B-Estradiol Mobilizes Bone Marrow – Derived Endothelial Progenitor Cells to Tumors

Neovascularization is critical for tumor growth and development. The cellular mediators for this process are yet to be defined. We discovered that bone marrow–derived endothelial progenitor cells (BM-EPC), having the phenotype (CD133+, CD34+, VEGFR-2+), initiate neovascularization in response to TG1-1 mammary cells implanted in the inguinal mammary gland of Tie-2 GFP transgenic mice. The fluorescence tag allowed for tracing the migration of green fluorescent protein–tagged endothelial progenitor cells to tumor tissues. We discovered that 17-B estradiol supplementation of ovariectomized mice significantly enhanced BM-EPC–induced neovascularization and secretion of angiogenic factors within the tumor microenvironment. Cell-based system analyses showed that estrogen-stimulated BM-EPCs secreted paracrine factors which enhanced TG1-1 cell proliferation and migration. Furthermore, TG1-1 cell medium supplemented with estrogeninduced BM-EPC mediated tubulogenesis, which was an experimental in vivo representation of the neovasculature. Our data provide evidence of BM-EPC mammary tumor cell interactions and identify a novel cellular mediator of tumor progression that can be exploited clinically. [Cancer Res 2008;68(15):6038–42]


Introduction
Neovascularization is a critical process needed for tissue repair (1) and is indispensable for tumor growth and metastases (2,3).This process requires assistance from bone marrow-derived endothelial progenitor cells (BM-EPC), phenotypically characterized as CD133 + , CD34 + , and VEGFR-2 + (4-6).Mobilization and homing of EPCs from the bone is influenced by molecules such as vascular endothelial growth factor and proteases such as matrix metalloproteinase-9 (7,8).Interestingly, estrogen (E 2 ) has been implicated in the proliferation and mobilization of EPCs by stimulating the expression of EPC nitric oxide synthetase (9).
Despite the evidence that breast cancers initiate as E 2 -responsive and 60% of breast cancers are E 2 -dependent, no studies have examined the possible involvement of E 2 on EPC mobilization and their contribution to neovascularization.Clinically, circulating EPCs were increased in patients with stage III and IV breast cancers, suggesting that they may correlate with disease (10).In this study, we examined the contribution of EPCs in breast cancerresponsive neovascularization using the well characterized Tie2/ green fluorescent protein (GFP) murine model, in which GFP expression is under the endothelial cell-specific receptor tyrosine kinase Tek, ( formerly Tie2) promoter.To test our hypothesis of whether E 2 mobilizes circulating EPCs that home to implanted breast carcinomas and participate in tumor neovascularization, we used ovariectomized Tg (TIE2GFP)287SATO/J female mice supplemented with E 2 .We also examined EPC and breast cancer cell interactions, as well as the effect of E 2 in both in vivo-based and in vitro-based experimental systems.
EPC mobilization experiments.Mononuclear cells, isolated by using a Histopaque-1083 (Sigma) density gradient, were stained with FITC anti-CD133 antibody (PharMingen) with isotype-matched antibodies (negative control).Immunofluorescence-labeled cells were fixed with 2% paraformaldehyde and analyzed using FACStar flow cytometer (Becton Dickinson) and Cell Quest Software counting 10,000 events per sample.
EPC characterization experiments.Mononuclear cells were cultured in EBM2 (Clonetics; ref. 8).Four days after culture on four-well glass slides coated with rat plasma vitronectin (Sigma) in 0.5% gelatin solution, EPCs were assayed by costaining with acetylated LDL-DiI (Biomedical Technologies) and FITC conjugated BS lectin-1 (Vector Laboratories), both of which are markers characteristic of endothelial lineage (8).
