Introduction
Breast cancer is the leading cause of cancer-associated death in women worldwide [
1]. Despite recent improvements in early detection and effective adjuvant chemotherapies, about one-third of patients with early disease will relapse with distant metastasis [
2]. Metastasis of breast cancer remains a largely incurable disease and is the major cause of mortality among breast cancer patients [
3]. Cancer metastasis is a complex process comprising dissociation of cancer cells from the bulk tumor, invasion of the neighboring tissue, intravasation, transport through the vascular system, extravasation, engraftment of disseminated cells and, finally, outgrowth of micrometastases [
4]. In our previous study, orthotopically grafted human breast cancer cells expressing high levels of IL-6, but not those with low levels of IL-6, spontaneously metastasized to the lung and liver in immunocompromised NOD/scid/γ
c-deficient (NOG) mice [
5]. IL-6 signaling in cancer cells themselves imbued them with cancer stem cell properties and epithelial-to-mesenchymal transition (EMT) phenotypes, which facilitate cancer cell invasion into the surrounding tissue and blood vessels, and cause distant metastasis [
5,
6]. In addition, IL-6 is known to be an important mediator of the expansion and recruitment of myeloid-derived suppressor cells (MDSCs) [
7,
8].
MDSCs are a heterogeneous population of cells comprising immature cells of monocyte or granulocyte lineage. They expand dramatically under conditions such as trauma, tumor growth and various chronic inflammatory disorders, including infection, sepsis and immunization [
7,
8]. Originally described as suppressive myeloid cells, thus-expanded MDSCs negatively regulate immune responses through multiple contact-dependent and -independent pathways [
8,
9]. Nitrosylation of T cell receptors (TCRs) and CD8 molecules leads to defective cytotoxic T cell (CTL) responses, rendering the cells unresponsive to antigen-specific stimulation [
10]. Shortage of
L-arginine due to arginase I activity in MDSCs inhibits T cell proliferation by several mechanisms [
11]. Nitrous oxide (NO) and transforming growth factor-β (TGF-β) produced by MDSCs induced further immunosuppressive microenvironments favoring tumor growth [
7‐
9]. In addition to the abovementioned immunosuppressive functions, MDSCs actively formulate microenvironments favoring the generation and survival of cancer cells in association with chronic inflammation. Induced expression of IL-1β in gastric epithelial cells induces the recruitment of MDSCs and leads to gastric inflammation and cancer, while activation of nuclear factor-kappa B (NF-κB) in MDSCs is strongly associated with carcinogenesis [
12]. MDSCs have been suggested to facilitate cancer cell metastasis through their immunosuppressive activities [
8,
13,
14]. Recently, cancer-derived remote signals were shown to induce the accumulation of myeloid cells including MDSC populations in putative metastatic sites before migrating cancer cells arrived, forming a 'pre-metastatic niche', which aided extravasation of migrating cancer cells and facilitated new blood vessel formation [
15‐
17]. Accumulating evidence shows that tumor-derived factors and tumor-cell-signaling mediators, such as Hsp72 and S1pr1, activate MDSCs to potentiate their immunosuppressive functions or increase the recruitment and colonization of these cells into pre-metastatic tissues [
18,
19]. Increased circulating MDSCs in breast cancer patients has been shown to be correlated with clinical cancer stage and metastatic tumor burden [
20,
21]. However, the evidence for the direct roles of cancer cell-exposed MDSCs in enhancing cancer cell aggressiveness, leading to spontaneous metastasis of these cells, from their invasion into the surrounding tissue and vascular system to their colonization of the target organ and the underlying mechanisms is either missing or merely circumstantial.
We questioned whether MDSCs activated by cancer cells directly increase breast cancer aggressiveness leading to spontaneous distant metastasis. To adequately evaluate the mutual interaction of breast cancer cells and inflammatory cells including MDSCs, we utilized murine models in which breast cancer cells were orthotopically grafted into immunocompetent syngeneic mice [
22]. We found that murine breast cancer cells with high IL-6 expression recruited more MDSCs and that the metastasizing capacity of cancer cells paralleled MDSC recruitment in tumor-bearing mice. Depletion and addition of MDSCs from tumor-bearing mice, respectively, reduced and increased the distant metastasis of breast cancer cells. Metastasizing, but not non-metastasizing, cancer cells activated MDSCs, increasing their expression and secretion of both IL-6 and soluble IL-6Rα, and facilitated breast cancer cell invasiveness and distant metastasis through IL-6 trans-signaling, acting both in afferent and efferent metastatic pathways. Thus, we provide evidence that breast cancer cells and MDSCs formed a synergistic mutual feedback loop and that thus-potentiated MDSCs directly affect breast cancer cell aggressiveness, leading to spontaneous metastasis.
