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Published as doi: 10.1096/fj.07-8832com.
 (The FASEB Journal. 2007;21:3763-3770.)
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Interleukin-6 is a potent growth factor for ER-{alpha}-positive human breast cancer

A. Kate Sasser*, Nicholas J. Sullivan*, Adam W. Studebaker{dagger}, Lindsay F. Hendey{dagger}, Amy E. Axel{dagger} and Brett M. Hall*,{dagger}

* Integrated Biomedical Science Graduate Program, Department of Pediatrics, School of Medicine & Public Health, The Ohio State University, Columbus, Ohio, USA; and

{dagger} Center for Childhood Cancer, Children’s Research Institute, Columbus, Ohio, USA

1Correspondence: Center for Childhood Cancer, WA5015, Children’s Research Institute, 700 Children’s Dr., Columbus, OH 43205, USA. E-mail: hallb{at}ccri.net


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bone is the primary anatomical site of breast cancer metastasis, and bone metastasis is associated with increased morbidity and mortality. Mesenchymal stem cells (MSC) are a predominant fibroblast cell population within the bone marrow, and metastatic breast cancer cells that seed within bone would predictably encounter MSC or their soluble factors. Therefore, we examined the impact of primary human MSC on a panel of estrogen receptor-alpha (ER{alpha})-positive (MCF-7, T47D, BT474, and ZR-75–1) and ER{alpha}-negative (MDA-MB-231 and MDA-MB-468) human breast tumor cell lines. All ER{alpha}-positive breast tumor cell lines displayed low basal activation of signal transducer and activator of transcription 3 (STAT3) until exposed to MSC, which induced chronic phosphorylation of STAT3 on tyrosine-705. Paracrine IL-6 was found to be the principal mediator of STAT3 phosphorylation in coculture studies, and MSC induction of STAT3 phosphorylation was lost when IL-6 was depleted from MSC conditioned media or the IL-6 receptor was blocked on tumor cells. Enhanced tumor cell growth rates were observed in the ER{alpha}-positive mammary tumor cell line MCF-7 after paracrine and autocrine IL-6 exposure, where MCF-7 growth rates were enhanced by >2-fold when cocultured with MSC in vitro and even more pronounced in vivo with autocrine IL-6 production.—Sasser, A. K., Sullivan, N. J., Studebaker, A. W., Hendey, L. F., Axel, A. E., Hall, B. M. Interleukin-6 is a potent growth factor for ER-{alpha}-positive human breast cancer.


Key Words: tumor microenvironment • IL-6 • metastasis • mesenchymal stem cells • ER-alpha • STAT3 • MSC


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BONE IS THE PRIMARY SITE OF METASTASIS IN WOMEN with breast cancer (1) , and clinical studies have demonstrated that hormone-responsive (i.e., estrogen receptor-alpha (ER{alpha})-positive) tumors have a much stronger metastatic predilection for bone than their ER{alpha}-negative counterparts (2 , 3) . Although hormone-responsive breast cancer patients tend to have a more favorable clinical prognosis than hormone unresponsive patients (4) , those presenting with bone metastasis or increased serum interleukin-6 (IL-6) levels, regardless of ER status, face high mortality rates (5) . Furthermore, a single nucleotide polymorphism (SNP) located within the IL-6 promoter –174 G>C has been linked to elevated serum levels of IL-6 (6) ; it significantly reduced overall survival in women with ER{alpha}-positive breast cancer (7) . These clinical data and the observation that activated fibroblasts (8) produce elevated levels of IL-6 prompted us to evaluate a possible connection between bone marrow fibroblasts, IL-6, and more aggressive ER{alpha}-positive tumor cell behavior within the bone microenvironment.

