Simultaneous blockade of IL-6 and CCL5 signaling for synergistic inhibition of triple-negative breast cancer growth and metastasis
Metastatic triple-negative breast cancer (TNBC) is a heterogeneous and incurable disease. Numerous studies have been conducted to seek molecular targets to treat TNBC effectively, but chemotherapy is still the main choice for patients with TNBC. We have previously presented evidence of the important roles of interleukin-6 (IL-6) and chemokine (C-C motif) ligand 5 (CCL5) in TNBC tumor growth and metastasis. These experiments highlighted the importance of the crosstalk between cancer cells and stromal lymphatic endothelial cells (LECs) in tumor growth and metastasis.
We examined the viability and migration of MDA-MB-231-LN, SUM149, and SUM159 cells co-cultured with LECs when treated with maraviroc (CCR5 inhibitor) and tocilizumab (anti-IL-6 receptor antibody). To assess the anti-tumor effects of the combination of these two drugs in an athymic nude mouse model, MDA-MB-231-LN cells were implanted in the mammary fat pad and maraviroc (8 mg/kg, orally daily) and cMR16-1 (murine surrogate of the anti-IL-6R antibody, 10 mg/kg, IP, 3 days a week) were administrated for 5 weeks and effects on tumor growth and thoracic metastasis were measured.
In this study, we used maraviroc and tocilizumab to confirm that IL-6 and CCL5 signaling are key pathways promoting TNBC cell proliferation and migration. Further, in a xenograft mouse model, we showed that tumor growth was dramatically inhibited by cMR16-1, the mouse version of the anti-IL6R antibody. The combination of maraviroc and cMR16-1 caused significant reduction of TNBC tumor growth compared to the single agents. Significantly, the combination of maraviroc and cMR16-1 abrogated thoracic metastasis.
Taken together, these findings show that IL-6 and CCL5 signaling, which promote crosstalk between TNBC and lymphatic vessels, are key enhancers of TNBC tumor growth and metastasis. Furthermore, these results demonstrate that a drug combination inhibiting these pathways may be a promising therapy for TNBC patients.
KeywordsTriple negative breast cancer Tumor microenvironment Secretome Drug repurposing Maraviroc Tocilizumab
Blood endothelial cell
Chemokine (C-C motif) ligand 5
CCL receptor 5
Cell invasion and migration
Hepatocyte growth factor receptor
Immature dendritic cells
Epidermal growth factor
Enzyme-linked immunosorbent assay
Fetal bovine serum
Fibroblast growth factor
Hepatocyte growth factor
Insulin-like growth factor
Lymphatic endothelial cells
Platelet-derived growth factor
Signal transducer and activator of transcription 3
Triple-negative breast cancer
Vascular endothelial growth factor
Vascular endothelial growth factor receptor
Lymphangiogenesis plays a critical role in tumor invasion, distal metastasis, and immune unresponsiveness. Crosstalk between tumor-associated lymphatic endothelial cells (LEC) and cancer cells enhances the recruitment of cancer cells to the lymphatic system from primary tumors. This recruitment is increased by various secreted factors such as CCL21, CXCL12, CCL27, IL6 and KAI1 , which also induce lymphangiogenesis in tumor-draining lymph nodes (LNs). Lymphangiogenesis, which promotes tumor metastasis, is induced in the pre-metastatic niche by lymphangiogenic growth factors such as vascular endothelial growth factor (VEGF)-C, VEGF-D, angiopoietins, platelet-derived growth factor (PDGF)-BB/AA and basic fibroblast growth factor (bFGF) [2, 3, 4, 5, 6]. The recruitment of immune cells such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and immature dendritic cells (DCs) contribute to tumor-induced immunosuppression in the lymph nodes . Due to a paucity of lymphatic markers the role of lymphangiogenesis in tumor growth and metastasis has not been studied as well as the role of blood endothelial cells (BEC). We have previously demonstrated that the crosstalk between LEC secreting CCL5 and triple-negative breast cancer (TNBC) cells expressing CCR5, the CCL5 receptor, promotes the recruitment of TNBC cells towards the lymphatic vessels, induces lymphangiogenesis, and facilitates subsequent lung metastasis. Consistent with these finding, we showed that maraviroc, a CCR5 inhibitor with anti-retroviral activity, inhibited TNBC lymphangiogenesis and lung metastasis. Furthermore, we discovered that IL-6, which is secreted by TNBC cells, is a key factor in upregulating CCL5 expression in LECs by activating the IL6 receptor and subsequently STAT3, which enhances transcription of the CCL5 gene. In our earlier study, we showed that inhibiting the IL-6 signaling pathway by depleting IL-6 levels using an anti-IL-6 antibody or a STAT3 inhibitor decreased CCL5 expression and consequently lymphogenous metastasis . Here, we present more evidence that both IL-6 and CCL5 are key factors in TNBC lymphangiogenesis, tumor growth, and thoracic metastasis. Furthermore, by using maraviroc (CCR5 inhibitor) and cMR16-1 Ab (murine surrogate of the anti-IL-6 receptor antibody) we showed that simultaneous blockade of CCR5 and IL-6 receptor signaling strongly inhibits TNBC tumor growth and profoundly inhibits TNBC tumor metastasis.