Methods
Animals
BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, MA, USA). Experiments involving mice were approved by the Institutional Animal Care and Use Committee of Seoul National University (authorization no. SNU05050203).
Cell lines
The mouse breast carcinoma cell lines 4T1 (ATCC CRL-2539) and EMT6 (ATCC CRL-2755) were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI 1640 (WelGENE, Daegu, South Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (GIBCO, Grand Island, NY, USA) and 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37°C in a humidified 5% CO2 atmosphere. IL-6-expressing EMT6 cells (EMT6_IL-6 cells) were established by transfection with the pcDNA3.1_IL-6 construct using PromoFectin (PromoKine, Heidelberg, Germany), according to the manufacturer's instructions. Control cells were transfected with the pcDNA3.1 vector only. Stably transfected clones were established by selection with G418 (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 500 μg/ml for three weeks. IL-6-expressing EMT6 (EMT6_IL-6) clones were selected by ELISA. IL-6-knockdown 4T1 cells (4T1_shIL-6 cells) and Stat3-knockdown 4T1 cells (4T1_shSTAT3 cells) were established using the lentiviral vectors containing the shRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cells were infected with the shIL-6 or shSTAT3 virus and cultured in the presence of puromycin, and IL-6-knockdown 4T1 (4T1_shIL-6) and Stat3-knockdown 4T1 (4T1_shStat3) clones were selected by ELISA and Western blotting, respectively.
Tumor models
4T1 and EMT6 cells (1×105/mouse) were injected into the mammary fat pads of BALB/c mice. Tumor growth was monitored every other day thereafter. At 26 or 29 days after injection, the mice were euthanized and primary tumor masses, livers and lungs were fixed in 4% paraformaldehyde (PFA) for 24 hours and embedded in paraffin. Sections (5 μm) were stained with H & E for histopathological analysis. Numbers of tumor masses in the liver and lungs were determined under a dissecting microscope before fixing with 4% PFA. To deplete MDSCs, mice were intraperitoneally injected with 100 μg of anti-Gr-1 antibody (RB6-8C5, eBioscience, San Diego, CA) or control Rat immunoglobulin G 1 (IgG1) twice a week, starting three days after 4T1 cell injection. To block IL-6 trans-signaling in the afferent pathways of metastasis, 4T1 cells were injected into the mammary fat pads and gp130-Fc (R&D Systems, Minneapolis, MN, USA) was administered continuously using an osmotic mini-pump (5 or 10 μg for 14 days; Alzet, Cupertino, CA, USA). To block IL-6 trans-signaling in the efferent pathways of metastasis, 4T1 cells were injected intravenously into BALB/c mice and mice were intravenously injected with gp130-Fc (2.5 μg/mouse) 4 four days after cell injection.
Flow cytometry and MDSC isolation
To analyze MDSCs, mice were sacrificed 19 or 21 days after cancer cell injection. Mononuclear cells from the liver, lungs and primary tumor masses were isolated by Percoll (Amersham, GE healthcare, Buckinghamshire, UK) gradient centrifugation. Tissues were digested with collagenase D (1 mg/ml, Invitrogen, Carlsbad, CA, USA) and DNase I (0.5 U/ml, Sigma-Aldrich) for one hour at 37°C. Cells were suspended in 30% Percoll, layered onto the top of a 70% Percoll gradient and centrifuged (800 × g, 20 minutes). The interface was retained. Next, the cells were incubated with mAbs to mouse CD11b, Ly6C and Gr-1 that were conjugated to phycoerythrin, PerCP-Cy5.5, or allophycocyanin (eBioscience). They were then analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and FlowJo software (Tree Star, Ashland, OR, USA). To isolate splenic MDSCs from naïve or tumor-bearing mice, splenocytes were prepared and labeled with phycoerythrin-conjugated anti-CD11b mAb (BD Pharmingen, San Jose, CA, USA) and allophycocyanin-conjugated anti-Gr-1 mAb (eBioscience). CD11b+Gr-1+ MDSCs were purified using a FACS Aria cell sorter (BD Biosciences). The purity after sorting was greater than 95%.