A growing body of evidence demonstrates that stromal cell fibroblasts are influential players in tumor progression and metastasis (9) . Tumor-promoting events such as angiogenesis, epithelial-to-mesenchymal transition, and progressive genetic instability have now all been linked in part to fibroblastic stromal cells (9 10 11 12 13) . Mesenchymal stem cells (MSC), also referred to as bone marrow stromal cells, are a common fibroblastic cell population within bone that regulates hematopoiesis, has a robust ability to self-renew, can differentiate into multiple mesenchymal cell lineages, and constitutively produces IL-6 (14 , 15) . As a predominant fibroblast population within the bone marrow, MSC would predictably interact with newly seeding metastatic breast cancer cells through direct physical interactions or soluble paracrine mechanisms. In hematogenous malignancies, MSC can influence tumor cell survival through both soluble and physical interactions (16) . MSC also have the capacity to acquire phenotypic profiles similar to activated fibroblasts and can preferentially engraft within mammary tumor xenografts (17) .

The pleiotropic cytokine IL-6 has many homeostatic functions including roles in B cell development, myeloid lineage maturation, acute-phase immune responses, hepatic function, and bone absorption (18) ; in cancer, it can serve as a growth factor for several cancers including multiple myeloma (19) , prostate cancer (20) , and cholangiocarcinoma (21) . With regard to breast cancer, elevated IL-6 serum levels directly correlate with disease staging and unfavorable clinical outcomes in women with metastatic breast cancer (5) . Yet a mechanistic link between IL-6 and progressive disease in breast cancer patients remains poorly understood (5) . Signal transducer and activator of transcription 3 (STAT3) is one of the primary intracellular targets activated after exposure to IL-6 (18) , and STAT3 activation has been connected with enhanced tumor cell growth, survival, and immune evasion in breast cancer (22 23 24) . Upon ligation of IL-6 to the heterodimeric IL-6 receptor (composed of the IL-6 receptor and the ubiquitously expressed signaling subunit gp130, a 130 kDa transmembrane signaling glycoprotein), recruitment and activation of downstream signaling partners result in phosphorylation of STAT3 on tyrosine residue 705 (pTyr705). STAT3 is typically maintained in the cytoplasm as an inactive monomer; once phosphorylated, STAT3 forms homodimers, enters the nucleus, and activates multiple progrowth and prosurvival genes (18) .

We hypothesized that bone marrow fibroblasts, IL-6, and ER{alpha}-positive breast tumor cells were biologically linked in a way that would support clinical observations of increased morbidity and mortality in patients with elevated IL-6 serum levels or ER{alpha}-positive bone metastasis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mammary epithelial tumor cell lines
MDA-MB-231, MDA-MB-468, MCF-7, T47D, BT474, and ZR-75–1 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). All lines were maintained in humidified incubators at 37°C and 5% CO2. All breast cancer cell lines were cultured in RPMI 1640 media containing 5% characterized FBS (HyClone; Logan, UT, USA), 2 mM L-glutamine, 10 U/ml penicillin, and 10 µg/ml streptomycin, hereafter referred to as RPMI complete media. The MCF-7 tumor cell line that stably expresses ectopic human IL-6 (MCF-7IL-6) was a kind gift from Mercedes Rincóne at University of Vermont (25) .

Mesenchymal stem cells
Human
Primary human mesenchymal stem cells (hMSC) from five independent donors (labeled hMSC-02 through hMSC-06) were obtained from Dr. Darwin Prockop’s group at Tulane University (http://www.som.tulane.edu/gene_therapy/distribute.shtml). Primary hMSC were cultured in {alpha}-MEM media containing 10% defined FBS (HyClone), 2 mM L-glutamine, 10 U/ml penicillin, 10 µg/ml streptomycin.

Mouse
Primary murine MSC (mMSC) were isolated from two independent inbred mouse strains (i.e., C57BL/6, and FVB/N) as described (26) and maintained in RPMI 1640 complete media.

