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Microenvironment and Immunology

Proangiogenic Factor PlGF Programs CD11b+ Myelomonocytes in Breast Cancer during Differentiation of Their Hematopoietic Progenitors

Julien Laurent, Eveline Faes-van't Hull, Cedric Touvrey, François Kuonen, Qiang Lan, Girieca Lorusso, Marie-Agnès Doucey, Laura Ciarloni, Natsuko Imaizumi, Gian Carlo Alghisi, Ernesta Fagiani, Khalil Zaman, Roger Stupp, Masabumi Shibuya, Jean-François Delaloye, Gerhard Christofori and Curzio Ruegg
DOI: 10.1158/0008-5472.CAN-10-3684 Published June 2011

Abstract

Tumor-mobilized bone marrow–derived CD11b+ myeloid cells promote tumor angiogenesis, but how and when these cells acquire proangiogenic properties is not fully elucidated. Here, we show that CD11b+ myelomonocytic cells develop proangiogenic properties during their differentiation from CD34+ hematopoietic progenitors and that placenta growth factor (PlGF) is critical in promoting this education. Cultures of human CD34+ progenitors supplemented with conditioned medium from breast cancer cell lines or PlGF, but not from nontumorigenic breast epithelial lines, generate CD11b+ cells capable of inducing endothelial cell sprouting in vitro and angiogenesis in vivo. An anti–Flt-1 mAb or soluble Flt-1 abolished the generation of proangiogenic activity during differentiation from progenitor cells. Moreover, inhibition of metalloproteinase activity, but not VEGF, during the endothelial sprouting assay blocked sprouting induced by these proangiogenic CD11b+ myelomonocytes. In a mouse model of breast cancer, circulating CD11b+ cells were proangiogenic in the sprouting assays. Silencing of PlGF in tumor cells prevented the generation of proangiogenic activity in circulating CD11b+ cells, inhibited tumor blood flow, and slowed tumor growth. Peripheral blood of breast cancer patients at diagnosis, but not of healthy individuals, contained elevated levels of PlGF and circulating proangiogenic CD11b+ myelomonocytes. Taken together, our results show that cancer cells can program proangiogenic activity in CD11b+ myelomonocytes during differentiation of their progenitor cells in a PlGF-dependent manner. These findings impact breast cancer biology, detection, and treatment. Cancer Res; 71(11); 3781–91. ©2011 AACR.

Introduction

The formation of new blood vessels within the tumor microenvironment, a process known as tumor angiogenesis, promotes tumor growth and metastatic spreading (1, 2). Although newly formed tumor vessels originate mostly by sprouting angiogenesis from locally preexisting vessels, increasing evidence indicates that tumor-mobilized bone marrow–derived (BMD) cells significantly contribute to tumor angiogenesis. Two mechanisms have been reported. On the one side, tumors mobilize endothelial progenitors capable of differentiating into mature endothelial cells and incorporating into the nascent tumor endothelium (3). On the other side, tumors mobilize myeloid cells, which accumulate in the tumor microenvironment and promote tumor angiogenesis through paracrine mechanisms (4, 5). A number of elegant studies combining cell surface phenotyping, in vivo cell tracing, and functional experiments identified multiple angiogenesis-promoting CD11b+ BMD cell populations in mice and human (6–11). Little is known, however, on the mechanisms by which tumor-mobilized CD11b+ myeloid cells are programmed to acquire proangiogenic properties. One proposed mechanism is that programming is induced by the tumor on recruitment to the tumor microenvironment (4). Alternatively, programming may occur early on during myeloid cell differentiation from hematopoietic progenitors. In this work, we have addressed this second hypothesis through experimental studies in vitro and in vivo and correlative analyses in cancer patients.

Materials and Methods

Proteins and chemicals

Human recombinant placenta growth factor (PlGF) was kindly provided by Prof. Kurt Ballmer, Paul Scherrer Institute, Villigen, Switzerland. Human VEGFA, VEGFB, interleukin (IL) 8, and IL1β and mouse FGF (fibroblast growth factor) were purchased from PeproTech EC Ltd. Human stem cell factor (SCF), Flt-3, IL6, and TPO (thrombopoietin) were purchased from R&D Systems. 4′,6-Diamidino-2-phenylindole, dihydrochloride (DAPI) was purchased from LuBioscience GmbH. Ficoll Histopaque Plus 1.077 were from Sigma-Aldrich Chemistry.

Antibodies

Anti-human antibodies.