Tumor and MDSC conditioned medium preparation
EMT6 cells (1×104) and 4T1 cells (1×104) were incubated for 72 hours on a 24-well plate and the culture supernatants were collected. To obtain MDSC CM, FACS-sorted splenic MDSCs (4×105) were cultured for 24 hours. 4T1/MDSC-CM and EMT6/MDSC-CM were prepared by cultivating MDSCs (4×105) in 50% (v/v) 4T1-CM or EMT6-CM for 24 hours. A volume of 4T1/MDSC-CM containing 1 ng of IL-6, or the same volume of EMT6/MDSC-CM or MDSC-CM, was added to the 4T1 and EMT6 cell cultures. To some cultures, the following signaling inhibitors were added; Stat3 inhibitor peptide (1 μM; Millipore, Billerica, MA, USA), PI3K inhibitor (LY294002, 10 μM; Calbiochem, Billerica, MA, USA), NF-κB inhibitor (Bay-117082, 10 μM; Calbiochem), JNK inhibitor (SP600125, 10 μM; Calbiochem), p38 MAPK inhibitor (SB403250, 10 μM; Cell Signaling, Danvers, MA, USA), and ERK inhibitor (PD98059, 10 μM; Calbiochem).
Immunofluorescence microscopy
Tissues were fixed in 4% PFA and embedded in paraffin. Sections were stained with H & E for histopathological analysis. To investigate IL-6, IL-6Rα, and Adam17 expression levels in MDSCs, sections were stained with anti-Gr-1 mAb and other appropriate antibodies. The following primary antibodies were used: anti-mouse IL-6 (Abcam, Cambridge, MA, USA), anti-mouse IL-6Rα (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-mouse Adam17 (Abcam) and anti-mouse Gr-1 (eBioscience). The following secondary antibodies were used: Alexa 488-conjugated anti-rabbit IgG (Invitrogen) and Alexa 594-conjugated anti-rat IgG (Invitrogen). Image acquisition and processing was performing using a confocal fluorescence microscope (Olympus, Center Valley, PA, USA) and an FV10-ASW 2.0 Viewer (Olympus).
ELISA
EMT6 and 4T1 cells were plated on a 24-well plate (1 × 104/well). The cells were permitted to grow for 24 or 48 hours. Supernatants were collected and assayed for IL-6 and soluble IL-6Rα levels by ELISA. For IL-6 detection, anti-mouse IL-6 (eBioscience) was used as the capture antibody, biotinylated anti-mouse IL-6 (eBioscience) in 0.1% BSA in PBS/T as the detection antibody and recombinant IL-6 (eBioscience) as the standard. To detect soluble IL-6Rα, we used anti-mouse IL-6Rα (R&D Systems) as the capture antibody, biotinylated anti-mouse IL-6Rα (R&D Systems) as the detection antibody and recombinant IL-6Rα (R&D Systems) as the standard.
RNA analysis
RNA was isolated from sorted splenic MDSC using the RNeasy kit (QIAGEN; 74104, Hilden, Germany). cDNA was generated from 1 μg of total RNA by reverse transcriptase from Moloney Murine Leukemia Virus (M-MLV) (TAKARA, Shiga, Japan), and subjected to PCR. The following primer pairs were used for PCR: GAPDH, 5'-GTCAGTGGTGGACCTGACCT-3' and 5'-AGGGGTCTACATGGCAACTG-3'; IL-6, 5'-GACAAAGCCAGAGTCCTTCAGAG-3' and 5'-CTAGGTTTGCCGAGTAGATCTC-3'. PCR products were analyzed by 1.5% agarose gel electrophoresis.
Western blot analysis
Cells were harvested in lysis solution containing 50 mM Tris/HCl (pH 7.6), 1% NP40, 150 mM NaCl, 2 mM EDTA, 100 μM PMSF, a protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland), and a phosphatase inhibitor (Sigma-Aldrich). After incubation on ice for 30 minutes, cellular debris was removed by centrifugation (10 minutes, 4°C). Proteins (10 μg) were separated by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane. After blocking with 5% skim milk, the membranes were probed with an appropriate antibody. Blots were developed with an enhanced chemiluminescence Western blotting detection system (Amersham, GE Healthcare, Buckinghamshire, UK). The following antibodies were used: anti-β-actin (Sigma-Aldrich), anti-phospho-Stat3 (Tyr705) (Cell Signaling, Danvers, MA, USA), anti-Stat3 (Santa Cruz Biotechnology), anti-I-κB (Santa Cruz Biotechnology), anti-phospho-JNK (Santa Cruz Biotechnology), anti-phospho-ERK (Santa Cruz Biotechnology), and anti-phospho-p38 (Cell Signaling).