Recombinant growth factors and MSC conditioned media
Recombinant human IL-6 (rIL-6) (Peprotech, Rocky Hills, NJ, USA) was used at a concentration of 50 ng/ml unless stated otherwise. Hepatocyte growth factor (HGF) 10 ng/ml, vascular endothelial growth factor (VEGF) 100 ng/ml, and epidermal growth factor (EGF) 100 ng/ml were all purchased from R&D Systems (Minneapolis, MN, USA). Human or murine MSC conditioned media (hMSC-CM or mMSC-CM) was prepared by plating MSC onto 10 cm or 15 cm plates. Once cells became confluent, RPMI complete media was reduced to 6 ml (in a 10 cm plate) or 15 ml (15 cm plate) and conditioned for 48 h by MSC before being 0.2 µm sterile filtered. MSC-CM was used immediately or stored at –80°C for later use.

In vitro coculture studies
Stable red fluorescent MCF-7 (MCF-7RE) cells were generated using DNA plasmid pDsRed-Express-C1 (Clontech; Palo Alto, CA, USA). After G418 selection (400 µg/ml), stable red fluorescent MCF-7RE lines were cocultured with primary hMSC at 10:1 ratios for 48 h in RPMI complete media. Red fluorescent MCF-7RE cells were then harvested and sterile cell-sorted from nonfluorescent hMSC using a Becton Dickinson FACS-Vantage/DiVa flow cytometer (Becton Dickinson; Mountainview, CA, USA). Whole cell lysates of MCF-7RE cells were subsequently analyzed for pTyr705 STAT3 levels by Western blot analysis. MCF-7RE cells grown alone were also sterile cell-sorted and served as a baseline pSTAT3 control for Western blot analysis.

Western blot analysis
Breast tumor cells and hMSC were plated in 6-well plates at a concentration of 500,000 and 100,000 cells per well, respectively, and allowed to adhere overnight. Cells were incubated in hMSC-CM, rIL-6 (in RPMI complete media), or RPMI complete media for 48 h. After treatment, cells were washed once with 1 x PBS and lysed in SDS lysis buffer (62.5 mM Tris-HCl, 2% w/v SDS, 10% glycerol, 50 nM DTT, 0.01% w/v bromphenol blue). Whole cell lysates were boiled at 100°C in water for 5 min and separated on 10% glycine-based SDS-PAGE gels. Samples were transferred to PVDF, blocked in 5% milk/PBS-0.1%Tween-20 (PBS-T) for 1 h at room temperature, then incubated with primary antibody as indicated below. Primary antibodies included: antiphospho (Tyr705) STAT3 (Cell Signaling, Danvers, MA, USA) [1:1000] dilution in 5% milk/PBS-T incubated overnight at 4°C, antiactin (clone AC-15, Sigma, Saint Louis, MO, USA) 1:5,000 dilution in 5% milk/PBS-T for 1 h at room temperature, and antitotal STAT3 (Cell signaling, Danvers, MA, USA) 1:1000 dilution in 5% milk/PBS-T overnight at 4°C. Secondary reagents included goat anti-rabbit-HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and sheep anti-mouse-HRP (Santa Cruz Biotechnology), both used at a 1:1000 dilution in 5% milk/PBS-T.

Soluble IL-6 protein quantification
Cellular supernatants were harvested from human or murine cells as indicated and assayed for IL-6 protein levels using the DuoSet® Human IL-6 ELISA (R&D Systems) or the Biosource Murine IL-6 ELISA (Invitrogen, Carlsbad, CA, USA). 30,000 tumor cells or 10,000 MSC were plated onto 96-well plates and allowed to adhere overnight. The next morning, cells were thoroughly rinsed with 1 x PBS and fresh RPMI complete media was added to cells. Cell cultures were incubated for 48 h and the soluble supernatants were collected and assayed for IL-6 protein by ELISA according to the manufacturer’s instructions.