TG1-1 tumor cell migration and proliferation.Primary cultures of EPCs from mice were cultured with or without estradiol (10 À8 mol/L) 7 days in complete EBM2 medium.Medium from EPC cultures, with or without E 2 and ERa inhibitor (ICI 182780, 1 Amol/L) or ERh inhibitor (MJM II-17B, 1 Amol/L), was harvested and added to TG1-1 tumor cells for 48 h.TG1-1 proliferative activity was measured by tritiated thymidine ([ 3 H]TdR) incorporation for 18 h and migration of TG1-1 cells was measured using a modified Boyden chamber (11).
In vitro EPC tubulogenesis.Primary cultures of EPCs were isolated from intact mouse bone marrow and plated on growth factor-reduced Matrigel-coated (300 AL; Becton Dickinson) dishes in EBM2 medium.Cells (50,000 cells/slide) were plated on Matrigel chambers and tubulogenesis monitored 24 h after the addition or not of E 2 -supplemented TG1-1 cellconditioned medium.tumor-bearing mammary gland sections (Fig. 1A), in neovasculature (merged fields, Fig. 1A), and in TG1-1 cells (Fig. 1B) using immunofluorescence and Western blotting.TG1-1 cells being derived from HER-2/neu background also express HER-2/neu (Fig. 1C).E 2 induces the mobilization of BM-EPCs into the circulation and homing to tumor tissues.Implanting 2 Â 10 6 or 4 Â 10 6 TG1-1 cells/mouse resulted in 100% tumor incidence for all groups of mice with the histology of the tumors consistent through groups and characteristic of TG1-1-induced tumors (Supplementary Fig. S1; Supplementary Table S1).Double-positive cells for GFP (endothelial lineage) and CD133 (hematopoietic stem cell marker) were counted as a representative population of circulating EPCs and E 2 significantly increased the number of circulating EPCs in the blood, peaking at day 3 and returning to baseline by day 7 (Fig. 2A).The presence of authentic EPCs was validated by costaining with markers of endothelial lineage acetylated LDL-DiI and FITC-conjugated BS lectin-1 (Fig. 2B).Vascular structures or capillaries were detected through GFP expression of EPCs.The presence of tumors increased the number of capillaries (Fig. 2C) and E 2 -mediated effect on capillary enhancement (Fig. 2D).Quantitation of the data revealed a 2-fold enhancement suggesting that E 2 not only mediates EPC migration but also participates in capillary formation in response to implanted tumors.E 2 -induced homing of EPCs to tumor tissues is accompanied by enhanced expression of angiogenic and matrix metalloproteinase RNA transcripts.Further evidence supporting the homing of EPCs to tumor sites, thus leading to neovascularization, was confirmed by immunofluorescence staining of tumors with anti-CD133 (Fig. 3A).The number of CD133-positive cells was significantly higher in OVX + E 2 mice (156 F 2.9) compared with OVX À E 2 mice (65 F 3.1).Also, colocalization of CD133-positive endothelial cells with GFP-positive endothelial cells (red arrows) was observed, suggesting that not only do EPCs home to the tumor-bearing gland but physically associate with new blood vessels, which was considerably enhanced in response to E 2 .Consistent with previous published literature (12)(13)(14)(15)(16)(17), during cardiac injury, we observed an E 2 -induced enhanced expression of secretory angiogenic paracrine cytokines such as angiopoetin-1, angiopoetin-2, thrombospondin-1, vascular endothelial growth factor, matrix metalloproteases 2 and 9, and basic fibroblast growth factor.Expression of these cytokines indicates that E 2 participates not only in neovascularization by recruiting EPCs but also by inducing biochemical components which promote a microenvironment conducive to tumor survival and growth (Fig. 3B).