Invasion assay
Matrigel matrix solution (200 μg/ml, Matrigel™ Basement Membrane Matrix, BD Bioscience) was applied to each Transwell (Falcon, Franklin Lakes, NJ, USA). 4T1 cells (5×104) were seeded into the upper chamber of the Transwell and then the lower chamber was filled with collagen matrix (5 μg/ml). Invasion assays were carried out for 18 hours. Non-invading cells on top of the matrix were removed by rubbing with a moistened cotton swab. Invaded cells on the lower surface of the Matrigel matrix were fixed with 4% PFA and stained with 0.2% crystal violet. Cells were counted using ImageJ software (version 1.46).
Statistical analyses
The two-tailed Student's t-test was used to compare measurements for pairs of samples. Two-way analysis of variance (ANOVA) and Bonferroni post hoc testing were used to compare the tumor volumes of the two groups. The SPSS software (SPSS Inc.) was used for all statistical analyses.
Discussion
In this study, we showed that IL-6 derived from metastasizing murine breast cancer cells recruited MDSCs and tumor-expanded MDSCs expressed Adam-family proteases, which facilitated shedding of IL-6 receptors, thereby providing sIL-6Rα. In addition, factors other than IL-6, released from the cancer cells, promoted IL-6 production from recruited MDSCs in the vicinity of cancer cells. MDSC-derived IL-6 and sIL-6Rα induced persistent activation of STAT3 and increased invasiveness of breast cancer cells via an IL-6 trans-signaling mechanism. This IL-6 trans-signaling also increased distant metastasis
in vivo. From these experiments, we provide novel information regarding potential tumor-MDSC synergistic axis involving IL-6 and soluble IL-6Rα [see Additional file
1, Figure S12].
MDSCs have been suggested to constitute tumor-favoring microenvironments largely through their suppressive effects on innate and adaptive immunity and promotion of angiogenesis [
8,
9,
13,
14]. In our murine breast cancer cell model, 4T1 breast cancer cells recruited more MDSCs and metastasized more strongly compared to EMT6 cells, not only in syngeneic immunocompetent BALB/c mice, but also in immunodeficient NOG mice, in which T, B, and NK cells are defective (our unpublished data and [
27]). This implies that MDSCs in 4T1 cell-bearing mice induced spontaneous distant metastasis of cancer cells independently of their suppressive effects on adaptive and natural-killer cell anti-tumor immunity. Thus, in this study, we provide evidence that MDSCs potentiated by metastasizing breast cancer cells directly enhance the aggressiveness of cancer cells though trans-signaling by upregulating both IL-6 and sIL-6Rα secretion in primary tumor sites and the metastatic lung.
Induced expression of IL-6 in EMT6 cancer cells caused recruitment of MDSCs in lymphoid organs, metastatic target organs and primary tumor sites comparable to that caused by metastasizing 4T1 cancer cells, which implies that IL-6 or downstream signaling may be involved in this process. However, recruitment of MDSCs
per se was not enough to guarantee that non-metastasizing breast cancer cells fully adopted metastatic capability. Transfer of splenic MDSCs from metastasizing 4T1 cell-bearing mice increased distant metastasis of non-metastasizing EMT6 cells but did not imbue them with the full metastatic capability of 4T1 cells. Based on the above findings, we assume that additional factors from metastasizing breast cancer cells affected the homing of MDSCs into the tumor sites and increased the potency of recruited MDSCs [
18,
19,
28,
29]. Our
in vitro co-culture experiments showed that recruited MDSCs in the spleens of tumor-bearing mice required additional activation in the vicinity of metastasizing cancer cells, predominantly through contact-independent mechanisms. The outcome of activation of MDSC by metastasizing cancer cells
in vitro can be summarized as exaggerated augmentation of IL-6 production by MDSCs [
18,
19]. Immunofluorescence microscopy of different tissues from 4T1 cell-bearing mice indeed showed that MDSCs in the primary tumor mass and metastatic lung, but not in the spleen, expressed high levels of IL-6. These findings suggest that recruited MDSCs may have different roles or function through different mechanisms depending on the recruited sites (lymphoid organs
versus tumor sites, primary or metastatic) [
30,
31].