IL-6 immunoprecipitation and IL-6 receptor neutralization
To remove IL-6 from hMSC-CM, conditioned media was incubated with 1 µg/ml anti-IL-6 monoclonal antibody (Mab206; R&D Systems) for 4–6 h at 4°C with constant rotation. After incubation, 20 µl/ml protein G PLUS-agarose beads (Santa Cruz Biotechnology) was added and incubated overnight at 4°C with rotation. Conditioned media containing agarose beads was centrifuged at 350 g for 10 min, and the supernatant above the pelleted agarose beads was collected and used immediately. To verify that IL-6 had been removed from the conditioned media, an aliquot of conditioned media stripped of IL-6 via immunoprecipitation was tested for IL-6 concentration by ELISA; in all cases, IL-6 concentrations were below the limit of ELISA detection (data not shown).

Three-dimensional (3-D) tumor growth assay (TGA) to monitor MCF-7 growth rates in vitro
The TGA is a fluorescence-based 3-D in vitro assay designed to noninvasively monitor tumor cell growth for up to 10–12 days after cells are embedded in basement membrane extract (BME) at 3 mg/ml. Briefly, red fluorescent MCF-7RE tumor cells were placed in a black-walled, clear-bottom 96-well plate at 25,000 tumor cells per well (±2500 hMSC, hMSC-CM, hMSC-CM(–IL-6; i.e., hMSC-CM with IL-6 removed via immunoprecipitation) or 5 ng/ml recombinant IL-6, as indicated. Each experimental condition was set up in triplicate within a 100 µl plug of 3 mg/ml BME, then overlaid with phenol-free, serum-free media. Individual well fluorescence intensities were monitored daily for 1 wk, and relative MCF-7 growth rates were documented and graphed as relative fluorescence units. For a full description of this assay and its utility for evaluating growth rate kinetics of breast cancer cells in complex 3-D tumor-like microenvironments, see our published study (27) .

MCF-7 xenograft studies
Immunocompromised athymic nude (nu/nu) female mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) at 3–4 wk of age. MCF-7 or MCF-7IL-6 tumor cells were suspended in 100 µl of BME [6 mg/ml] (Cultrex® BME; Trevigen Inc.; Gaithersburg, MD, USA) before injection, and all mice received an orthotopic inoculum of 2 x 106 MCF-7 or 2 x 106 MCF-7IL-6 tumor cells into the mammary fat pad. Tumor volumes were calculated by length x width measurements twice a week for the times indicated (formula: lxw2x0.512) (28) . All experiments involving animals were approved by the Columbus Children’s Research Institute’s Animal Care and Usage Committee (Columbus, OH, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MSC induce STAT3 activation in breast tumor cells
Cocultures of hMSC and the red fluorescent ER{alpha}-positive breast tumor cell line MCF-7RE induced STAT3 activation in MCF-7RE tumor cells, as assessed by phosphorylation on tyrosine residue-705 (pTyr705) (Fig. 1 A). MSC-soluble factors (hMSC-CM) induced similar levels of pTyr705 STAT3, suggesting that one or more MSC-derived soluble factors were responsible for the induction of STAT3 phosphorylation (Fig. 1A ). We subsequently screened a panel of four ER{alpha}-positive breast tumor cell lines including MCF-7, BT474, T47D, and ZR-75–1, and in all four ER{alpha}-positive breast tumor cell lines we observed a robust induction of STAT3 phosphorylation after exposure to hMSC-CM (Fig. 1B ). In contrast, exposure to hMSC-CM had little effect on already elevated levels of pTyr705 STAT3 in the ER{alpha}-negative breast tumor cell lines MDA-MB-231 and MDA-MB-468 (Fig. 1C ). As MCF-7 and T47D are the two most commonly studied and cited ER{alpha}-positive human breast cancer cell lines, the remaining mechanistic studies focused on these two lines.