Fluorescein isothiocyanate (FITC)-conjugated anti-CD11b, anti-CD14, anti-Ki67; phycoerythrin (PE)-conjugated anti-CD14, PerCP-conjugated anti-CD34; allophycocyanin (APC)-conjugated anti-CD11b; and Pacific Blue–conjugated anti-CD45 were purchased from BD Pharmingen (Becton Dickinson). PE-conjugated anti-Flt-1, APC-conjugated anti–Flt-1, and IgG1 isotype control were purchased from R&D Systems. The function-blocking anti-human Flt-1 antibody KM1732 was described before (12).

Anti-mouse antibodies.

FITC-conjugated anti-CD11b and APC-conjugated anti-CD45 were purchased from BD Pharmingen (Becton Dickinson). Unconjugated anti-F4/80 and anti-CD31, PerCP-conjugated anti-F4/80, and PerCP-conjugated rat anti-IgG2a isotype control were purchased from Biolegend.

Cell lines and primary cells

The human breast cancer cell line MDA-MB-231 and human nontransformed breast epithelium-derived cell line MCF10A were purchased from American Type Culture Collection (HTB-26D and CRL-10317) and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (P/S; Invitrogen). The primary human mammary epithelial cell (HMEC) line, isolated from normal human breast tissue obtained from reduction mammoplasties and the tumorigenic Wnt-1–transformed HMEC line (Wnt-1-HMEC) were provided by Dr. C. Brisken, ISREC-EPFL (13). Cell-conditioned medium (CCM; 2–4 days old) was collected from cell lines at near-confluence conditions, filtered at 0.2 μm, and stored at −20°C. The mouse breast cancer cell line 4T1 was provided in 2008 by Dr. Fred R. Miller (Michigan Cancer Foundation, Detroit, MI), who originally generated them (14). We have not tested/authenticated these cell lines after reception in our laboratory. 4T1 cells were cultured in DMEM (Dulbecco's modified Eagle' medium) high glucose supplemented with 10% FCS and 1% P/S. Human umbilical vein endothelial cells (HUVEC) were prepared and cultured as described previously (15) and used between passage 3 and 5. BALB/c murine lung endothelial cells (MLEC) were generously provided by Dr. K. Hodivala Dilke. CD34+ progenitor cells were purified from cord blood using anti-CD34 magnetic beads separation kit (StemCell Technologies Inc.) and cultured in RPMI 1640 medium supplemented with 8% human AB plasma, SCF, Flt-3, IL6, and TPO (basal hematopoietic medium, BHM; ref. 16).

Umbilical cord blood

CD34+ progenitors cells were isolated using EasySep selection kit (StemCell Technologies Inc.) from umbilical cord blood obtained from newborn babies of healthy volunteer pregnant women for whom a cesarotomy was preprogrammed, on approval by the ethic committee of the University Hospital of Lausanne (protocol 236/07) and according to the declaration of Helsinki.

In vitro CD34+ progenitor differentiation assay

Purified CD34+ cord blood hematopoietic progenitor cells were cultured in a 24-well tissue plate in BHM ± 10% filtered CCM from MDA-MB-231, Wnt-1-HMEC, MCF10A, and HMEC. Cell cultures were harvested at day 7. The morphology of CD11b+ cells generated in culture was determined by Wright–Giemsa staining.

Cell sorting

CD11b+ cells and/or Flt-1+CD11b+ cells from CD34+-initiated in vitro cultures or from peripheral blood mononuclear cells (PBMC) were isolated using EasySep selection kit for labeled cells (StemCell Technologies Inc.) or using FACSAria cell sorting.

In vitro angiogenesis sprouting assay

HUVEC or MLEC cells were trypsinized and cultured during 24 hours in U-bottom 96-well plates to form spheroids (17) Spheroids were collected and transferred to 1% collagen. Cells or cytokines to be tested for angiogenic sprouting activity were added on top of the gel. After 48 (HUVEC) or 24 (MLEC) hours, sprouting was scored by measuring the length of the sprouts using Axiovision Software (Zeiss). Data are presented as mean of cumulated sprout lengths from 10 spheroids ± SD.

Cytokine measurement

Measurements of human or mouse PlGF were conducted in duplicate using the Quantikine ELISA systems (R&D Systems). Results represent means of duplicate determinations ± SD.

Tumor model

4T1 cells were injected (5 × 104 cells in 50 μL PBS/20% Matrigel per injection, respectively) in the fourth right inguinal mammary gland of 4- to 6-week-old BALB/c female mice (Harlan or Charles River Laboratories). Tumor growth was measured twice a week with a caliper and tumor volume was calculated with the equation: volume = π/6 (length × width2). Single-cell suspensions from tumors were obtained as recently reported (18). Mouse experiments were approved by the cantonal veterinary service of Canton Vaud.