MDA-MB-231-luc-D3H2LN (MDA-MB-231-LN) cells were purchased from Caliper and propagated in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin (Sigma). SUM149 and SUM159 breast cancer cells were cultured in F-12 medium supplemented with 5% FBS, 1 ng/ml hydrocortisone, 5 μg/ml insulin (Sigma, St. Louis, MO, USA), and 0.1 mM HEPES (ThermoFisher Scientific, Waltham, MA, USA). LECs were purchased from Lonza, and grown in EGM-2MV. Cells were maintained under standard conditions of 37 °C and 5% CO2. Cells were cultured for a maximum of 4 weeks after thawing fresh, early passage cells and confirmed to be Mycoplasma negative (Hoechst stain).
When TNBC cells had grown to confluence in T175 tissue culture flasks, the normal cancer cell growth medium was replaced with 8 ml serum-free medium (SFM) after extensive washing. After 24 h incubation in a tissue culture incubator, the supernatant was centrifuged and filtered through 0.2-μm syringe filters (Corning). The resulting tumor-conditioned medium (TCM) was stored in aliquots at − 80 °C. When LECs reached 30–40% confluence in T75 tissue culture flasks, the growth medium (GM) was replaced with 30% TCM in GM (TCM:GM = 3:7) to allow the TCM to activate the LECs. For education of LECs by TCM, the cells were allowed to grow in the medium for 3–4 days at which point the medium was replaced with 3 ml SFM containing 2% FBS. After 48 h, the supernatant was centrifuged and filtered. The resulting tumor-educated LEC conditioned medium, (TCM-LEC)CM, was stored in aliquots at − 80 °C to avoid multiple freeze thaws.
Cell migration and proliferation assays
Cancer cell migration was assessed using the Oris™ cell migration kit (Platypus), as previously described . The (TCM-LEC)CM (100 μl) with or without 20 μM maraviroc (R&D Systems) or 200 μg/ml tocilizumab (Genentech) was added once the cancer cells had attached. Migration and proliferation assays using CIM (cell invasion and migration) plates and the RTCA system (ACEA Bioscience) were performed as previously described .
Mouse xenograft studies
Before tumor inoculation, athymic nude mice (female, 5–6 weeks, 18–20 g) were pre-treated by injecting 50 μl TCM or SFM subcutaneously for 2 weeks daily as described previously . MDA-MB-231-LN cells were grown to 90% confluence, trypsinized, resuspended in serum-free medium, and mixed 1:1 with Matrigel (BD Biosciences) and 2 × 106 cells were injected into the upper inguinal mammary fat pad of the animals induced for 2 weeks with TCM as described above. Tumor sizes were measured using calipers, and the volume was calculated, using the formula: V = 0.52 × (length) × (width)2. Animals were imaged every week to track anterior tumor metastases, using the IVIS Xenogen 200 optical imager (Xenogen) after intraperitoneal (i.p.) injection of D-luciferin (Caliper, 150 mg/kg body weight). After 5 weeks, organs were harvested and bathed in D-luciferin solution for 5–10 min and placed in the IVIS imager to detect metastases ex vivo. Luciferase-mediated photon flux was quantified using Living Image® 3D Analysis (Xenogen). Maraviroc (8 mg/kg body weight, R&D systems) was administered orally daily; cMR16-1 (10 mg/kg, Genentech) was administered intraperitoneally 3 days per week for 5 weeks.
