Tumor-associated vacuolar ATPase subunit promotes ... - Nature

Oncogene (2014) 33, 5649–5654 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

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Tumor-associated vacuolar ATPase subunit promotes tumorigenic characteristics in macrophages GK Katara, MK Jaiswal, A Kulshrestha, B Kolli, A Gilman-Sachs and KD Beaman Macrophage polarization contributes to distinct human pathologies. In tumors, a polarized M2 phenotype called tumor-associated macrophages (TAMs) are associated with promotion of invasion and angiogenesis. In cancer cells, vacuolar ATPase (V-ATPase), a multi-subunit enzyme, is expressed on the plasma/vesicular membranes and critically influences the metastatic behavior. In addition, the soluble, cleaved N-terminal domain of a2 isoform of V-ATPase (a2NTD) is associated with in vitro induction of pro-tumorigenic properties in monocytes. This activity of a2 isoform of V-ATPase (a2V) caused us to investigate its role in cancer progression through the evaluation of the immunomodulatory properties of a2NTD. Here, we present direct evidence that surface expression of V-ATPase is associated with macrophage polarization in tumor tissue. Macrophages from BALB/c mice (peritoneal/ bone marrow derived) were stimulated with recombinant a2NTD in both ex vivo and in vivo systems and evaluated for TAM characteristics. a2V was highly expressed in tumor tissues (breast and skin) as well as on the surface of tumor cell lines. The a2NTDstimulated macrophages (a2MF) acquired TAM phenotype, which was characterized by elevated expression of mannose receptor-1, Arginase-1, interleukin-10 and transforming growth factor-b. a2MF also exhibited increased production of other tumorigenic factors including matrix metalloproteinase-9 and vascular endothelial growth factor. Further, a2MF were cocultured with mouse B-16F0 melanoma cells for their functional characterization. The coculture of these a2MF subsequently increased the invasion and angiogenesis of less invasive B-16F0 cells. When cocultured with naive T cells, a2MF significantly inhibited T-cell activation. The present data establish the role of V-ATPase in modulating a macrophage phenotype towards TAMs through the action of a2NTD, suggesting it to be a potential therapeutic target in cancer. Oncogene (2014) 33, 5649–5654; doi:10.1038/onc.2013.532; published online 23 December 2013 Keywords: a2V-ATPase; a2NTD; tumor-associated macrophages; M2 macrophages; invasion assay; angiogenesis

INTRODUCTION The modulation of the cellular microenvironment towards favorable conditions is essential for tumor growth and progression. For this purpose, tumor cells produce various factors, which interact with the neighboring cells and modulate their functions for their own advantage. Vacuolar ATPases (V-ATPases) are multisubunit enzymes present on the various endomembranes of cells and are required for many functions and cytosolic pH regulation. Tumor cells express V-ATPases on their surface to maintain a lowpH extracellular environment, and are required for growth and survival of these cells.1 Besides pH regulation, V-ATPases are also involved in drug resistance, invasion and metastasis of tumor cells.1–3 Previous studies have shown that the a2 isoform of the ‘a’ subunit of V-ATPase (a2V) has a role in cancer-related inflammation and maintenance of pregnancy.4–7 The soluble N-terminus domain of a2V (a2NTD) is cleaved from the a2 subunit, secreted through micro-vesicles from cancer cells, and stimulates monocytes to produce interleukin (IL)-1b and IL-10, which are important for cancer related inflammation.6,8 Macrophages can change their functional profiles according to environmental stimuli and can be classified as M1 and M2 macrophages.9,10 M1 macrophages are induced by lipopolysaccharides and interferon-g, and have antitumor activities. They produce pro-inflammatory cytokines including tumor necrosis factor-a, IL-1, IL-6, IL-12 or IL-23. In contrast, IL-4/ IL-13 stimulation induces M2 macrophages, which are involved in

the response to parasites, tissue remodeling, angiogenesis and tumor progression and in producing anti-inflammatory cytokines IL-10 and transforming growth factor-b. In addition, the M2 subtype is characterized by the expression of Arginase-1, CD206 (mannose receptor) and CD204 (scavenger receptor A).11 Among the cells in leukocyte infiltrates of tumors, macrophages are a major component and referred to as tumor-associated macrophages (TAMs). TAMs have been proposed to exhibit many similarities in terms of gene expression and function with alternatively activated M2 macrophages.12 TAMs produce a host of growth factors that affect tumor-cell proliferation, angiogenesis and degradation of the extracellular matrix. These factors include epidermal growth factor, members of the fibroblast growth factor family, transforming growth factor-b, vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs) and several distinct chemokines.13 In this study, we established an association between V-ATPase and host immune responses in cancer. We demonstrate that V-ATPases are present in abundance during cancer and the a2V subunit is highly expressed on the surface of tumor cells. Murine macrophages were stimulated with a2NTD, referred to as a2MF, and cocultivation of these a2MF with tumor cells induced invasion and angiogenesis of tumor cells. Further, a2MF were able to inhibit T-cell activation when cocultured with naive T cells. To our knowledge this is the first study establishing the direct involvement of V-ATPases in modulating host immune responses, through the signal of a2NTD, in tumor progression.