Tumor perfusion measurement by power Doppler

Measurements were conducted 21 days after tumor cell line injection using the VEVO 770 Micro Three-dimensional High-Frequency Doppler Ultrasound system (VisualSonics) equipped with a 30 MHz transducer. Mice were anesthetized by inhaling a mixture of isoflurane (1.5%) and oxygen (98.5%). The anesthetized animals were placed on a warmed examination table, and the tumors were covered with ultrasound gel. An ultrasound transducer mounted on a 3-dimensional (3D) motor on a rail system above the animal, moved perpendicular to the beam axis, thereby acquiring consecutive 2-dimensional images in B mode and power Doppler mode with a slice thickness/interval of 500 μm. A region of interest (ROI) was drawn within every 2D image. Two-dimensional ultrasound images were used to compile a 3D volume using VisualSonic software. Intratumoral perfusion was determined by measuring the Doppler-derived pixels over a constant surface placed over the most perfused area of each of 3 sections per tumor using ImageJ free software (http://rsbweb.nih.gov/ij/). Five to 7 tumors per each experimental condition were measured (4T1 wild type, 4T1 nonsilenced, and 4T1 sh-PlGF).

Corneal angiogenesis assay

Corneal angiogenesis assay was conducted on the basis of a modification of the method originally reported (19). CD11b+ cells purified by immunomagnetic beads (StemCell Technologies Inc.) were resuspended at 20 × 106 cells/mL in RPMI 1640 medium with 2% FCS. NOD/SCID (severe combined immunodeficient)/IL2 receptor common γ2 chain knockout mice were anesthetized with ketamine and xylazine and 5 μL of cell aliquots were injected into the corneas using a 35-gauge NanoFil injection kit (WPI). Eyes' vascularization was monitored with a stereomicroscope and mice were euthanized 20 days postinjection.

Breast cancer patients and healthy donors

Peripheral blood from healthy donors and cancer patients was obtained according to the declaration of Helsinki and on approval by the ethic committee of the University Hospital and University of Lausanne. Breast cancer patients aged from 32 to 75 years (mean: 56.7 ± 13.4) were all previously untreated and presenting at first diagnosis with ductal invasive carcinoma without evidence of distant metastasis (T1 and T2, N0 and N1, M0), estrogen receptor (ER) positive (85%–95%), and scheduled for tumor resection (10 patients). Blood was collected the day before surgery. Healthy volunteers were individuals without history of cancer, chronic disease, or medication other than hormonal contraception. PBMCs were isolated using Ficoll Histopaque Plus 1.077 (Becton Dickinson).

Human samples processing

Blood was collected in Lithium heparin anti-coagulated Vacutainer tubes (Becton Dickinson). Plasma phase was collected and centrifuged 5 minutes at 1,500 × g at room temperature. Plasma was stored at −80°C. PBMC were isolated using Ficoll Histopaque Plus 1.077 (25 minutes centrifugation at 680 × g at room temperature). The PBMC fraction was collected, washed in PBS, counted, aliquoted at 10 × 106 cells/mL, and frozen at −80°C in 90%FCS/10%DMSO (dimethyl sulfoxide) medium. Twenty-four hours later, samples are transferred into liquid nitrogen.

Statistical analysis

In all experiments of this study, values are expressed as mean ± 95% CI. Statistical analyses were conducted by paired Student's t test unless indicated otherwise. Differences were considered statistically significant at P < 0.05.

Results

Breast cancer CCM induces the generation of proangiogenic CD11b+ myelomonocytic in CD34+ hematopoietic progenitor–initiated culture