Figure 1
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  		Figure 1. Human MSC induce phosphorylation of STAT3 on tyrosine residue-705 (pSTAT3). A) MCF-7 cells express low levels of pSTAT3 until exposed to MSC (in coculture: MCF-7RE + hMSC; in MSC-soluble factors: hMSC-CM). Note: to best illustrate changes in phosphorylation of STAT3, total STAT3 levels are shown as internal lane loading controls when large changes in STAT3 phosphorylation were observed; otherwise actin served as our internal lane loading control. B) STAT3 phosphorylation is induced in ER{alpha}-positive tumor cell lines MCF-7, BT474, T47D, and ZR-75–1 after 48 h exposure to hMSC-soluble factors (hMSC-CM). C) hMSC-CM does not alter pTyr705 STAT3 levels in the ER{alpha}-negative tumor cell lines MDA-MB-231 and MDA-MB-468. A) *Red fluorescence MCF-7 cell line was sterile-sorted (alone or after coculture with MSC) before running Western blots. Sterile cell sorting events may have slightly elevated baseline phosphorylation of STAT3 in MCF-7RE* but fell far short of MSC induction of STAT3 phosphorylation).

MSC and soluble IL-6 induce similar patterns of STAT3 activation in breast tumor cells
Several factors known to be produced by MSC were considered as potential candidates for induction of STAT3 activation. We screened the ER{alpha}-positive T47D tumor cell line for pTyr705 STAT3 induction when exposed to several recombinant human growth factors including HGF, VEGF, EGF, and IL-6. Only IL-6 from this group was found to induce pTyr705 STAT3 (Fig. 2 A). Next, we evaluated the kinetics of pSTAT3 induction and found that both IL-6 at 10 ng/ml (Fig. 2B ) and hMSC-CM (Fig. 2C ) induced a similar biphasic pattern of STAT3 activation, with one peak of activity centered at ~30 min and a chronic induction phase established by 12–24 h. To verify that MSC from independent bone marrow donors could induce STAT3 activation, we examined five primary human MSC lines for their ability to induce pTyr705 STAT3 in ER{alpha}-positive breast tumor cell lines. MSC conditioned media (from hMSC-02 through hMSC-06) and recombinant IL-6 readily induced STAT3 phosphorylation in each of the four ER{alpha}-positive breast tumor cell lines examined (Fig. 3 ).


Figure 2
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  		Figure 2. Interleukin-6 and hMSC-CM phosphorylate STAT3 with similar kinetics. A) T47D tumor cells exposed to hMSC-02-CM, hMSC-06-CM, and IL-6 (50 ng/ml) for 48 h resulted in elevated pSTAT3 levels. In contrast, no induction of STAT3 phosphorylation was observed after exposure to HGF (10 ng/ml), VEGF (100 ng/ml), or EGF (100 ng/ml). A similar biphasic pattern of pTyr705 STAT3 induction was observed after T47D tumor cell exposure to B) recombinant IL-6 (10 ng/ml) or C) hMSC-CM.


Figure 3
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  		Figure 3. Western blot analysis demonstrated that recombinant IL-6 and hMSC-CM, isolated from 5 independent donors (hMSC-02 through hMSC-06) induced pSTAT3 in the ER{alpha}-positive cell lines MCF-7, BT474, T47D, and ZR-75–1.

Paracrine MSC-derived IL-6 induces STAT3 phosphorylation in ER{alpha}-positive breast tumor cells
MSC conditioned media (hMSC-CM) contains a complex array of soluble factors. Therefore, we assessed hMSC-CM (stripped of IL-6 protein) for its ability to induce pTyr705 STAT3 levels in ER{alpha}-positive breast tumor cells. Removal of IL-6 from hMSC-CM substantially diminished the ability of hMSC-CM to phosphorylate STAT3 in ER{alpha}-positive breast tumor cells (Fig. 4 A). Likewise, neutralizing antibodies against the 80 kDa IL-6 receptor on ER{alpha}-positive tumor cells inhibited in a concentration-dependent manner the ability of hMSC-CM to induce STAT3 phosphorylation (Fig. 4B ). Restoration of pTyr705 STAT3 levels was observed when recombinant IL-6 was added back into hMSC-CM previously stripped of IL-6 via immunoprecipitation (Fig. 4C ). Finally, we demonstrated that murine MSC (mMSC) isolated from femoral bone marrow of two independent strains of inbred mice were unable to induce phosphorylation of STAT3 in human ER{alpha}-positive breast tumor cells (Fig. 4D ). Although FVB/N and C57BL/6 mMSC secrete IL-6 protein in basal media (data not shown), it has been shown that murine IL-6 is not recognized by the human IL-6 receptor (29) .


