Monocytes/macrophages support mammary tumor invasivity by co-secreting lineage-specific EGFR ligands and a STAT3 activator
© Vlaicu et al.; licensee BioMed Central Ltd. 2013
Received: 29 December 2012
Accepted: 8 April 2013
Published: 18 April 2013
Tumor-associated macrophages (TAM) promote malignant progression, yet the repertoire of oncogenic factors secreted by TAM has not been clearly defined. We sought to analyze which EGFR- and STAT3-activating factors are secreted by monocytes/macrophages exposed to tumor cell-secreted factors.
Following exposure of primary human monocytes and macrophages to supernatants of a variety of tumor cell lines, we have analyzed transcript and secreted protein levels of EGFR family ligands and of STAT3 activators. To validate our findings, we have analyzed TAM infiltration levels, systemic and local protein levels as well as clinical data of primary breast cancer patients.
Primary human monocytes and macrophages respond to tumor cell-derived factors by secreting EGFR- and STAT3-activating ligands, thus inducing two important oncogenic pathways in carcinoma cells. Tumor cell-secreted factors trigger two stereotype secretory profiles in peripheral blood monocytes and differentiated macrophages: monocytes secrete epiregulin (EREG) and oncostatin-M (OSM), while macrophages secrete heparin-binding EGF-like growth factor (HB-EGF) and OSM. HB-EGF and OSM cooperatively induce tumor cell chemotaxis. HB-EGF and OSM are co-expressed by TAM in breast carcinoma patients, and plasma levels of both ligands correlate strongly. Elevated HB-EGF levels accompany TAM infiltration, tumor growth and dissemination in patients with invasive disease.
Our work identifies systemic markers for TAM involvement in cancer progression, with the potential to be developed into molecular targets in cancer therapy.
KeywordsTAM Macrophage Monocyte Breast Carcinoma Patient EGFR STAT3 Marker Progression
Molecular targeting of cancer cells has proven to be an extremely effective approach in tumor therapy. However, the inherent genomic instability of tumor cells promotes the establishment of therapy-refractory cancer cell clones during the course of treatment .
In addition, recent studies of the tumor microenvironment have emphasised that cancer cells effectively reprogram infiltrating immune cells, to the point where they support malignant progression . Tumor-associated macrophages (TAM) are key participants in the crosstalk between tumor and immune cells. Thus, these genomically stable cells represent promising targets for anticancer therapy. TAM density correlates with poor prognosis in a considerable number of human cancers . Their best studied contribution to malignancy is the involvement in tumor progression. In a mouse breast carcinoma model, Pollard and colleagues have shown that macrophage (MΦ) ablation inhibits metastasis . MΦ exert important functions as trophic agents by secreting a plethora of growth factors and metalloproteases [5, 6]. With TAM being the most thoroughly studied components of the innate immune system, less is known about the impact of tumor-associated monocytes on tumor progression. Yet, populations of Tie2+ infiltrating monocytes promote angiogenesis in a mouse tumor model .
We studied the influence of tumor cells on ligand transcription and secretion patterns of monocytes and differentiated macrophages. Given the pivotal roles the oncogenic STAT3 and EGFR pathways play in tumor development [8, 9], we focused primarily on the repertoire of interleukin-6-like STAT3 activators and EGFR agonists secreted by tumor-primed monocytes and MΦ. Apart its founder protein, the interleukin-6 cytokine family includes interleukin-11 (IL-11), leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1) and oncostatin-M (OSM). The group of epidermal growth factor-like ligands contains seven EGFR agonists: amphiregulin (AREG), betacellulin (BTC), epidermal growth factor (EGF), epigen (EPGN), epiregulin (EREG), heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor alpha (TGFα). Several lines of evidence point to a role of TAM-secreted EGFR ligands in malignant progression. McGee and colleagues provided clues for EGF production by TAM in breast carcinoma biopsies . TAM number and EGFR expression levels correlate in human breast tumors , and TAM induce the EGFR-dependent migration of tumor cells in a murine breast carcinoma model . However, the exact repertoire of TAM-secreted EGFR agonists is unknown, as are the related regulatory pathways.