To test the effect of tumor cells on the generation of proangiogenic CD11b+ myeloid cells, we cultured human CD34+ progenitors in a BHM (16) alone or in the presence of CCM obtained from the tumorigenic metastatic breast cancer–derived cell line MDA-MB-231 or from the normal breast epithelium-derived nontumorigenic cell line MCF10A. In this model, CD34+CD45dull cells progressively disappear to give rise to CD34−CD45dull and CD34−CD45high populations. CD11b+ cells are present within CD34−CD45high populations (>70% CD11b+) and virtually absent from CD34+CD45dull and CD34−CD45dull populations (Supplementary Fig. S1A). The CD34−CD45highCD11b+ subpopulation is not cycling (Ki67neg) compared with CD11b− populations and has myelomonocytic (nonmacrophage) morphology (Supplementary Fig. S1B and C). CD11b+ cells isolated from 7-day-old cultures expanded in the presence of MDA-MB-231–derived CCM-induced robust sprouting in an in vitro endothelial cell–sprouting assay (17), whereas CD11b+ cells isolated from cultures expanded in BHM alone or BHM supplemented with MCF10A-CCM did not (Fig. 1A). In a second experiment, CD11b+ generated from CD34+-initiated cultures exposed to CCM derived from normal primary HMECs did not induce sprouting, whereas those exposed to CCM derived from the tumorigenic Wnt-1–transformed HMEC line (Wnt-1-HMEC; ref. 13) did (Fig. 1B).

Figure 1.
Figure 1.

Conditioned medium from breast cancer cell cultures programs proangiogenic activity in CD11b+ myelomonocytic cells differentiating from CD34+ progenitors. A, endothelial sprouting activity of CD11b+ cells isolated from CD34+ hematopoietic progenitor–initiated cultures expanded in the presence of BHM or BHM supplemented with cell culture medium (CCM) conditioned by MCF10A (normal breast epithelium derived) or MDA-MB-231 (breast cancer derived) cells. B, endothelial sprouting activity of CD11b+ cells isolated from CD34+ hematopoietic progenitor–initiated cultures expanded in the presence of BHM or BHM supplemented with CCM derived from HMEC (normal breast epithelium derived) primary cells or Wnt-1–transformed HMEC cell cultures (Wnt-1-HMEC). Only CCM derived from tumor cell lines induced angiogenic programming of CD11b+ cells. CSL, cumulative sprout length. Bar graphs represent mean values and SD (95% CI).

These results show that human breast cancer cell lines confer sprouting-promoting activity to CD11b+ myelomonocytic cells generated from CD34+ progenitor–initiated cultures.

PlGF programs proangiogenic activity in CD11b+ myelomonocytic cells derived from CD34+ progenitor–initiated cells cultures

Several tumor-derived cytokines associated with BMD cell mobilization and angiogenesis were expressed in MDA-MB-231 cells (data not shown). Among these, we tested 5 for their ability to confer proangiogenic properties to CD11b+ cells: VEGFA, VEGFB, IL8, IL1β, and PlGF. Only CD11b+ cells derived from CD34+-initiated cultures supplemented with PlGF induced endothelial sprouting (Fig. 2A). This effect was not due to a direct effect of PlGF possibly carried over to the sprouting assay, as PlGF, in contrast to VEGF, did not induce endothelial sprouting even at the highest concentration tested (100 ng/mL; Supplementary Fig. S2A). PlGF levels were significantly elevated in CCM of Wnt-1-HMEC and MDA-MB-231 cell lines compared with MCF10A and HMEC CCM (Supplementary Fig S2B). Addition of the function-blocking anti–Flt-1 mAb KM1732 (20) and an Flt-1 trap (sFlt-1-Fc) to CD34+ cell–initiated cultures exposed to MDA-MB-231–derived CCM or PlGF prevented the generation of CD11b+ cells with sprouting-inducing capacity (Fig. 2B and Supplementary Fig. S2C). CD11b+ cells derived from PlGF and MDA-MB-231 CCM–stimulated cultures induced a robust angiogenic response in vivo in the mouse corneal angiogenesis assay, whereas CD11b+ cells derived form BHM cultures did not (Fig. 2C). Addition of PlGF a later time points in CD34+ cell–initiated cultures resulted in a progressive decline in the endothelial sprouting activity of generated CD11b+ cells (Supplementary Fig. S3). In CD34+ cell–initiated cultures, Flt-1 was expressed on a minority of CD34−CD45dullCD11b− cells (≈15%–20%) on CD34−CD45highCD11b+ cells (≈10%), whereas the majority of CD11b+ cells were negative (Supplementary Fig. S4). Differentiated Flt-1+/CD11b+ cells issued from CD34+ initiated BHM culture and further cultured in the presence of PlGF did not acquire proangiogenic activity (Fig. 2D).

Figure 2.
Figure 2.