Figure 4
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  		Figure 4. IL-6 is the primary soluble factor from hMSC that induces pSTAT3 in ER{alpha}-positive breast tumor cells. A) Immunoprecipitation of IL-6 from hMSC-CM greatly diminishes the ability of hMSC-CM to induce pSTAT3 in ER{alpha}-positive tumor cells. B) Neutralizing antibody to the IL-6 receptor inhibits hMSC-CM induction of pSTAT3 in a concentration-dependent manner. C) The addition of recombinant IL-6 into hMSC-CM previously stripped of IL-6 via immunoprecipitation restores pSTAT3 induction in ER{alpha}-positive tumor cells. D) Even though murine MSC produce IL-6 (data not shown), mMSC from two inbred strains do not induce pSTAT3 in ER{alpha}-positive tumor cell line MCF-7.

MSC and ER{alpha}-negative tumor cells express and secrete IL-6 protein
We evaluated all primary MSC and breast tumor cell lines for the production of IL-6 protein (Fig. 5 ). All of the primary human MSC lines (i.e., hMSC-02 through hMSC-06) concentrated basal media in excess of 3000 pg/ml of IL-6 protein within 48 h. However, we found a clear delineation between ER{alpha}-positive and ER{alpha}-negative human breast tumor cell lines with respect to IL-6 production (Fig. 4) . The ER{alpha}-positive cell lines MCF-7, T47D, BT474, and ZR-75–1 had undetectable levels of IL-6 (limit of ELISA detection≤0.70 pg/ml; R&D Systems) whereas the ER{alpha}-negative cell lines MDA-MB-231 and MDA-MB-468 produced 2067 and 433 pg/ml of IL-6, respectively (Fig. 5) .


Figure 5
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  		Figure 5. Human MSC and ER{alpha}-negative breast tumor cells produce IL-6 but ER{alpha}-positive tumor cells do not produce IL-6. A panel of 5 human MSC lines (hMSC-02 through hMSC-06) each concentrated media to >3000 pg/ml of IL-6. IL-6 was not detected in the ER{alpha}-positive cell lines MCF-7, BT474, T47D, and ZR-75–1, whereas the ER{alpha}-negative cell lines MDA-MB-231 and MDA-MB-468 both produced IL-6. IL-6 concentrations from all ER{alpha}-positive tumor cell lines were below the limit of ELISA detection (i.e., <0.70 pg/ml); SD of three replicates shown.

