffer and dehydrated through graded series of ethanol followed by critical point dryer (CPD), Samdri-PVT-3B (Tousimis, Rockville, MD, USA), in a Touimis CPD machine and 5 minutes sputter coating in a Hummer V with a gold-palladium target. Samples were analyzed in a Leo 1450VP scanning electron microscope.
Leukemia cell survival BM-EC were cultured in 6-well plates until confluent layers were formed. Viable leukemia cells (4–6 3 106) were added to the BMEC layers and the coculture was carried in B-cell media [27]. As control, ALL cells were cultured in the absence of BM-EC. After 3 to 5 days, cells were harvested, stained with annexin V and propidium iodide (PI) using the Apoptosis Detection Kit (R&D Systems), and analyzed by flow cytometry using a Coulter-XL or a Cytomics FC500 cytometer (Beckman-Coulter, Fullerton, CA, USA). In some experiments, soluble CD40L plus IL-4 was added to the cocultures (day 5) to stimulate the leukemia cells, as previously described [32]. Expression of anti-apoptotic molecules ALL cells were harvested from the BM-EC–ALL cocultures (day 3–5), counted, and stained with anti-CD19-PE antibodies (BD Pharmingen). Cells were then permeabilized with Cytoperm (BD Pharmingen) and intracellular staining was performed using FITC-conjugated anti-bcl-2 mAb (Dako) or purified anti-bcl-xL mAb (Santa Cruz Biotechnology) plus FITC-conjugated goat anti-rabbit (Southern Biotechnology Associates, Birmingham, AL, USA) as secondary antibody. Irrelevant isotype-matched antibodies were used as negative controls. Samples were analyzed by flow cytometry using a Coulter-XL or a Cytomics FC500 (Beckman-Coulter). Results were expressed as the ratio of mean fluorescence intensity (MFI) of the specific antibody stain over the MFI of the negative control antibody. Blockade of bcl-2 expression bcl-2 was abrogated using a specific antisense oligonucleotide (Biomol Research, Plymouth Meeting, PA, USA), which has been shown to effectively inhibit the expression of bcl-2 [33]. Briefly, viable ALL cells were plated at 2 3 106 cells/mL in 24-well plates and bcl-2 antisense or scrambled control oligonucleotides (200 nM) were added with Lipofectin (Life Technologies, Gaithersburg, MD, USA) and incubated for 4 hours at 37 C. Cells were harvested, washed in media, and plated on BM-EC monolayers (as described above), in the presence of the same concentration of the respective oligonucleotides. Additional controls included untreated leukemia cells plated on BM-EC layers or without BM-EC. ALL cell viability and bcl-2 protein expression was assessed after 5 days of culture. Statistical analysis The statistical significance was determined using the two-tailed Student’s t-test (with the Welch correction, when appropriate), the paired Student’s t-test, or the two-tailed nonparametric Mann-Whitney test. Differences were considered as statistically significant when a # 0.05.
Results Leukemia development in the bone marrow of NOD/ SCID mice recapitulates the angiogenic phenotype observed in the human disease The marked neovascularization observed in the BM of leukemia patients suggests that it plays a significant role in this malignancy, and indicates that ALL cells stimulate BM
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endothelial cells or their precursors to form new vessels. To investigate the pro-angiogenic potential of ALL cells, viable primary leukemia cells were transplanted into NOD/ SCID mice. Leukemia cells from ALL patients were used and animals were sacrificed when moribund. Engraftment was observed in 18 out of 20 ALL patients tested, with the mice developing a full-blown leukemia (terminal status at 34 to 276 days; mean, 154 days). Leukemia cells, identified as human CD45D cells, were present in the animal’s BM, spleen, liver, and kidneys. Histological analyses of femurs and tibias showed that the BM was largely replaced with human blasts (Fig. 1A). Importantly, these BMs
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showed evidence of active angiogenesis, as demonstrated by the presence of neovessels and endothelial sprouts (Fig. 1A,C,D). This angiogenic phenotype is in striking contrast with the BM morphology of age-matched NOD/ SCID mice (Fig. 1B), or of mice injected with normal human B-cell precursors (data not shown). Immunohistochemistry (IHC) analyses using an antibody specific for murine CD31 confirmed the endothelial origin of the cells forming these ‘‘angiogenic’’ structures (Fig. 1C,D). Importantly, the neovascularization induced by leukemia cells in the animals’ BM is comparable to the phenotype observed in the BM of ALL patients, as exemplified by
Figure 1. Development of human ALL cells in the BM of NOD/SCID mice recapitulates the neovascularization phenotype observed in the human disease. H&E stainings of the BM of NOD/SCID mice comparing animals transplanted with human leukemia cells (A) to age-matched control (B). Inset in (A) represents a 4003 magnification. Immunohistochemistry staining of the BM of transplanted NOD/SCID mice, using an antibody for murine CD31 (C,D) at magnifications of 1003 and 4003 respectively; area magnified in (D) is indicated in (C). Representative BM biopsy of an ALL patient, stained with anti-vWF antibody (E,F), at magnifications of 1003 and 6003 respectively. Arrows indicate blood vessels.