PlGF programs angiogenic activity in CD11b+ myelomonocytic cells differentiating from CD34+ progenitors. A, endothelial sprouting activity of CD11b+ cells isolated from CD34+ hematopoietic progenitor–initiated cultures in the presence of BHM alone or BHM supplemented with recombinant VEGFA, VEGFB, IL1β, IL8, or PlGF. Only PlGF induced proangiogenic programming. B, Flt-1 function-blocking mAb KM1732 and Flt-1 trap (sFlt-1-Fc) inhibited angiogenic programming of CD11b+ cells derived from CD34+ hematopoietic progenitor–initiated cultures stimulated with PlGF or MDA-MB-231 CCM (CCM-MDA). C, in vivo corneal angiogenesis assay. CD11b+ cells generated in vitro from CD34+ hematopoietic progenitors in the presence of BHM, MDA-MB-231 CCM, or recombinant PlGF were injected into the mouse cornea. Angiogenic response was evaluated 20 days later. Cells exposed to MDA-MB-231 CCM or PlGF, but not BHM, induced cornea angiogenesis. D, angiogenic programming of mature CD11b+ cells. CD11b+ cells generated from BHM-CD34+ cultures were further cultivated for 4 days in the absence or presence of PlGF and their proangiogenic activity was determined in comparison to CD11b+ cells generated from CD34+ initiated culture in the continuous presence of PlGF. PlGF did not program differentiated (diff.) CD11b+ cells. Bar graphs represent mean values and SD (95% CI).

From these results, we conclude that PlGF programs proangiogenic activity in CD11b+ myeloid cells during the early stages of differentiation from CD34+ hematopoietic progenitors in vitro.

Presence of proangiogenic CD11b+ myeloid cells and elevated levels of PlGF in the circulation of breast cancer patients

These results suggest the possibility that in cancer patients PlGF may program CD11b+ myeloid cells to acquire proangiogenic properties while they are generated during hematopoiesis. To test whether circulating CD11b+ are proangiogenic, CD11b+ myeloid cells were isolated from PBMC collected from the peripheral blood of first diagnosed ER+ breast cancer patients (T1 and T2, N0 and N1, M0) and healthy donors and tested in the endothelial sprouting assay. Indeed, CD11b+ cells isolated from the blood of breast cancer patients, but not CD11b+ cells isolated from healthy individuals, induced endothelial sprouting in vitro (Fig. 3A). In the same cohort of patients, we observed significantly increased levels of PlGF compared with healthy individuals (Fig. 3B). Exposure of mature CD11b+ myeloid cells isolated from healthy donors to PlGF, however, did not result in proangiogenic programming, whereas they did after 1-week culture in presence of MDA-MB-231 CCM (Supplementary Fig. S5A). Sprouting activity was observed in whole CD11b+ population generated from CD34 progenitors, but not in Flt-1+/CD11b+ cells (Supplementary Fig. S5B), and differentiated CD11b+ cells were insensitive to PlGF-induced proangiogenic programming (Fig. 3C).

Figure 3.
Figure 3.

Peripheral blood CD11b+ myeloid cells in breast cancer patients are proangiogenic. A, micrographs and quantification of angiogenic sprouting activity of CD11b+ cells isolated from the peripheral blood of healthy donor (HD; n = 10) and nonmetastatic breast cancer (BC) patients (n = 10). BC-derived, but not HD-derived, CD11b+ cells promoted endothelial sprouting. B, elevated levels of plasma PlGF were observed in nonmetastatic BC patients compared with HD. C, CD11b+ cells isolated from peripheral blood of HD and cultured for 4 additional days in the presence of BHM or BHM-PlGF do not acquire proangiogenic activity (n = 3). VEGF, induction of sprouting by addition of recombinant VEGFA during the sprouting assay. Bar graphs represent mean values and SD (95% CI).

These results show that breast cancer patients have elevated levels of PlGF and proangiogenic CD11b+ cells circulating in their blood compared with healthy individuals.

Tumor-derived PlGF promotes proangiogenic programming of CD11b+ myelomonocytes and tumor growth