Department of Microbiology and Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA. Correspondence: Professor KD Beaman, Department of Microbiology and Immunology, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA. E-mail: [email protected] Received 1 July 2013; revised 21 October 2013; accepted 6 November 2013; published online 23 December 2013

a2V-ATPase promotes tumor-associated macrophages GK Katara et al

5650 RESULTS AND DISCUSSION In comparison with other subunits of V-ATPase, the role of a2 has been less investigated in association with cancer. Here, we examined the expression of a2V in two different tumor tissues (breast tumors and melanomas) as well as on tumor cell lines. Both of the tumor tissues were highly positive for a2V staining compared with their respective normal tissues (Figure 1a). In breast tumor tissue, most of the a2V-positive cells were located in the lumen, where hyperplasia exists. By immunoblotting, the elevated protein expression of both a2V and a2NTD was observed in tumor tissue compared with control (Figure 1b). In cell lines, a2V was highly expressed on the surface of all cell lines with varying intensity, which was correlated with their reported invasive potential (Figure 1c). 4T1 cells showed the highest a2V expression, whereas, B-16F0 showed the lowest a2V expression (Figure 1d). The above results suggest that the elevated expression of a2V in cancer cells might contribute to both tumor growth and aggressiveness. To determine the role of the soluble fragment of a2V (a2NTD) in cancer, we investigated the effect of a2NTD stimulation on the induction of macrophage phenotype (M1or M2) in ex vivo and in vivo systems. In both systems, mRNA expression of M2 markers was significantly upregulated with a2NTD treatment compared with untreated cells. No significant difference was observed between the groups for M1 marker gene expression (Figures 2a and d). Further, mRNA expression was validated at the protein level by flow cytometry and immunofluorescence assay. In both ex vivo and in vivo flow cytometric analyses, elevated numbers of F4/80 þ CD206 þ (M2) cells were observed (Figures 2b and e) and the difference was significant with a2NTD treatment compared with controls (Figures 2c and f). Interestingly, more M2 cells were observed in in vivo compared with ex vivo a2NTD treatment,

suggesting a possible influence of a2NTD on other cells, which further contributed to increased M2-polarized macrophage levels. However, no significant difference was observed for the various treatment groups with regard to the number of F4/80 þ CD11c þ (M1) cells. Immunofluorescence assay also confirmed an increase in the number of M2 cells after in vivo a2NTD stimulation (Figure 2i). As TAMs are functionally M2 macrophages,12 the above results strongly suggest that a2NTD modulates the macrophage phenotype towards TAMs. Many observations indicate that TAM express several pro-tumoral proteins, including those that promote angiogenesis, matrix remodeling and the suppression of adaptive immunity by producing factors like chemokines and cytokines (CCL17, CCL18, CCL22, IL-10), Arginase-1, MMPs, and VEGF.13,14 The mRNA expression of Arginase-1, IL-10 (Figures 2a and d), MMP-9, VEGF, CCL17 and CCL22 (Figure 3a) was significantly increased with a2NTD treatment compared with untreated macrophages. Further, higher Arginase-1 activity and IL10 protein levels were observed in a2MF (ex vivo: 27.02±2.267, in vivo: 113.2±21.46) compared with untreated cells (ex vivo: 5.625±1.169, in vivo:10.57±2.575) (Figures 2g and h). Similarly, protein levels of MMP-9 and VEGF were found significantly elevated with a2NTD treatment (MMP-9: 2.26±0.28, VEGF: 68.39±13.95) compared with untreated cells (MMP-9: 0.679±0.15, VEGF: 22.17±4.64, Po0.01 for both) (Figures 3c and d). Zymographic analysis also revealed that a2MF have enhanced MMP-9 activity compared with untreated cells (Figure 3b). These results confirmed that a2MF not only expressed the TAM markers but also secreted the characteristic tumorigenic factors of TAMs. After determining that a2NTD stimulation polarized macrophages towards TAMs, we further investigated whether a2MF have functional properties of TAMs. TAMs contribute to tumor