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a representative biopsy (Fig. 1E,F; endothelial cells stained with an anti-vWF antibody). These findings demonstrate that the growth of human leukemia cells in the BM of NOD/SCID mice recapitulates the morphology of the human leukemic BM, namely the leukemia-associated angiogenic phenotype in the BM microenvironment. Leukemic soluble microenvironment induces neovascularization Soluble factors play a critical role in the tumor-triggered ‘‘angiogenic switch’’ that stimulates endothelial cells to form new vessels [34,35]. To evaluate the pro-angiogenic properties of the leukemic soluble microenvironment, BM plasma from ALL patients was tested in vivo using a modification of the Matrigel-Plug assay. Leukemia BM plasma promoted neoangiogenesis as evidenced by the infiltration of the implanted Matrigel by vascular structures and endothelial sprouts (Fig. 2A), in the majority of ALL cases tested (12 out of 17 patients). Equivalent results were obtained using plasma collected from the peripheral blood of ALL patients (25 out of 42 patients). In contrast, plasma from normal controls did not promote vascularization or significant cellular infiltration of the implanted Matrigel (Fig. 2B). IHC analysis using a Tie-2-specific antibody confirmed that the cells form-
ing the vessels as well as most cells infiltrating the Matrigel were of endothelial origin (Fig. 2C,D). These vascular structures were functional, as shown by the presence of erythrocytes within their lumen (Fig. 2A,B) and the detection of hemoglobin in the Matrigel-Plug (data not shown). These findings demonstrate that the development of ALL in the human BM is associated with the production of soluble factors that promote neovascularization. We next screened the BM plasma from ALL patients for the presence of VEGF and bFGF, which are critical factors in the angiogenic phenotype associated with many hematological malignancies [36,37]. Different studies have shown that most ALL patients exhibit increased levels of circulating and urinary bFGF [1,2,12,13]. Using a competitive ELISA, we observed that the BM plasma from most ALL patients (58%) contained elevated levels of bFGF compared to BM plasma from normal donors (Fig. 3A; p ! 0.05). In contrast, the mean levels of VEGF did not significantly differ between leukemic and normal BM plasma (Fig. 3B; p O 0.1). These findings show that, in most cases, the leukemic BM contained increased levels of bFGF, but not of VEGF, extending previous observations that increased levels of bFGF are present in the peripheral blood and in the urine of most ALL patients [2,13].
Figure 2. Leukemia BM plasma promotes neoangiogenesis. Matrigel pellets containing BM ALL plasma (A,C,D) or normal plasma (B) were explanted from animals at day 7, and processed for histology (H&E; A,B) and immunohistochemistry. Arrows indicate blood vessels. Inset in (A) represents a 6003 magnification of a vessel, showing in detail the presence of intraluminal erythrocytes. Staining of ALL plasma pellets using an antibody specific for Tie-2 confirmed the endothelial origin of most cells infiltrating the Matrigel and vessel formation (C, 1003 magnification; D, 4003 magnification).
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Figure 3. Leukemia BM plasma contains elevated levels of bFGF but not VEGF. Competitive enzyme immunoassays were used to measure the levels of bFGF (A) and VEGF (VEGF165; VEGF121) (B) in the BM plasma of ALL patients or normal BM donors (p ! 0.05 for bFGF; p O 0.1 for VEGF). Measurements were performed in duplicate and as described in Materials and Methods. Mean (dotted lines) 6 2 S.E. (gray areas) are shown for each test group.