To test whether PlGF induced proangiogenic programming of CD11b+ myelomonocytes in vivo, we silenced PlGF expression in the murine mammary carcinoma–derived cell line 4T1 by PlGF short hairpin RNA (shRNA) expression (Supplementary Fig. S6A). As control 4T1 cells were transduced with a nonsilencing shRNA (21), which did not affect PlGF expression (Supplementary Fig. S6B). Silencing of PlGF significantly lowered plasma PlGF levels in 4T1 tumor–bearing mice (Fig. 4A) and slowed tumor growth compared with wild-type 4T1 cells or 4T1 cells transduced with the nonsilencing shRNA as measured by tumor volume (Fig. 4B) or weight (Supplementary Fig. S7). The frequency of circulating CD11b+ myelomonocytes in 4T1 tumor–bearing mice was not affected by PlGF silencing (Fig. 2C). However, circulating CD11b+ cells isolated from mice-bearing PlGF-silenced tumors did not induce sprouting in an in vitro MLEC sprouting assay, compared with CD11b+ cells isolated from control mice bearing wild-type or nonsilenced tumors (Fig. 4D). Importantly, circulating CD11b+ cells isolated from the blood of tumor-free mice did not induce endothelial sprouting (Supplementary Fig. S6C). PlGF silencing profoundly modified the morphology of the tumor vasculature. While in wild-type and nonsilenced 4T1 tumors, the tumor vessels were highly heterogeneous, ranging from larger vessels with well-formed lumen to microvessels without detectable lumen, in PlGF-silenced tumors vessels were uniformly small without detectable lumen (Fig. 5A). Power Doppler analysis revealed a significant reduction of blood perfusion in PlGF-silenced 4T1 tumors compared with wild-type or nonsilenced tumors (Fig. 5B). These results are consistent with a reduced maturation and functionality of tumor vessels in mice-bearing PlGF-silenced tumors.

Figure 4.
Figure 4.

PlGF silencing abrogates the proangiogenic properties of circulating CD11b+ myelomonocytes in 4T1 tumor–bearing mice. A, plasma levels of PlGF in BALB/c mice bearing orthotopic tumors derived from parental (4T1), nonsilenced (NS), and PlGF-silenced 4T1 cells (shPlGF). Mice bearing PlGF-silenced 4T1 tumors have significantly reduced plasma PlGF levels. B, PlGF silencing inhibits 4T1 tumor growth. The volume of tumors derived from 4T1, NS, and shPlGF was measured clinically at the indicated days. Representative volumes of 3 tumors measured by ultrasound are illustrated in a 3D reconstitution. C, frequency of CD11b+ cells measured by flow cytometry in the peripheral blood of tumor-bearing BALB/c mice at 17 days after tumor cell injection. D, tumor-derived PlGF programs proangiogenic activity in CD11b+ cells in vivo. CD11b+ cells isolated from peripheral blood of tumor-bearing BALB/c mice 17 days after tumor cell injection were tested in the sprouting assay. CD11b+ cells derived from mice bearing PlGF-silenced 4T1 tumors did not induce endothelial sprouting. 4T1, wild type.

Figure 5.
Figure 5.

PlGF silencing inhibits 4T1 tumor growth and perfusion and decreases the production of proangiogenic cytokines in the tumor microenvironment. A, CD31 and F4/80 immunohistochemistry staining of tumor biopsies from tumor-bearing BALB/c mice 21 days after tumor cell injection. PlGF-silenced tumors lack vessels with lumen. Endothelial cell (EC) surface and vessels with lumen were quantified (bottom). B, Power Doppler–based perfusion analysis of orthotopically implanted tumors in BALB/c mice. The images show an ultrasonographic tumor cross-section (black and white) overlaid with the perfusion signal (color). PlGF silencing in 4T1 tumors decreased tumor perfusion. C, frequency of F4/80+CD11b+ cells within CD45+ cells recovered from tumors and analyzed by flow cytometry 17 days after tumor injection (top). Dot plot representation of CD11b+ versus F4/80+ cells in a wild-type tumor (bottom). Infiltrating CD11b+ cells are F4/80+/−. D, PlGF silencing modulates expression of the angiogenic factors VEGFA, HBEGF, and MMP9 and proinflammatory cytokines CCL17, IL10, and TNF in whole tumors. mRNA was measured by real-time RT-PCR. Bar graphs represent mean values and SD (95% CI).

PlGF silencing did not affect the fraction of F4/80+CD11b+ or F4/80−CD11b+ tumor-recruited cells (Fig. 5C and Supplementary Fig. S8; ref. 21). Real-time reverse transcriptase PCR (RT-PCR) analysis of selected transcripts encoding for proangiogenic [i.e., VEGF, HBEGF (heparin-binding epidermal growth factor), and MMP9 (matrix metalloproteinase)] and inflammation-modulatory factors (i.e., CCL17, IL10, and TNF) in whole tumor explants, revealed a downregulation of VEGF, HBVEGF, MMP9, CCL17, and IL10 and an upregulation of TNF expression in PlGF-silenced tumors, compared with control tumors, consistent with decreased angiogenic activity (Fig. 5D).

These data show that in the 4T1 tumor model, silencing of tumor-derived PlGF reduces proangiogenic activity of circulating CD11b+ myelomonocytic cells, decreases tumor growth, decreases the maturation of the tumor vasculature and perfusion. and impinges on the production of proangiogenic cytokines in the tumor microenvironment.