Figure 1. Tumor cells expressed high levels of a2vATPase on their surface. Immunohistochemical analysis of a2V expression in (a) human breast and, skin tumor and in the respective normal tissues (n ¼ 4). Tumor tissues have increased number of a2V-positive cells (brown) ( 100; inset 400). (b) Total protein from breast tumor and respective normal tissues were immunoblotted with anti-a2V (2C1) and anti-a2NTD. (c) Immunoflorescence analysis of a2V in mouse melanoma B-16F10 and B-16F0 cell lines. 5 104 cells were cultured in chamber slides, fixed with paraformaldehyde and incubated with 2C1 antibody (green) and examined microscopically ( 200). Representative images from four independent experiments are shown. (d) Representative surface a2 expression on mouse B-16F0, B-16F10 melanoma and 4T1 breast cancer cells examined by flow cytometry. Values are mean fluorescence intensities of 2C1-stained cells divided by isotype±s.e.m. of n ¼ 6. Oncogene (2014) 5649 – 5654

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Figure 2. a2NTD treatment enhanced M2 marker gene expression. Ex vivo: Mouse peritoneal macrophages (PMF) were isolated and stimulated with a2NTD (500 ng/ml). After 48 h cells were analyzed for ex vivo gene and protein expression of TAM markers. In vivo: Mice were injected with a2NTD (5 mg/mice) or PBS. After 48 h PMF were harvested and analyzed for in vivo gene and protein expression of TAM markers. (a, d) Mannose receptor-1 (MRc-1), Arginase-1, IL-10, CD11c and TNF-a expression was assessed by quantitative real-time PCR. Results were obtained from three independent experiments with 4–5 mice per group and data are reported as means±s.d. *Po0.05, as compared with unstimulated macrophages. (b, e) Unstimulated or a2NTD-stimulated MF were stained with F4/80, CD11c and CD206 and analyzed using flow cytometry. F4/80-positive cells were further analyzed with anti-CD11c and anti-CD206 antibodies. Representative flow cytometry results. (c, f ) the cell numbers in the F4/80-positive fraction. The cell numbers in the CD11c-positive/CD206-negative and CD11c-negative/CD206-positive fractions are shown according to the percentage of each fraction per total F4/80-positive cells. (g) Arginase activity was determined in cell lysates from both in vivo and ex vivo stimulated macrophages by measuring the amount of urea released from cell lysates. Data represent enzyme activity from individual samples (in vivo) or experiments (ex vivo). Bar represents mean±s.e. **Po0.01. (h) Protein levels of IL-10 were measured in supernatants from macrophages by ELISA. Values from individual samples or experiments are indicated as mean±s.e. **Po0.01. (i) Mice were injected with a2NTD (5 mg/mice) or PBS (n ¼ 10). After 48 h, PMF were harvested and immunoflorescence analysis was performed for anti-F4/80 (green) and anti-CD206 (red) stained cells.

progression in multiple ways and one of them is enhancement of the invasive potential of tumor cells. To assess whether a2MF could increase the invasiveness of tumor cells, a low-invasive mouse melanoma cell line B-16F0 was cocultured with a2MF in a non-cell to cell contact system and tumor invasion assays were performed. The cocultured B-16 cells showed an increased number of invading cells compared with B-16 cells alone or cells cocultured with control macrophages (Figure 4b). Similarly, in a wound-healing assay, cocultured B-16 migratory cells extended protrusions and ultimately invaded and closed the wound field faster than the control cells (Figure 4a). These data confirmed that interaction of tumor cells with a2MF enhanced their invasive potential. Another contribution of TAMs in the process of tumor progression is to support the angiogenic potential of tumor cells.14 To determine whether a2MF can stimulate the angiogenic properties of B-16F0 cells, an in vitro angiogenesis assay was performed using mouse C166 umbilical vein endothelial cells.15 The supernatants derived from B-16 cells that had been cocultured with a2MF showed an increase in the numbers of tube-like structures of C166 compared with C166 only or non-cocultured B-16 cells (both Po0.05; Figures 4c and d). TAM plays a major role in suppression of T-cell activation in tumors.12 To investigate the immunosuppressive properties of a2MF, naive T cells were cocultured with a2MF, and T-cell activation (CD25 & 2014 Macmillan Publishers Limited