Our work demonstrates that in response to tumor cell-derived factors, mononuclear cells in turn secrete oncogenic ligands. Subsequent to priming with tumor cell-secreted factors, human primary MΦ and peripheral blood monocytes (PBMC) both secrete OSM, but each cell type in combination with one specific EGFR agonist. These findings reveal characteristic secretory patterns of tumor-primed MΦ and PBMC in vitro, regardless of tumor etiology.
This study was approved by the responsible ethics committee of the Ludwig-Maximilians-University, Munich, project nr. 189-11.
HL-60, MDA-MB-231, T-47D, MCF7, MDA-MB-468, MDA-MB-435S, SCC-9 and MCF 10A cell lines were obtained from the American Type Culture Collection (Manassas, VA). Primary HMEC were obtained from Clonetics (Basel, Switzerland) and cultured in MEGM® medium (Lonza, Walkersville, MD). HL-60, MDA-MB-231, MCF7, MDA-MB-468 and MDA-MB-435S cells were cultured in RPMI1640 containing 10% heat-inactivated fetal bovine serum (Invitrogen, Grand Island, NY). T-47D cells were cultured in RPMI1640 containing 10% heat-inactivated fetal bovine serum and 10 μg/ml bovine insulin (Sigma, St. Louis, MO). SCC-9 cells were cultured in DMEM/Ham’s F-12 (1:1) containing 10% heat-inactivated fetal bovine serum and 0.4 μg/ml hydrocortisone (Sigma). MCF 10A cells were cultured in DMEM/Ham’s F-12 (1:1) containing 5% heat-inactivated horse serum (Invitrogen), 0.5 μg/ml hydrocortisone, 20 ng/ml EGF (PeproTech, Hamburg, Germany), 10 μg/ml bovine insulin, 0.1 μg/ml cholera toxin (Sigma). All culture media contained 100 Units/ml Penicillin and 0.1 mg/ml Streptomycin (PAA, Pasching, Austria). Cells were cultured at 37°C in humidified air under 5% CO2 and were confirmed to be devoid of mycoplasma.
Monocyte isolation and macrophage differentiation
Peripheral blood was collected from healthy human donors after obtaining informed written consent. 15 ml of EDTA-treated blood were layered over a Ficoll-Paque™ PLUS column (GE Healthcare, Piscataway, NJ) and centrifuged at 400 g for 30 minutes, at 18°C. Leukocytes were washed twice with ice-cold PBS and transferred into RPMI containing 10% heat-inactivated fetal bovine serum and 5% active human serum. Following adherence to gelatine-coated culture plates (Sigma) that had been freshly precoated with human plasma, monocytes were washed 5 times with warm washing buffer (RPMI and PBS; 1:1), then detached using 5 mM EDTA in washing buffer. Cells were collected, centrifuged at 400 g for 15 minutes at 4°C then washed twice with ice-cold washing buffer. PBMC were plated at a density of 1.0 × 106 cells/ml in 6 well culture plates (Corning, Corning, NY) in monocyte culture medium (RPMI supplemented with 5% normal human male AB serum, Sigma). After 4 days of cultivation, cells were washed 3 times with RPMI and received fresh culture medium. Between day 4 and 10, medium was changed every third day. After 7 days of culture, macrophages had completed spontaneous differentiation.
Unless otherwise stated, cells were plated into transwell migration inserts (BD Biosciences, Bedford, MA) using RPMI + 0.1% BSA. The lower compartments contained either serum-free MΦ supernatants, culture media or RPMI + 0.1% BSA with or without OSM/HB-EGF. Where indicated, a 1 hr preincubation with neutralising antibodies, DMSO or AG1478 was performed prior to cell plating. At the end of the migration period, cells were fixed and stained with 20% methanol, 0.5% crystal violet and cotton swabs were used to remove the cells from the upper side of the membrane. An Axio Observer.A1 microscope (Carl Zeiss, Jena, Germany) was used to record 5 micrographs per well. Photoshop CS4 (Adobe, San Jose, CA) and a proprietary script were used for automated quantification of migration 5 × 104 SCC-9 cells were allowed to migrate for 6 hrs (chemotaxis) or 17 hrs (intrinsic motility). 1.5 × 105 MCF 10A cells were allowed to migrate for 17 hrs. 1.5 × 105 MCF7 cells were allowed to migrate for 48 hrs.