Metalloproteinase activity is required for sprouting induced by PlGF-programmed CD11b+ myelomonocytic cells

Next, we searched for factor(s) mediating the endothelial sprouting activity of PlGF-programmed CD11b+ cells. Endothelial sprouting was still induced when CD11b+ cells and endothelial spheroids were physically separated using a Transwell system, thus implying the involvement of soluble factors (Supplementary Fig. S9A). We analyzed the expression of selected candidate genes by real-time RT-PCR in CD34+ progenitor–initiated cultures. ITGAM mRNA, the transcript encoding for the CD11b protein, was induced in both BHM- and PlGF-supplemented cultures. MMP9 mRNA was induced 30- to 40-fold by PlGF, whereas other potentially relevant transcripts (i.e., VEGFA, TGFB-1, SEMA4D, IF44L, ANG1, or PROK2) were either unaffected or only marginally affected by PlGF (Fig. 6A). Addition of MMI270, a synthetic hydroxomate-type MMP inhibitors, previously shown to inhibit MMP9 and to suppress tumor angiogenesis (22), during the sprouting assay fully blocked endothelial sprouting induced by PlGF-programmed CD11b+ cells (Fig. 6B). Sprouting induced by VEGF was unaffected (Supplementary Fig. S9B). The anti-VEGF blocking antibody bevacizumab caused only a minor, statistically nonsignificant reduction of endothelial cell sprouting induced by PlGF-programmed CD11b+ cells (Supplementary Fig. S9C). Bevacizumab inhibited VEGF-induced endothelial sprouting, as expected (Supplementary Fig. 9C).

Figure 6.
Figure 6.

Metalloproteinase activity is required for sprouting activity in PlGF-programmed CD11b+ myelomonocytic cells. A, expression of ITGAM, TGFB-1, SEMA4D, IF44L, ANG1, MMP9, VEGFA, and PROK2 mRNA in CD34+ hematopoietic progenitor–initiated cultures was monitored by real-time RT-PCR. PlGF induced MMP9 expression compared with BHM cultures. The time course is coded by gray shading of the bars (days 2 to 7 from left to right). B, the MMP inhibitor MMI-270 inhibited endothelial cell sprouting activity induced by PlGF-programmed CD11b+ cells. C, the anti-VEGFA antibody bevacizumab (BV) added during the sprouting assay did not inhibit endothelial sprouting induced by PlGF-educated CD11b+ cells. Bar graphs represent mean values and SD (95% CI).

From these results, we conclude that PlGF induces MMP9 expression during programming of CD11b+ cells and that metalloproteinase activity is required for sprouting activity of PlGF-programmed CD11b+ cells.

Discussion

Different myeloid cell subsets with proangiogenic and tumor-promoting properties have been recently reported including VEGFR1+CD11b+ cells (8), VEGFR1+CXCR4+CD11b+ cells (6, 7), Gr1+CD11b+ cells (9), and Tie-2+CD11b+ monocytes (23). Although it is well accepted that CD11b+ cells and subsets thereof promote tumor angiogenesis once recruited at tumor sites (12), the question of whether proangiogenic education may already occur during CD11b+ myeloid cell generation from progenitors has not been formally addressed. Here, we provide experimental and clinical evidence showing that breast cancer can program CD11b+ myeloid cells to acquire proangiogenic activity while differentiating from CD34+ hematopoietic progenitors though a PlGF-dependent mechanism, and that proangiogenic CD11b+ cells circulate in the blood of tumor-bearing mice and cancer patients. Although blood-circulating TEM (Tie-2–expressing monocytes) in tumor-bearing mice were reported to stimulate tumor angiogenesis when coinjected together with tumor cells in mice (24), we show for the first time that circulating CD11b+ monocytes in tumor-bearing mice and breast cancer patients induce endothelial cell sprouting. PlGF programs only myeloid cells while they differentiate from progenitor cells. It is unable to program differentiated CD11b+ cells even if expressing Flt-1, whereas tumor cell–derived conditioned medium does. These results suggest that further angiogenic programming of CD11b+ cells might occur on recruitment to the tumor microenvironment.

Two important corollaries stem from these observations. First, endothelial cell sprouting–inducing activity of circulating CD11b+ cells might be a potential biomarker of angiogenesis (25). Second, circulating CD11b+ monocytes possessing proangiogenic capacity and homing to normal tissues (e.g., lung and bone) might promote metastatic spreading by facilitating initial angiogenesis and thereby contributing to tumor cell extravasation and formation of the premetastatic niche (26, 27). A translational study aimed at testing these hypotheses is currently in progress in our breast cancer clinic.