Leukemic soluble microenvironment promotes the proliferation, migration, and morphogenesis of human bone marrow endothelial cells We then evaluated the effects of leukemia BM plasma on the proliferation, migration, and morphogenesis of human BM endothelial cells, which are critical processes in neovascularization. BM plasma from the majority of ALL patients tested induced proliferation of BM-EC (Fig. 4A; S.I. 5 5.1 6 1.6), which was significantly higher (p ! 0.001) than that promoted by BM plasma from normal donors (Fig. 4A; S.I. 5 1.6 6 0.2). For chemotaxis studies, BM-EC were placed in Transwell inserts and tested for their responsiveness to plasma from ALL patients or normal donors. As shown in Figure 4B, leukemia BM plasma induced significant migration of BM-EC (2.2% 6 0.7%; n 5 15), which was significantly higher (p ! 0.001) than that promoted by control plasmas (0.5% 6 0.1%; n 5 5). These observations demonstrate that the leukemic microenvironment contains soluble factors capable of stimulating the proliferation and migration of human BM endothelial cells.
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Figure 4. Leukemia BM plasma stimulates BM endothelial cell proliferation (A) and migration (B). Mean (dotted lines) 6 2 S.E. (gray areas) are shown for each test group. For proliferation assays, S.I. was calculated as described, and the basal media used was EBM-2. For migration experiments, percentage of migrating cells was calculated as described. Leukemia BM plasma was significantly more effective than normal BM plasma in promoting BM-EC proliferation (p ! 0.001) and chemotaxis (p ! 0.001).
To determine the effect of the leukemic soluble microenvironment on BM endothelial cell morphogenesis, we assessed the ability of ALL plasma to promote the differentiation of this endothelium into capillary-like structures in Matrigel. BM endothelial cells from leukemia patients or normal donors were cultured in Matrigel in the presence of either BM plasma, a combination of angiogenic cytokines, or control media. In all cases tested, leukemia plasma (n 5 18; 17 B-cell and 1 T-cell ALL) promoted the formation of capillary-like BM endothelial structures (Fig. 5A), with an efficacy comparable to that observed using a cocktail of recombinant pro-angiogenic cytokines (Fig. 5B). No structures were formed in the absence of cytokines or plasma (Fig. 5C). In 2 out of 10 cases tested, BM plasma from normal donors also induced the formation of endothelial networks in Matrigel, but these were considerably less complex than those promoted by leukemia plasma (reduced branching and fewer nodal points; data not shown).
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Figure 5. Leukemia soluble milieu stimulates BM endothelium to assemble as capillary-like structures. Experiments were performed in growth factor– reduced Matrigel, and BM-EC were rested in growth factor–free media for 12 hours prior to use. Images show assembly of BM endothelium at 12 hours in response to BM leukemia plasma (A), a cocktail of pro-angiogenic cytokines (bFGF, VEGF, EGF, and IGF-1) (B), and control media (C). At later time points (18–24 hours) structures promoted by recombinant cytokines evolve to form more complex networks.