Two-tailed Mann–Whitney U-Tests were used for qPCR and ELISA experiments. For immunoblot, motility and chemotaxis experiments we used the two-tailed Student’s T-Test. Differences were considered significant when p < 0.05. Data are presented as means ± SEM or SD.
Tumor cells and primary mononuclear cells interact via secreted factors
Tumor cell-derived factors trigger divergent patterns of transcriptional activity and ligand secretion in PBMC and MΦ
Together, these data indicate that that EREG, TGFA and OSM transcription is induced in PBMC upon priming with MDA-MB-231 supernatants. However, PBMC only secrete functionally active EREG and OSM proteins.
Priming of differentiated macrophages significantly increased HBEGF, AREG and OSM transcript levels (Figure 2D). EREG levels, though upregulated in primed MΦ, generally remain low in this cell type (Additional file 1: Figure S1C). Upon priming, MΦ supernatants significantly upregulated pEGFR levels in the reporter cell line, an effect which was completely blocked by an inhibitory antibody against HB-EGF. Though AREG transcription was highly upregulated, a neutralising antibody directed against AREG had no effect (Figure 2E). Supernatants of primed MΦ significantly elevated pSTAT3 levels in the reporter cell line, and an antibody blocking OSM function neutralised this effect (Figure 2E). Priming of differentiated MΦ for seven days provoked secretion of EGF family ligands and OSM to a similar extent (data not shown). Quantitation of secreted HB-EGF and OSM levels confirmed that secretion is significantly increased by priming (Figure 2F). In contrast, AREG was not secreted by primed MΦ (data not shown).
Taken together, these data indicate that, even though various EGFR ligands are transcriptionally induced, primed macrophages only secrete functionally active HB-EGF and OSM.
Tumor-primed PBMC and MΦ display specific secretory patterns
Tumor-primed PBMC and MΦ secrete one specific EGFR agonist, respectively, in combination with OSM, the relevant STAT3 activator. We next asked whether priming with an expanded panel of tumor cell supernatants provokes similar response patterns in PBMC and MΦ. The panel contained MDA-MB-231 (231), T-47D (T47), MCF7 (7), MDA-MB-468 (468) invasive mammary and SCC-9 (SCC) squamous carcinoma cell lines. MCF 10A (10A) are derived from a benign breast lesion and primary HMEC (HM) represent healthy breast epithelium.
Tumor-secreted factors induce specific secretory patterns in PBMC and MΦ
MΦ primed by 231, T47, 7 and SCC co-secreted considerable amounts of HB-EGF and OSM (Table 1, Additional file 1: Figure S6B), which were shown to be the only EGFR and STAT3 activators released by primed MΦ (Additional file 1: Figure S6C/D).
We observed clearly distinct priming capacities of tumor cell lines on primary mononuclear cells: primary breast epithelial cells (HM) derived from healthy donors neither convey a secretory response in PBMC nor in MΦ (Table 1, Additional file 1: Figure S5/S6). Selective PBMC priming was achieved by 10A and 468 cells, while 7 and SCC cells exclusively primed MΦ. 231 and T47 breast carcinoma cells primed PBMC as well as MΦ (Table 1). Thus, a number of tumor cell lines triggers PBMC and MΦ to co-secrete an EGFR agonist and a STAT3 activator. The secretory patterns of tumor-primed mononuclear cells are lineage-specific: PBMC co-secrete EREG and OSM, while MΦ co-secrete HB-EGF and OSM.