Immunofluorescence and immunohistochemical analysis
Immunofluorescence and immunohistochemical analysis (IHC) were performed using monoclonal antibodies against CD31 and pan-cytokeratin (Sigma). For immunofluorescence, after blocking with 5% normal goat or normal chicken serum (Jackson Immunoresearch) in PBS/Tween (PBST) (0.3% Triton) for 1 h at room temperature (RT), the sections were treated with anti-CD31 primary antibodies overnight at 4 °C. After three rinses with PBST, the sections were incubated for 1 h at RT with FITC-conjugated goat anti-rabbit secondary antibodies (1:500). After three rinses with PBST, the samples were counterstained with 4’ ,6-diamidino-2-phenylindole (DAPI) (1:10,000, Roche) (5 min at RT). The samples were washed with PBST once and mounted with the ProLong Gold anti-fade reagent (Invitrogen) in the dark. Fluorescent signals were visualized and digital images were obtained using the Zeiss LSM-700 confocal microscope (Carl Zeiss). For IHC, after blocking with 5% goat serum in PBST for 1 h at room temperature, the sections were treated with the pan-cytokeratin antibody overnight at 4 °C, then the peroxidase conjugated streptavidin complex method was performed, followed by the 3, 3′ diaminobenzidine (DAB) procedure according to the manufacturers’ protocols (VECTASTAIN Elite ABC Kit, Vector Lab).
Error bars represent the SEM. All statistical tests were two sided, and differences were considered statistically significant at P < 0.05. The synergy calculation was performed using the Chou-Talalay method as previously described [10, 11, 12].
Maraviroc and tocilizumab block TNBC cell proliferation
Decrease of TNBC cell migration by inhibition of IL-6 and CCL5 signaling
The combination of maraviroc and cMR16-1, an anti-mouse anti-IL6 receptor antibody, inhibits growth of MDA-MB-231-LN tumors
Inhibition of IL-6 and CCL5 signaling prevents TNBC metastasis
Tumor cells are commonly spread to tumor-draining lymph nodes in many types of cancer. There is ample evidence that lymphangiogenesis is actively induced by tumor cells and lymphangiogenic growth factors play an important role in lymphangiogenesis and distal organ metastasis [15, 16, 17]. For example, the proteolytic matured VEGF-C and VEGF-D interact with VEGFR-3 and upregulate lymphangiogenic activity [18, 19]; the VEGF-A-VEGFR-2 axis also induces tumor lymphangiogenesis by inducing LEC proliferation [20, 21]. Angiogenin 1 (Ang1) and Ang2 were also shown to be important for stimulating lymphangiogenesis and lymphatic metastasis in a tumor xenograft model [22, 23]. Hepatocyte growth factor receptor (HGF c-MET) is also known to be an inducer of lymphangiogenesis in vivo [24, 25, 26]. Studies by our laboratory and others showed that fibroblast growth factor (FGF), epidermal growth factor (EGF), PDGF, and insulin-like growth factor (IGF) play critical roles in lymphangiogenesis [13, 27, 28, 29, 30]. In our previous studies, we have shown that crosstalk between MDA-MB-231-LN cells and LECs induce upregulation of EGF to promote MDA-MB-231-LN cell proliferation and LECs co-injected with MDA-MB-231-LN cells into nude mice enhanced tumor growth. In addition, we demonstrated that an interaction of MDA-MB-231-LN and LEC stimulated PDGF-BB expression and recruited pericytes to the neovasculature in a xenograft mouse model; SU16f, a PDGFRβ inhibitor, inhibited the recruitment of pericytes in this model . It has been reported that the CCL5-CCR5 axis enhanced metastasis of basal breast cancer cells [31, 32, 33], but detailed mechanisms of how this happens are not yet clear. We have tried to clarify these mechanisms in this and earlier studies. In a previous study, we discovered that IL-6 secreted by TNBC cells upregulates CCL5 and VEGF in LECs through the IL-6-STAT3 signaling pathway. Additionally, we showed that the secreted CCL5 recruited CCR5-positive TNBC breast cancer to LNs resulting in LN angiogenesis and lung metastasis. In addition, the increased levels of VEGF induced LN angiogenesis and supported tumor cell extravasation into the lung. In agreement with these results, we have also found a significant inhibitory effect on TNBC metastasis by the following manipulations: depleting IL-6 in the tumor-conditioned medium injected into mice prior to tumor inoculation using an anti-IL-6 antibody, inhibiting CCL5 by maraviroc, inhibiting VEGF by an anti-VEGF antibody, and inhibiting STAT3 by S3I-201 . Maraviroc exerts its anti-retroviral activity by inhibiting the CCR5 receptor and it is commonly utilized as a treatment for HIV infection. Our results show that maraviroc inhibits TNBC metastasis so we propose repurposing this drug to treat patients with TNBC. In order to produce even more effective treatments for TNBC using repurposed drugs, we tested tocilizumab, a Food and Drug Administration (FDA)-approved anti-inflammatory IL-6 receptor inhibitor drug for the treatment of rheumatoid arthritis. Application of these two drugs allowed us to directly test the hypothesis that drugs against both IL-6 and CCL5 could be used to treat TNBC. We discovered that co-cultures of MDA-MB-231-LN cells with LECs enhanced proliferation of MDA-MB-231-LN cells. It has been reported that autocrine IL-6 is a key promoter of TNBC cell proliferation [34, 35]. In support of our hypothesis, in our experiments, tocilizumab significantly decreased the viability of TNBC cells and the combination of tocilizumab and maraviroc decreased cellular viability even more (Fig. 1). Consistent with this result, we observed no significant difference in the viability of TNBC cells in the presence of maraviroc. Our data demonstrate that IL-6 signaling in the crosstalk between TNBC cells and LECs is a major determinant of TNBC cell proliferation and viability.