BM endothelial networks induce directional motion of primary ALL cells To evaluate potential cross-talk between ALL cells and BM endothelium, leukemia cells were plated in Matrigel containing assembled capillary-like networks of human BM endothelium (induced by leukemia plasma, as in Fig. 5A). The movement and kinetics of leukemia cells in Matrigel were studied at sequential time points using either light microscopy or time-lapse video microscopy (TLVM). Leukemia cells actively moved through the Matrigel and, after 4 to 8 hours, most tumor cells surrounded the BM-EC structures (Fig. 6A). As shown in this figure, small, round leukemia cells trafficked and adhered to the outline of the BM endothelial structures. TLVM studies showed that the leukemia cell movement towards the BM endothelial structures is directional, suggesting the involvement of chemotaxis (see Film 1A, Supplemental Data available at http://www.research.dfci.harvard.edu/veiga). In the absence of BM endothelium, leukemia cells cultured in the presence of leukemia BM plasma did not cluster, remaining dispersed in the gel (Fig. 6B), and displayed random motion (Film 1B, Supplemental Data). Interestingly, the directional movement is not observed or is markedly reduced when leukemia cells are added to Matrigel containing organized clusters or structures of BM mesenchymal stem cells or BM stromal myofibroblasts (data not shown). Scanning electron microscopy showed that leukemia cells in direct contact with assembled BM-EC exhibited pseudopodia and were often polarized (Fig. 6C). This is in agreement with the continuous movement of leukemia cells in contact with the assembled BM endothelium observed in TLVM. Ultrastructural analysis using transmission electron microscopy showed endothelial cellular processes and abluminal differentiation with deposition of matrix elements. Importantly, it revealed the existence
of junctional complexes between the plasma membrane of the leukemia cells and the BM endothelial cells (Fig. 6D), suggesting that active cross-talk between these cells may occur. BM endothelium promotes leukemia cell survival To determine whether BM endothelial cells impact on leukemia cell survival, primary ALL cells (n 5 6) were cultured in monolayers of BM endothelial cells and assessed for viability using an annexin V/PI assay. Coculture with BM-EC significantly (p ! 0.05) reduced leukemia cell apoptosis. In contrast, leukemia cells cultured in control conditions underwent spontaneous cell death (Fig. 7A). The mean viability of leukemia cells in contact with BM endothelium was 68%, in comparison to 47% in control conditions (n 5 6 patients). This anti-apoptotic effect was observed in both B-cell and T-cell leukemia specimens. To evaluate whether the leukemia cells rescued by BM endothelium maintain their functional properties, we examined their capacity to respond to physiologic signals. We have previously shown that, in most patients, stimulation of B-cell precursor ALL by CD40L/CD154DIL-4 results in cell clustering, proliferation, and induction/ upregulation of MHC and B7 family molecules [32,38]. Leukemia cells cocultured with BM-EC responded to sCD40L/IL-4 by forming homotypic clusters of leukemia cells (Fig. 7B) and upregulating MHC class I and class II molecules as well as CD80/B7-1 and CD86/B7-2 (data not shown). In contrast, leukemia cells cultured for identical periods without BM-EC did not demonstrate either cluster formation (Fig. 7B) or upregulation of MHC or B7 family molecules (data not shown). These results show that BM endothelium provides a survival signal to ALL cells that preserves their capacity to respond to physiological stimuli.
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Figure 6. Assembled BM endothelium promotes leukemia cell directional motion. Leukemia cells were added to Matrigel containing assembled tube-like endothelial structures promoted by ALL plasma (A) or, as control, to Matrigel containing ALL plasma (B). Cell motion was tracked using time-lapse video microscopy. Snapshots in (A) document the leukemia cell movement towards endothelium. The movies of the experiments depicted in (A) and (B) are included as Supplemental Data. Leukemia cells were plated on preassembled BM endothelium in Matrigel (C; for scanning electron microscopy) or a BM endothelial cell layer (D; for transmission electron microscopy). Images in (C) show ALL cells adhering to multicellular, branching endothelial structures, with cell polarization and pseudopodia (image on the right shows magnification of the indicated area). Images in (D) show the contact interface between BM endothelial cells (EC) and leukemia cells (LC), indicating the existence of junctional complexes.
Human BM endothelium promotes leukemia cell survival through modulation of bcl-2 To examine the molecular mechanisms involved in the BM-EC–mediated leukemia cell survival, we evaluated the expression in ALL cells of the anti-apoptotic molecules bcl-2 and bcl-xL, which have been implicated in the regulation of leukemia cell viability. Coculture with BM-EC promoted the maintenance of high levels of both bcl-2 and bcl-xL in ALL cells, supporting their potential involvement in the BM-EC–promoted ALL survival (Fig. 7C; 4 B-ALL patients). In contrast, ALL cells cultured in control conditions showed downregulation of bcl-2 and bcl-xL (Fig. 7C). To confirm that BM-EC–induced ALL survival was mediated through an anti-apoptotic signal, experiments were performed using anti-bcl-2–specific antisense oligonucleotides, which have been shown to effectively and specifically abrogate bcl-2 expression [24,33]. As shown in Figure 7D, bcl-2 anti-sense oligonucleotides significantly inhibited the BM-EC–promoted leukemia cell survival, in contrast to control missense oligonucleotides (p ! 0.001). These results demonstrate that the viability effect
promoted by BM-EC involves the maintenance of antiapoptotic genes and is mediated, at least in part, through modulation of bcl-2.