IL-6 enhances MCF-7 growth rates in vitro
We recently demonstrated that soluble hMSC-derived factors significantly enhanced the growth rates of a panel of ER{alpha}-positive breast cancer cell lines including MCF-7, BT474, T47D, and ZR-75–1 (P<0.05) (27) . Given that IL-6 induces chronic phosphorylation of STAT3 (Fig. 2B ) and the established role of chronic STAT3 activation in ER{alpha}-negative breast cancer growth rates in vitro and in vivo (23 , 24) , we next evaluated the impact of STAT3 induction by IL-6 on the growth rates of the ER{alpha}-positive breast cancer cell line MCF-7. First, we compared the growth rate of MCF-7 alone or in the presence of 10:1 hMSC-02, hMSC-CM, hMSC-CM (–IL-6) (i.e., hMSC-CM previously stripped of IL-6) or 5 ng/ml recombinant human IL-6 (Fig. 6 A). As described earlier (27) , hMSC enhanced MCF-7RE growth in excess of 2-fold over baseline growth in 8 days. Since hMSC produced on average 3–7 ng/ml of IL-6 (Fig. 5) , we evaluated the growth rate of MCF-7RE in the presence of 5 ng/ml IL-6 and found that the addition of IL-6 alone could enhance the growth rate of MCF-7RE to the same extent as observed for hMSC-02 cocultures or hMSC-CM (Fig. 6A ). Finally, when we removed IL-6 from hMSC-CM, the remaining factors found in hMSC-CM were unable to enhance MCF-7RE growth rates above that of baseline (Fig. 6A ). IL-6 concentrations in the range of 1–200 ng/ml were found to enhance the growth rate of MCF-7 cells in a dose-dependent manner, and IL-6 concentrations at or below 100 pg/ml did not result in discernable enhancement of MCF-7 growth rates within 8 days (data not shown).


Figure 6
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  		Figure 6. IL-6 enhances the growth rate of the ER{alpha}-positive MCF-7 cell line in vitro and in vivo. A) Human IL-6 is necessary and sufficient for hMSC-induced enhancement of MCF-7 growth. 25,000 MCF-7RE cells were embedded in 3 mg/ml BME (±10:1 hMSC-02, hMSC-CM, 5 ng/ml IL-6, or hMSC-CM stripped of IL-6). Triplicate wells of a 96-well plate were read daily for 1 wk, and relative fluorescence units corresponding to MCF-7RE cell growth for each well are shown. Similar results were observed for 3 independent experiments (B) 2 x 106 MCF-7 cells or 2 x 106 MCF-7IL-6 cells were injected into the mammary fat pads of athym ic nude mice (cells were mixed with 100 µl of BME [6 mg/ml], Cultrex® BME; Trevigen Inc., Gaithersburg, MD, USA) prior to injection). MCF-7IL-6 cells rapidly formed tumor xenografts (averaging 8.2x7.4 mm by 6 wk postinjection) whereas MCF-7 cells did not expand beyond an average xenograft size 3 x 3 mm over the same period. (n=12 xenografts for each cell line; tumor volume=lxw2x0.512).

IL-6 enhances MCF-7 growth rates in vivo
To determine whether IL-6 could enhance the growth rate of MCF-7 in vivo, we examined whether ectopic expression of human IL-6 in MCF-7 cells (MCF-7IL-6) would alter tumor xenograft growth rates in immunocompromised mice. In agreement with our in vitro data, we found that orthotropic MCF-7IL-6 xenografts rapidly engrafted and expanded within 6 wk. In contrast, MCF-7 xenografts, which are not exposed to human IL-6, failed to develop beyond small palpable tumor nodules (Fig. 6B ). This observation is consistent with other reports demonstrating that ER{alpha}-positive MCF-7 cells do not readily form xenografts in immunocompromised mice unless they are supplemented with estrogen tablets, coinjected with fibroblasts in MatrigelTM BME, or mutant Ras is introduced into MCF-7 cells before injection (30 , 31) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
After evaluating the biological impact of primary human MSC on a panel of human breast cancer cell lines, we found a clear and reproducible delineation between ER{alpha}-positive and ER{alpha}-negative breast cancer cell lines with respect to STAT3 phosphorylation. Human MSC induced robust phosphorylation on tyrosine 705 (p-Tyr705) of STAT3 in the estrogen receptor-alpha (ER{alpha})-positive breast cancer cell lines MCF-7, T47D, BT474, and ZR-75–1 (Fig. 1B ). In contrast, baseline levels of p-Tyr705 STAT3 in the ER{alpha}-negative lines MDA-MB-231 and MDA-MB-468 were unaltered by MSC exposure (Fig. 1C ), suggesting that inherent genetic alterations or autocrine signaling events maintained p-Tyr705 levels on STAT3 in these ER{alpha}-negative lines. We also demonstrated that paracrine IL-6 from hMSC is a predominant factor that induced STAT3 phosphorylation in ER{alpha}-positive breast cancer cell lines (Fig. 4) . Finally, we demonstrated that IL-6 can act as a potent growth factor for the ER{alpha}-positive cell line MCF-7 in vitro and in vivo (Fig. 6) .