In contrast to the requirement for contact with metastasizing cancer cells for increased IL-6 production by MDSCs, the components necessary for increased soluble IL-6Rα production were increased in MDSCs in the remote sites of metastasizing tumor-bearing mice, but not those of non-metastasizing tumor-bearing mice. Expression levels of both IL-6Rα and the enzymes responsible for digesting the membrane form into soluble forms (Adam10 and Adam17) [
25,
26,
32,
33] were increased in the splenic MDSCs of 4T1 cell-bearing mice. Moreover, simple cultivation of splenic MDSCs from 4T1 cell-bearing mice increased the expression of soluble IL-6Rα compared to EMT6 cell-bearing mice. Thus, at least four remote signals were secreted by metastasizing 4T1 cancer cells; these induced (1) recruitment of MDSCs to various sites of tumor-bearing hosts, (2) increased expression of IL-6Rα, (3) increased expression of Adam family proteases, and (4) highly increased expression of IL-6 by MDSCs [
34,
35,
18,
19,
29]. Further studies are needed to clarify the critical roles of the various mediators that may be involved in MDSC modulation. In this study, we convincingly demonstrated that 4T1 cells responded to IL-6 trans-signaling by MDSCs. However, as we did not perform experiments specifically inhibiting expression of IL-6 and sIL-6Rα in MDSCs
in vivo, we cannot absolutely rule out the possibilities that IL-6 and sIL-6Rα responsible for metastasis could potentially be coming from other cell types
in vivo either the tumor cells themselves or other cells within the tumor microenvironment. Further studies are needed to clarify this aspect.
The importance of IL-6 signaling in promoting tumorigenesis is well-documented, particularly for tumors associated with chronic inflammation such as colitis-associated colon cancer, pancreatic cancer and hepatocellular carcinoma [
36‐
39]. In addition to these tumors, increased IL-6 signaling is preferentially found in basal-like breast cancers and high-grade tumors and is associated with a poor response to chemotherapy, increased distant metastasis in xenograft animal models and decreased metastasis-free survival in patients [
5,
6]. Thus, IL-6 signaling has been linked to tumor aggressiveness, including cancer stem cell phenotypes [
5,
6,
40,
41] and EMT phenotypes [
42], drug resistance [
43], and anoikis resistance, that is, contact-independent survival, which is required for travel through the vascular system [
44]. In addition to tumor-derived IL-6 autocrine signaling [
5,
6], paracrine IL-6 signaling within tumor microenvironments has been highlighted recently. Mesenchymal stem cells constitute the cancer stem cell niche by providing IL-6 and CXCL7 [
45,
46]. Paracrine IL-6 signaling from tumor-infiltrating inflammatory cells is more important because these cells have a greater inflammatory cytokine secretion capacity, including IL-6 [
47‐
49]. The caveat for this paracrine signaling is that cancer cells should express sufficient receptor machineries to recognize the increased IL-6 supply from the microenvironments. In this way, aggressive breast cancer cells exploited a trans-signaling mechanism by inducing the expression of molecules responsible for production of soluble IL-6 receptors (Adam proteases) from the recruited inflammatory cells [
50]. In terms of STAT3 signaling as a downstream of IL-6, previous reports suggested that knocking down STAT3 using the shRNA technique in 4T1 cells leads to decreased invasiveness of the cells
in vitro [
51]. The differences between this report and our current study may lie in the perfectness of STAT3 knockdown considering the total absence of phosphorylated STAT3 in the previous report compared to our current study (our unpublished data).
The site of MDSC function in the metastatic tumor-bearing mice requires further comment. In terms of sites of MDSC immunosuppressive activity, the available data are contradictory. Some authors suggest that lymphoid organs, including the liver, are the primary sites of MDSC accumulation and immunosuppression [
30,
52], while others emphasized effector sites, such as inflammatory sites and tumors, but not lymphoid organs, such as the spleen [
31,
53,
29]. In terms of MDSC function during the efferent phase of tumor metastasis and related angiogenesis, accumulation of MDSCs in the lung, a metastatic target organ, supported the effective engraftment of metastatic tumor cells at this site [
15,
19,
54]. Because the metastasis-promoting effects of MDSCs in this study occurred in the absence of adaptive immunity and natural-killer cell activity and there was no increase in IL-6 signaling in the spleen, MDSCs themselves must have directly increased tumor cell metastatic capability in the tumor sites, either primary tumors or metastases, but not in the lymphoid organs, affecting both the afferent and efferent phases of metastasis through exaggerated IL-6 trans-signaling. Lastly, although we clearly showed the direct effects of MDSCs on the
in vitro invasiveness and effector phase of lung metastasis, the co-occurrence of reduction in the primary tumor size and metastasis in some experiments prevented us to totally rule out the possibilities that differences in metastasis might be due to differences in tumor size rather than specific effects of MDSCs on metastatic capacity.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KO designed the research, performed the experiments, interpreted the data and wrote the manuscript; OYL performed most of the experiments; SYS, ON and PMR conducted RT-PCR and immunohistochemistry experiments; MWS generated the animal models; DSL designed the research, interpreted the data, wrote and edited the manuscript. All authors read and approved the final manuscript.