HB-EGF and OSM are promigratory in epithelial cells
Though originally reported as an antiproliferative factor , OSM failed to inhibit proliferation of several breast cancer cell lines (Additional file 1: Figure S7A). However, OSM induces the undirected motility of MCF7 cells . We incubated MCF7 cells for five days at increasing doses of recombinant OSM. The Boyden chamber assay confirmed a dose-dependent increase in undirected MCF7 cell motility (Figure 3B). Similarly, a 2.6-fold increase in undirected motility was observed in SCC-9 cells (Figure 3C).
10A, benign breast epithelial cells, are barely migratory under most assay conditions. OSM did not induce motility of 10A cells (data not shown). However, OSM-treated 10A cells migrated two to three times faster along an HB-EGF gradient (Figure 3D). For this cell line, HB-EGF is a potent mitogen (Additional file 1: Figure S7B).
Taken together, prolonged treatment of MCF7 and SCC cells with recombinant OSM induces undirected motility in these tumor cells. Priming of MΦ by tumor cells results in HB-EGF secretion at levels sufficient to drive cell migration, and HB-EGF is the relevant chemotactic factor for SCC cells secreted by primed MΦ. OSM and HB-EGF cooperate in promoting chemotaxis of MCF 10A epithelial cells. All three cell lines express receptors for OSM (Additional file 1: Figure S2) and EGFR (Additional file 1: Figure S6A), [22, 23].
TAM express HB-EGF/OSM in invasive breast carcinoma and HB-EGF plasma levels correlate with primary tumor growth and lymph node involvement
Given that HB-EGF levels were elevated in a larger proportion of patients’ plasma samples, we reasoned that HB-EGF plasma levels might correlate with growth and/or progression of mammary carcinomas. Mean plasma concentrations of HB-EGF reflect primary tumor size: for small tumors (pT1; 23 patients) we detected 29.6 pg/ml, medium sized tumors (pT2; 13 patients) displayed 62.1 pg/ml and large tumors (pT3; 9 patients) 131.8 pg/ml. In conclusion, HB-EGF plasma levels correlated with primary tumor size (Figure 4B).
In addition, elevated HB-EGF plasma levels also correlate with lymph node dissemination of mammary breast carcinomas (Figure 4C): healthy donors (Ctrl; n = 26) and lymph node negative invasive mammary carcinoma patients (pN0; n = 26) displayed mean HB-EGF plasma concentrations of 21.2 pg/ml and 24.6 pg/ml, respectively, whereas significantly elevated HB-EGF levels were detected in patients with disseminated tumors (97.5 pg/ml; pN > 0; n = 28).
We defined the mean plasma HB-EGF concentration of healthy donors (21.2 pg/ml; Figure 4A) as a threshold that grouped invasive breast carcinoma patients into an HB-EGF negative or HB-EGF positive cohort. In this scoring, 50% (27 of 54) of the patients were determined HB-EGF positive, and tumor cell positive lymph nodes were detected in 52% (28 of 54) of the patients. However, 70% (19 of 27) of HB-EGF positive patients were diagnosed with disseminated tumors, whereas only 33% (9 of 27) of the HB-EGF negative cohort were lymph node positive (Figure 4D). In conclusion, mammary carcinoma patients with disseminated tumors can be identified by elevated HB-EGF plasma levels.
To test a potential participation of monocyte-secreted EREG in the development of mammary tumors, we quantified EREG plasma protein levels in 59 donors with DCIS or invasive mammary carcinomas as well as in 10 healthy donors. In the control donors and in the majority of breast cancer patients, EREG plasma levels remained low (Additional file 1: Figure S9). We only detected elevated EREG levels in 5 of 59 cancer patients, rendering a significant contribution of monocyte-derived EREG to the progression of breast cancer implausible. A supplementary list containing ELISA and clinical patient data is available online (Additional file 2).
Confirming our in vitro interaction model, the presented immunohistochemical data identify TAM as sources of production of HB-EGF and OSM in primary tumors. Elevated HB-EGF and OSM levels in patients’ plasma indicate secretion of both ligands by TAM in mammary carcinomas in vivo.