Cumulating evidence point to a role of PlGF in promoting human cancer progression including breast cancer. PlGF expression is increased in breast cancer tissues compared with normal breast and elevated PlGF levels are associated with elevated risk for recurrence, metastasis, and reduced survival (28, 29). Elevated levels of PlGF also correlate with progression and reduced survival in mesothelioma (30), oral (31), non–small cell lung (32), colon (33), renal (34), brain (35), hepatocellular (36), and gastric (37) cancers. Genetic ablation of PlGF or antibody-based PlGF inhibition suppressed tumor angiogenesis and tumor growth and enhanced efficacy of anti-VEGF therapy (38–40). The angiogenic activity of PlGF appears indirect and mediated by BMD myeloid cells recruited to sites of angiogenesis (39, 41). Recently, the role of PlGF in promoting tumor angiogenesis has been challenged by a report (42) in which new anti-PlGF antibodies failed to suppress tumor angiogenesis. By showing that PlGF programs proangiogenic activity in differentiating CD11b+ cells, we provide further evidence for its role in cancer (38, 41).

Furthermore, we identified MMP9 as being strongly upregulated by PlGF and show that MMP inhibition suppresses the proangiogenic activity of programmed CD11b+ cells. MMP9 is thought to promote angiogenesis through the release of matrix-bound VEGF at tumor sites (43) and by mobilizing BMD cells (44). MMP9 is also induced by primary tumors in an Flt-1–dependent manner in endothelial cells and macrophages at premetastatic sites in the lung (45). Whether MMP9 expression in the lungs was due to a PlGF-mediated angiogenic programming and recruitment of monocytes was not addressed in that study. Thus, our results provide experimental evidence for a functional link between PlGF and MMP9 expression during proangiogenic programming of CD11b+ cells and requirement of MMP activity in promoting endothelial sprouting.

Taken together, our work unravels a previously unrecognized activity of PlGF, namely the proangiogenic programming of differentiating CD11b+ myelomonocytic cells, with relevant implications to breast cancer biology, detection, and treatment and reinforces published observations on the contribution of the PlGF/Flt-1 axis in cancer progression.

Disclosure of Potential Conflicts of Interest

C. Ruegg: commercial research support, Novartis; cofunder and stock owner, Diagnoplex. The other authors disclosed no potential conflicts of interest.

Grant Support

This work was funded by the Molecular Oncology Program of the National Centre for Competence in Research (NCCR; C. Ruegg and G. Christofori), a research instrument of the Swiss National Science Foundation; the Swiss National Foundation (M.-A. Doucey; 310030-120473), Oncosuisse (M.-A. Doucey; 02069-04-2007), a Collaborative Research Grant (CCRP) from Oncosuisse (C. Ruegg and G. Christofori; 01812-12-2005); the Geber Ruef Stiftung (C. Ruegg); the MEDIC foundation (C. Ruegg); and the TUMIC program of the 7th Frame Program of the European Union (C. Ruegg and G. Christofori).

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

Acknowledgments

The authors thank C. Brisken, Fred R. Miller, K. Hodivala-Dilke, and K. Ballmer for providing cells and reagents; T. Petrova and O. Hartle for helpful discussion; S. Monnier-Benoit for assistance with real-time PCR; M. Vicari, L. Ponsonnet, and J-C. Stehle for technical assistance; and A. Sarre for Power Doppler analyses.

Footnotes

  • Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

  • Received October 9, 2010.
  • Revision received March 15, 2011.
  • Accepted April 5, 2011.

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Cancer Research: 71 (11)
June 2011
Volume 71, Issue 11

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Proangiogenic Factor PlGF Programs CD11b+ Myelomonocytes in Breast Cancer during Differentiation of Their Hematopoietic Progenitors
Julien Laurent, Eveline Faes-van't Hull, Cedric Touvrey, François Kuonen, Qiang Lan, Girieca Lorusso, Marie-Agnès Doucey, Laura Ciarloni, Natsuko Imaizumi, Gian Carlo Alghisi, Ernesta Fagiani, Khalil Zaman, Roger Stupp, Masabumi Shibuya, Jean-François Delaloye, Gerhard Christofori and Curzio Ruegg
Cancer Res June 1 2011 (71) (11) 3781-3791; DOI: 10.1158/0008-5472.CAN-10-3684
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