Discussion The BM of most patients with ALL exhibit marked neovascularization and increased numbers of endothelial cells [1–3]. Little is known on how ALL cells induce the angiogenic phenotype observed in the BM, and whether the BM endothelial cells provide signals that are advantageous to the leukemia cells. Our data using in vivo and in vitro models provide compelling evidence that ALL cells are responsible for the development of the observed BM ‘‘angiogenic’’ microenvironment. Reciprocally, BM endothelial cells provide signals to ALL cells that promote their survival through the modulation of pro-apoptotic molecules. Taken together, these observations support the hypothesis that cross-talk between ALL cells and BM endothelium is critical to the development of ALL.
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Figure 7. BM endothelium promotes leukemia cell survival, which is abrogated by blockade of bcl-2 expression. (A): Cell viability and apoptosis were determined using the annexin V/PI assay; dead ALL cells were identified as strongly binding annexin V. Representative case from 6 different ALL patients (4 B-cell and 2 T-cell), and 5 independent experiments. (B): Responsiveness to stimulation by CD40LDIL-4 was assessed by the formation of homotypic clusters of ALL cells. ALL cells were incubated with BM endothelium for up to 5 days prior to addition of CD40L/IL-4. One representative case is shown from 3 individual experiments using cells from 5 ALL patients. (C): Expression of bcl-2 and bcl-xL was measured by flow cytometry. Black symbols indicate ALL cells cultured in BM-EC and open symbols indicate cells cultured in control conditions; solid and dotted lines indicate bcl-2 and bcl-xL respectively. Representative case from 4 B-cell ALL patients. (D): Leukemia cells were cultured with anti-sense bcl-2 oligonucleotides or control missense oligonucleotides prior to their addition to BM endothelial cells. Cell viability and apoptosis were determined using the annexin V/PI assay. The effects of anti-sense bcl2 oligonucleotides (black bars; coculture with BM-EC) and control media (gray bars; no BM-EC) were calculated relative to the maximal survival effect of BM-EC in the presence of missense oligonucleotides (white bars; normalized to 100% survival). n.d., not determined.
Human ALL cells transplanted into NOD/SCID mice promote a significant expansion of the murine BM endothelium with neovascularization that recapitulates the ALL microenvironment. These studies demonstrate that leukemia cells can, either directly or indirectly, stimulate BM endothelial cells. Since the BM contains multipotent stem cells and endothelial cell precursors [39–41], it is likely that leukemia cell transformation and expansion create local conditions leading to the recruitment of these BM precursors. Interestingly, myeloma and myeloid leukemia cells, developing in the BM of immunodeficient mice, also induced increased neovascularization [42–45], suggesting that the ability to stimulate BM endothelial cells is a common trait of malignant cells of different lineages and distinct genetic programs. In support of the biologic relevance of these observations to human ALL is the demonstration that comparable BM phenotype has been observed in patients with other hematological malignancies [2,6,46–49]. This is further reinforced by the recent observation that patients with breast cancer BM micrometastases have increased microvessel density [50]. Moreover, it has been recently reported that endothelial cells purified from the BM of
myeloma patients exhibit an activated, ‘‘angiogenic’’ phenotype [51]. To define the mechanisms by which ALL cells stimulate BM endothelium, we elected to use plasma isolated from the BM of leukemia patients since it more accurately represents the complexity of the soluble microenvironment of the leukemic BM, in contrast to the use of single recombinant pro-angiogenic factors. The observation that leukemia BM plasma is angiogenic and stimulates BM endothelial cells to proliferate, migrate, and assemble into capillarylike structures suggests that ALL cells secrete one or more soluble factors that induce the observed neovascularization. In support of the concept that multiple cytokines are responsible for this phenotype is our preliminary evidence that neutralization of individual cytokines (bFGF, VEGF, aFGF/FGF-1, TGF-a) failed to abrogate or significantly inhibit the effects of the leukemic BM plasma (Veiga JP and Cardoso AA; unpublished observations). Although others have not formally examined the angiogenic properties of BM soluble microenvironment in other hematological malignancies, studies have shown that conditioned media from malignant leukocytes possess pro-angiogenic activity