Given the strong correlation between TAM density, HB-EGF plasma levels and tumor size, increased HB-EGF plasma levels identify a substantial part of node positive patients, connecting TAM-secreted HB-EGF with tumor dissemination.
Tumor HBEGFexpression is predominantly localised in the stromal compartment
To further substantiate the clinical data, we analysed tumor microarray datasets available at Oncomine™. The breast carcinoma microarray dataset by Ambs and colleagues distinguished between genes expressed by tumor cells versus stromal cells . Our analysis of this dataset revealed that both HBEGF and the monocytic marker CD64 are predominantly expressed in the stromal compartment (Additional file 1: Figure S10A), suggesting that tumor-associated mononuclear cells express this EGFR agonist.
In our experiments, priming of differentiated MΦ was not restricted to mammary carcinoma cell lines. MΦ-derived factors are also functional in other carcinomas. Analysis of the bladder carcinoma microarray dataset published by Radvanyi and colleagues  showed HBEGF to be overrepresented in infiltrating carcinoma samples (Additional file 1: Figure S10B). These data corroborate our in vitro findings that HB-EGF is involved in the progression of a variety of carcinomas.
Recent scientific advances have established that monocytes and macrophages are not only critically involved in inflammatory processes such as pathogen defence and wound healing, but also play an active role in cancer development. Their functions are modulated by tumor cells, resulting in tolerant, trophic phenotypes indispensable for tumor progression . Among the factors secreted by tumor cells the chemokine CCL2 has been identified as an important priming factor of TAM in vivo. Based on a model that tumor cells and mononuclear cells communicate via secreted factors, we have used cell supernatants to study the reciprocal influence of both cell types. Our main focus was on the paracrine release of both EGFR and STAT3 activators by monocytes/macrophages to tumor cells . The presented data indicate that the differentiation process of monocytes to macrophages is of no importance for the activation of these two signaling pathways, even though the precise nature of the secreted ligands changes with differentiation.
Constitutive activation of STAT3 is common in melanoma, breast, head and neck, lung, pancreatic and prostate cancer . Tumor-primed primary PBMC and MΦ secrete oncostatin-M, and only OSM activates STAT3 in our reporter system. Both OSM receptors are exclusively found in epithelial and carcinoma cells. This underscores the unique paracrine role of mononuclear cell-derived OSM acting on tumor cells. This is the first report that shows tumor cell-derived activities to induce OSM secretion by primary human PBMC and MΦ, supporting an interaction model defined by mutually secreted factors. All of the tested tumor cell lines effectively primed human PBMC and/or differentiated MΦ. Priming induced rather complex transcriptional induction profiles of EGFR ligands in mononuclear cells. At protein level, however, priming resulted in co-secretion of EREG and OSM by PBMC, while tumor-primed MΦ co-secreted HB-EGF and OSM in vitro. Transcript analysis alone, however, would not reliably predict the functionally relevant EGFR agonist released by tumor-primed mononuclear cells . In summary, two secretory patterns emerge in PBMC and MΦ in response to tumor cell-secreted factors: a lineage-specific EGFR ligand in combination with OSM.