Taken together, our data suggest that IL-6 is necessary and sufficient for enhanced MCF-7 cell growth rates in the presence of hMSC; in conjunction with previous reports that have linked STAT3 activity to more aggressive breast tumor cell behavior (23 , 24 , 32) , we postulate that a biological link exists between ER{alpha} breast tumor cells, a common fibroblast cell population within the bone marrow (i.e., MSC), and the manifestation of a more aggressive form of disease in women with ER{alpha}-positive bone metastasis or elevated serum levels of IL-6. Of interest, the bone marrow microenvironment in postmenopausal women maintains stable levels of IL-6 protein, regardless of systemic estradiol levels (33) ; therefore, it is possible that the bone marrow microenvironment may represent a more favorable tissue for IL-6-responsive ER{alpha}-positive tumor cells (32 , 34) . Ongoing studies in the lab are testing this hypothesis and whether tissue-specific IL-6 levels contribute to the high rate of ER{alpha}-positive bone metastasis (2 , 3) .

Our findings also reaffirm the need for cancer biologists to consider that paracrine factors within the tumor microenvironment may not readily cross the species boundary from mice to humans (35) and that some of these factors may be involved in critical aspects of human cancer pathophysiology. It has been known for some time that the human IL-6 receptor does not recognize murine IL-6 (29) . In agreement with this earlier report, we demonstrated that IL-6 derived from murine MSC does not induce STAT3 phosphorylation in the ER{alpha}-positive human breast cancer cell line MCF-7 (Fig. 4D ), and thus it is likely that MCF-7 xenografts formed in immunocompromised mice would lack functional paracrine IL-6 signaling from the murine tumor microenvironment. The addition of human IL-6 to murine tumor microenvironments in a paracrine or autocrine fashion resulted in robust MCF-7 tumor cell growth in vitro and in vivo (Fig. 6) .

Although elevated serum levels of IL-6 are known to correlate with disease progression and poor clinical outcome in breast cancer patients (7 , 36) , the biological mechanism (or mechanisms) that account for these observations are not fully resolved (5) . It has been suggested that increased systemic IL-6 in postmenopausal women may drive ER{alpha}-positive tumorigenesis through induction of local breast tissue estradiol synthesis (37) , but the strength of this hypothesis on its own is weakened by several experimental and clinical observations. First, estradiol antagonizes IL-6 function by repressing both IL-6 and its signaling receptor, gp130 (38) . As such, elevated levels of breast tissue estradiol would predictably blunt the impact of IL-6 within the breast microenvironment, a site associated with a good clinical prognosis for ER{alpha}-positive tumors. Second, ovaries are the predominant source of estradiol in premenopausal women; after loss of ovarian function at menopause, the availability of systemic estradiol dramatically decreases, placing more pressure on a given tissue to maintain estradiol levels through local aromatase activity (38) . And finally, concurrent with declining systemic estradiol (and increasing IL-6) at menopause (38) , the incidence of ER{alpha}-positive breast cancer dramatically increases, suggesting that other factors, possibly IL-6, may contribute to ER{alpha}-positive tumor growth and metastasis (39) .


   ACKNOWLEDGMENTS
 
The authors thank Dr. Dawn Chandler for helpful discussions and critical review of this manuscript. This work was funded by the generous support of the Elsa U. Pardee Foundation (B.H.) and The American Cancer Society-Ohio Division (B.H.).

Received for publication April 18, 2007. Accepted for publication May 24, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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