Results and Discussion
E 2 enhances TG1-1 cells and EPCs to secrete paracrine cytokines which lead to cell tubulogenesis, migration, and proliferation.We also present evidence that the mobilized EPCs participate in tubulogenesis, which is a hallmark activity of EPCs in vitro (18).EPCs cultured in vitro with TG1-1-conditioned medium supplemented with E 2 displayed the formation of wellorganized and defined tubes (Fig. 4A).Examination of the proliferative effect of secreted EPC factors on TG1-1 tumor cells indicated that EPC-conditioned medium supplemented with E 2 significantly enhanced TG1-1 proliferation as measured by [ 3 H]TdR incorporation (Fig. 4B).Furthermore, TG1-1 cell migration increased towards E 2 -conditioned EPC medium as opposed to non-E 2conditioned EPC medium (Fig. 4C).This E 2 -enhanced migration and proliferative activity was abrogated in the presence of both inhibitors of ERa (ICI) and ERh (MJM II-17B; Fig. 4B and C), showing the ability of E 2 to orchestrate a synergistic interplay In summary, our data suggest that E 2 acts as a mobilizing agent for EPCs, which is tumor-responsive.This results in enhanced circulating EPCs that home to the tumor and initiate the neovascularization of tumors as well as participate in secreting endothelial and tumor-enhancing factors.The effect of antiestrogens on the E 2 responsiveness of EPCs suggests the possible use of selective antiestrogens to target EPC mobilization, and hence, neovascularization of primary and metastatic breast cancers.Furthermore, because E 2 -mediated signaling pathways cross-talk with other receptor signal transduction pathways such as HER-2/ neu, therapies directed against HER-2/neu may also serve as a novel target (19).Analogous to the use of liposomes and exosomes as delivery vehicles of toxic compounds to tumor tissue (20), EPCs can be perceived as a ''Trojan horse'', whereby their mobilization to the tumor environment can be exploited for the specific delivery of anticancer compounds for breast cancer therapy.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

Figure 2 .
Figure2.Estrogen enhances BM-EPC mobilization and homing to tumors.Isolated mononuclear cells were used to quantitate circulating EPCs.OVX + E 2 mice (E) displayed a significant increase in circulating double-positive EPCs (GFP+ and CD133+) when compared with OVX mice (n; A).Circulating EPCs were also authenticated by costaining using acetylated LDL-DiI and FITC-conjugated BS lectin-1 and appear in yellow after the merge (B ).A qualitative increase in neovascularization by GFP+ EPCs in tumor-bearing mammary glands of OVX + E 2 mice was observed compared with Intact and OVX mice (C ).Six random tissue fields/section were counted by two blinded investigators and a significant increase in GFP+ capillaries was observed in the tumor-bearing glands (gray columns ) between OVX + E 2 and OVX (D ); *, statistically significant differences.

Figure 3 .
Figure 3. Estrogen homes BM-EPCs to tumor tissues and enhances angiogenic and matrix metalloproteinase RNA transcripts.Immunofluorescent staining of mammary gland sections show an increased presence of EPCs in OVX + E 2 tumor-bearing glands as opposed to OVX and Intact tumor-bearing glands.Red arrows, colocalization of GFP+ and CD133+ endothelial EPCs.Overall, significant and consistent increases in the angiogenic and MMP RNA transcripts assayed were observed in OVX + E 2 mice (white columns ) compared with OVX mice (gray columns ).Columns , fold expression over the levels observed in control mammary glands without tumor; *, statistically significant differences.

Figure 4 .
Figure 4.Estrogen stimulates EPCs and TG1-1 cells to secrete paracrine factors that induce tubulogenesis, migration, and proliferation.E 2 -stimulated TG1-1 cell culture medium was added to EPC cultures which were monitored for tubulogenesis.Well-organized tubules were present in EPC cultures which received E 2 -supplemented TG1-1 medium, as opposed to cultures which received non-E 2 -supplemented TG1-1 medium, or EPC cultures grown in normal medium (A).Culture medium from estrogen (10 À8 mol/L) stimulated EPCs (white columns) was observed to increase TG1-1 tumor cell proliferation, quantitated by thymidine incorporation (B) and migration (C) when compared with non-E 2 -supplemented EPC culture medium (gray columns).The stimulatory effect of E 2 on EPCs was validated through the use of an ERa inhibitor, ICI 182780 (horizontally striped columns), and the ERh inhibitor, MJM II-17B (vertically striped columns), in which TG1-1 cell proliferation and migration was significantly decreased; *, statistically significant differences.