Functionally, recombinant OSM and HB-EGF strongly cooperate in inducing chemotaxis of benign breast cells in vitro. OSM enhances the intrinsic motility of carcinoma cells, while macrophage-produced HB-EGF is the key chemotactic factor for squamous cell carcinoma cells in our assays. Our in vitro data suggest that TAM-derived OSM and HB-EGF influence migratory processes of cancer cells. In breast cancer patients, HB-EGF and OSM are co-expressed by TAM and plasma protein levels of both ligands correlate strongly. Elevated HB-EGF plasma protein levels are strongly associated with primary tumor growth and lymph node dissemination in invasive breast cancer patients. In fact, the majority of lymph node positive patients display elevated HB-EGF plasma levels. Increased density of infiltrating TAM in patients with elevated HB-EGF plasma levels further supports the hypothesis that TAM-secreted HB-EGF promotes breast carcinoma growth and metastasis in vivo. HBEGF transcripts primarily detected in the stroma of mammary carcinomas correlate with the myeloid marker CD64, further supporting the assumption that TAM account for the expression of HBEGF in the tumor microenvironment. Our analysis of the Stransky bladder carcinoma microarray data  corroborated these findings inasmuch as HBEGF transcript levels increase in infiltrating tumors with respect to superficially growing lesions. In bladder carcinomas, HBEGF transcript levels inversely correlate with patients’ survival times . As Massagué and co-workers have shown in a mouse xenograft model, HBEGF sustains the specific dissemination of human mammary tumor cells to the brain, whereas dissemination to the lung is accompanied by EREG expression [29, 30]. Elevated EREG plasma protein levels, however, were detected in a minor fraction of our breast cancer patient data set, suggesting that EREG is not a primary contributor to breast cancer progression mediated by monocytes in humans. Immunohistochemical studies of HB-EGF expression in breast carcinoma tumor cells have found no  or an inverse correlation  to tumor progression parameters, whereas our study indicates a positive contribution of TAM-derived HB-EGF production in the primary tumor to invasive mammary carcinoma progression. TAM expression of HB-EGF has been reported for cholangiocarcinoma metastases. Additionally, this report linked the priming of mononuclear cells to HB-EGF expression in vitro .
Inflammatory processes have been studied in wound healing, and macrophages play a key role in skin repair . In this process, only infiltrating polymorphonuclear neutrophils have yet been described to secrete OSM , and HB-EGF is detected in wound exudates .
Our work focuses on paracrine factors secreted by mononuclear cells upon priming by tumor cells. This priming process induces secretory patterns that consistently feed back to activate oncogenic EGFR- and STAT3 pathways in tumor cells. We identify HB-EGF and OSM as the key EGFR and STAT3 activators secreted by tumor-primed MΦ, and EREG and OSM as the key activating factors secreted by tumor-primed PBMC. HB-EGF and OSM profoundly influence tumor cell behaviour and cooperatively promote cell motility. Our in vitro results indicate that these proteins effectively mediate macrophage-assisted cancer progression. The identified stereotype secretion patterns are elementary to design strategies to interfere with pro-tumorigenic mononuclear functions by inhibiting HB-EGF, EREG and OSM. In particular, HB-EGF function is apparently connected to tumor growth and dissemination. But first, a clear clinical characterisation of the subset of tumors infiltrated by pro-tumorigenic mononuclear cells is essential. In particular, HB-EGF serves as potential blood-bound marker allowing the minimally invasive identification of tumors supported by TAM. Our work describes pro-tumorigenic marker proteins that identify tumor-supportive mononuclear cells and establishes a basis for therapeutic intervention aimed at neutralising these STAT3 and EGFR activators at the molecular level.
Soluble tumor-cell-derived factors trigger mononuclear cells to co-secrete lineage-specific EGFR agonists together with a common STAT3 activator. Tumor-primed MΦ release HB-EGF and OSM, while tumor-primed PBMC co-secrete EREG and OSM. TAM co-express HB-EGF and OSM in primary mammary carcinoma samples. In these patients, HB-EGF plasma protein levels strongly correlate with TAM infiltration levels, primary tumor size and lymph node dissemination, indicating that TAM-derived HB-EGF supports carcinoma progression. Thus, HB-EGF plasma protein levels are a potential marker indicating a pro-tumor reaction of TAM in mammary carcinoma.
The authors thank all blood donors at the Department of Molecular Biology, Max Planck Institute of Biochemistry, as well as Monika Sommer and Joerg Reichert for collecting the blood samples. We thank Tatjana Knyazeva for preparing cDNA samples. We thank Esther Zwick-Wallasch at U3 Pharma, Martinsried, for kindly providing an anti-HB-EGF antibody. Furthermore, we thank Angela Gunesch and Kerstin Badlehner as well as Johanna Schuster, Red Cross Women’s Hospital, Munich, for retrieval of patient data, and Sebastian Kaiser, LMU Munich, for help with statistical analysis.
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