We next investigated the effect of maraviroc and tocilizumab on the migration of TNBC cells induced by (TCM-LEC)CM. The results indicate that the migration of TNBC cells is dependent on CCL5-CCR5 signaling and that IL-6 indirectly contributes to migration of TNBC cells by regulating CCL5 expression in LECs (Fig. 2). We tested agents targeting the IL-6 and CCR5 pathways in athymic mice with TNBC tumor xenografts to extend these findings. The growth of MDA-MB-231-LN tumors was inhibited by cMR16-1, whereas maraviroc had no effect on tumor growth, consistent with our observation that maraviroc had no effect on the proliferation of MDA-MB-231-LN cells. The combination of maraviroc and cMR16-1 resulted in greater inhibition of tumor growth compared to the single agents (Fig. 3). Next, we were interested in determining whether maraviroc and cMR16-1 could inhibit TNBC tumor metastasis. Thoracic metastasis was detected in all control group mice while mice treated with either maraviroc or cMR16-1 had low incidences of metastasis. Strikingly, we observed no metastasis in any of the mice treated with a combination of maraviroc and cMR16-1 (Fig. 4). These observations demonstrate that CCL5-CCR5 and IL-6-IL-6R signaling between TNBC cells and LECs plays a critical role in TNBC tumor growth and metastasis.
Taken together, these studies confirmed that IL-6 and CCL5 function as critical molecules in TNBC growth and metastasis. Furthermore, maraviroc and tocilizumab could be repurposed and provide a new clinical approach to treat TNBC.
We thank Pfizer for providing maraviroc, Genentech for providing tocilizumab and cMR16-1, and ACEA Biosciences and Dr. Yama Abassi for technical advice in using the RTCA system.
This work was supported by the National Institutes of Health grant R01 CA138264.
Conception and design: KJ, NBP, and ASP. Development of methodology: KJ and NBP. Laboratory experiments: KJ. Analysis and interpretation of data: KJ, NBP, and ASP. Writing and review of the manuscript: KJ, NBP, and ASP. Administrative, technical, or material support: KJ, NBP, and ASP. All authors read and approved the final manuscript.
All animal experiments were performed in accordance with protocols reviewed and approved by Johns Hopkins University Institutional Animal Care and Use Committee (IACUC).
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 3.Wakisaka N, Hasegawa Y, Yoshimoto S, Miura K, Shiotani A, Yokoyama J, Sugasawa M, Moriyama-Kita M, Endo K, Yoshizaki T. Primary tumor-secreted lymphangiogenic factors induce pre-metastatic lymphvascular niche formation at sentinel lymph nodes in oral squamous cell carcinoma. PLoS One. 2015;10(12):e0144056.CrossRefPubMedPubMedCentralGoogle Scholar
- 5.Von Marschall Z, Scholz A, Stacker SA, Achen MG, Jackson DG, Alves F, Schirner M, Haberey M, Thierauch KH, Wiedenmann B, et al. Vascular endothelial growth factor-D induces lymphangiogenesis and lymphatic metastasis in models of ductal pancreatic cancer. Int J Oncol. 2005;27(3):669–79.PubMedGoogle Scholar
- 19.Stacker SA, Stenvers K, Caesar C, Vitali A, Domagala T, Nice E, Roufail S, Simpson RJ, Moritz R, Karpanen T, et al. Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers. J Biol Chem. 1999;274(45):32127–36.CrossRefPubMedGoogle Scholar
- 34.Hartman ZC, Poage GM, den Hollander P, Tsimelzon A, Hill J, Panupinthu N, Zhang Y, Mazumdar A, Hilsenbeck SG, Mills GB, et al. Growth of triple-negative breast cancer cells relies upon coordinate autocrine expression of the proinflammatory cytokines IL-6 and IL-8. Cancer Res. 2013;73(11):3470–80.CrossRefPubMedGoogle Scholar
- 35.Lin C, Liao W, Jian Y, Peng Y, Zhang X, Ye L, Cui Y, Wang B, Wu X, Xiong Z, et al. CGI-99 promotes breast cancer metastasis via autocrine interleukin-6 signaling. Oncogene. 2017;36(26):3695-705.Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.