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in the CAM assay or have the ability to stimulate vascular endothelial cells in vitro [9,46,52]. The observation that bFGF is increased in the urine and peripheral blood of the majority of ALL patients suggests that bFGF is a key player in the leukemia-promoted stimulation of BM endothelium. We observed that bFGF is elevated in the BM plasma of ALL patients, at a frequency concordant with that reported in the blood and urine of these patients [2,12,13]. Whether bFGF is sufficient to promote the angiogenic phenotype observed in ALL remains to be determined. Relevant to this issue is the discrepancy between the frequency of ALL patients with BM neovascularization and the comparatively lower occurrence of patients who have elevated levels of bFGF and/or VEGF. Other studies have shown that serum VEGF, in contrast to other hematological cancers [2], is elevated in only a small fraction of ALL patients [13]. Interestingly, it has been reported that ALL patients with increased levels of VEGF at diagnosis have a poor prognosis[13] and that patients with higher numbers of VEGF transcripts have lower relapse-free intervals and overall survival [53]. The observation that only a fraction of ALL patients have elevated VEGF does not exclude, however, a role for VEGF in leukemia-associated neovascularization. It is possible that VEGF, produced by leukemia cells and the BM endothelium (Veiga and Cardoso; unpublished observations), is mostly bound to the extracellular matrix [54] and may act as a juxtacrine factor for the BM-EC. A second possibility is that although the levels of VEGF produced in ALL are lower than observed in other BM malignancies, they may be sufficient to stimulate the endothelium in the leukemic BM. Our findings also indicate that other factors may be involved in the leukemia-promoted stimulation of BM endothelium, and that a more comprehensive analysis of pro-angiogenic mediators and putative endogenous inhibitors in the ALL microenvironment is necessary. Likely candidates include CXCL8/IL-8 and CTGF/CCN2, which seem to be produced by ALL cells or the leukemia microenvironment [14–16]. The fact that BM endothelial cells, which are markedly increased in the leukemic BM, provide survival signals to leukemic cells supports the concept that cell nonautonomous signals play an important role in leukemia development. Many studies have shown that bcl-2 exerts an important role in lymphoid leukemogenesis through its cell-autonomous cooperation with oncogenic events [55– 57]. Here, we show that the leukemia microenvironment also modulates bcl-2 expression, thus promoting leukemia cell survival. Similarly, we and others have previously shown that IL-7, which is produced by BM endothelium and therefore is present in the leukemic microenvironment, mediates survival of leukemia cells through bcl-2 [23,24,58]. Additional evidence for the role of the microenvironment in leukemia cell survival is derived from the observation that BM-derived stroma can rescue ALL cells
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from apoptosis and protect them from the cytotoxic effects of conventional drugs [17,57,59,60]. Our in vitro observation that ALL cells move towards and interact with assembled BM endothelium in Matrigel provides additional support for the importance of leukemia-endothelium cross-talk. Although it remains to be formally demonstrated that BM endothelium plays an essential role in ALL pathogenesis and maintenance, studies support the biological relevance of tumor-promoted neovascularization in BM malignancies. Studies in animal models of myeloid leukemia showed that specific inhibition of the tumor-promoted angiogenesis resulted in significant suppression of tumor growth [43,61]. Since the therapeutic agents employed (neutralization of leptin-R; endostatin; PI-88) did not target the tumor cells, these studies formally demonstrated that abrogation of the endothelial component of the disease affected leukemia development [43,61]. These studies and our present findings suggest that targeting the BM-EC and tumor-promoted BM neovascularization will likely impact the outcome of leukemia therapy and may offer a rationale for therapeutic intervention in ALL.
Acknowledgments We thank Drs. W. Nicholas Haining, Nadia Carlesso, Ana Limo´n, and Gordon Freeman for critical review of the manuscript. We are indebted to Maria Ericsson and Rebecca Stearn for their assistance in the studies of scanning and transmission electron microscopy, and to Drs. Ravi Salgia and Patrick Ma for their help in the studies of time-lapse video microscopy. We thank Dr. Roderick Bronson for his kind assistance in interpreting the murine pathology slides. This work was supported by grants from the National Institutes of Health (P01-CA68484; to SES, LMN, AAC), the Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT-Portugal; SAU/13240; to AAC), and the National Leukemia Research Association (to AAC). JPV and LFC were supported by scholarships from Programa PRAXIS XXI, FCT-Portugal.
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