expression) and proliferation were measured. Coculture with a2MF significantly inhibited CD25 expression (Figures 4e and f) or T-cell proliferation (Figure 4g) in comparison to coculture with untreated MF (Po0.05). A number of studies have shown that V-ATPases are expressed on the surface of tumor cells in a tissue-specific manner and, particularly, a3 and a4 isoforms of the ‘a’ subunit are important for pH regulation and metastasis of tumor cells.1,16–18 Further, the inhibition of these subunits at the transcript level has a significant impact on related functions.18,19 In our study, the presence of a2V on tumor tissues or cancer cells indicates its potential role in cancer, which has not been investigated well before. Here, the elevated expression of TAM markers, after a2NTD stimulation, suggested the immunomodulatory capacity of a2NTD on macrophages. In tumor cells, we have shown that a2NTD is cleaved from a2V subunit and secreted in microvesicles from these cells. Also, the level of a2NTD correlated with the expression of a2V.6 This observation supported the possibility that tumor cells express high levels of a2V on their surface to modulate the phenotype of resident macrophages toward TAMs through the action of a2NTD. TAMs produce multiple factors like MMPs to degrade the extracellular matrix, VEGF to enhance angiogenesis at the tumor site, and several chemokines to recruit Th2 and Treg cells to the tumor site; all of them further contribute to tumor Oncogene (2014) 5649 – 5654


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Figure 3. a2NTD-treated macrophages secrete pro-tumoral factors: (a) Matrix metalloprotease-9 (MMP-9), vascular endothelial growth factor (VEGF), CCL17 and CCL22 mRNA expression levels were assessed by quantitative PCR in PMF harvested from a2NTD- or PBS-injected mice (n ¼ 10). Data are reported as mean±s.d. *Po0.05, as compared with unstimulated macrophages. (b) Gelatinase activity in MF media. MF were harvested as described above and cultured in serum-free media for 24 h. The media was concentrated 10-fold and used for zymography and ELISA. The white bands represents gelatin degraded by MMP-9. Representative results from three individual experiments. Secreted levels of MMP-9 (c) and VEGF (d) by MF were measured by ELISA (n ¼ 10). Individual values are presented as mean±s.d. *Po0.05.

survival and growth.13 Our investigation of tumorigenic factors confirmed that a2MF secreted these factors, confirming that a2MF are TAMs. It has been established that TAM makes a significant contribution to tumor-associated immunosuppression through the secretion of factors such as CCL17, CCL22, transforming growth factor-b, IL-10 and Arginase-1.14 In our study, the levels of all of these factors were elevated after a2NTD treatment and a2MF successfully inhibited T-cell activation when cocultured with naive T-cells. We speculate that a2MF mediates suppression of T-cell activation through these factors. The enhancement in the invasive as well as angiogenic potential of tumor cells and suppression of T-cell activation after cocultivation with a2MF confirmed that a2MF have a similar impact on cancer cells as TAMs and are phenotypically and functionally similar to TAMs. In summary, this study reveals that V-ATPase can promote tumor survival and growth. We have identified the following evidence to support this conclusion. First, the analytical results of cancer tissues and cells point out that there is an overexpression of a2V on tumors. Second, in response to a2NTD, macrophages expressed and secreted distinct TAM associated molecules. Third, a2MF directly contributes to T-cell suppression, and the invasion and angiogenesis of cancer cells. These findings demonstrate a crucial role of V-ATPase in the progression of cancer through the action of a2NTD. A drug-mediated inhibition of V-ATPase has been used before for therapeutic purpose in cancer, but development of drug resistance is a major obstacle in disease cure.20,21 siRNAbased targeting of V-ATPase has shown promising results for controlling drug resistance and metastasis in a human xenograft cancer model.22,23 Here, we suggest that a2V may represent an effective and potentially selective cancer gene for molecular targeted therapies to control tumor growth and survival by involving the host immune system.

MATERIALS AND METHODS Cells and tissue samples Mouse malignant melanoma B16-F0, B16-F10, mouse breast cancer 4T1 cells and mouse embryonic endothelial cells C166 (ATCC) were cultured in Oncogene (2014) 5649 – 5654

Dulbecco’s modified Eagle’s medium (DMEM) or DMEM-Ham’s F12 or RPMI 1640 (Invitrogen, Grand Island, NY, USA) media with 10% fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine (Sigma, St Louis, MO, USA). BALB/c mice, 6–8 weeks old, were purchased from Jackson Labs (Bar Harbor, ME, USA) and were maintained at the Rosalind Franklin University Animal Care Facility. All experiments were conducted with institutional animal care and use committee approval. The peritoneal cells were harvested by injecting 7 ml of ice-cold phosphatebuffered saline and peritoneal macrophages (PMF) were purified by isolating adherent cells and checked by flow cytometry for purity, yielding 80–85% F4/80 þ cells. For bone marrow-derived macrophages (BMF), femurs were obtained from mice and flushed with RPMI 1640 to extrude the bone marrow. The bone marrow was homogenized and cells were cultured in RPMI 1640 with macrophage colony-stimulating factor (50 ng/ ml) (R&D Systems, Minneapolis, MN, USA) for 15 days. Cell culture medium was subsequently changed every 3 days with fresh medium. For immunohistochemistry and western blot, human tumor or normal frozen tissue sections and protein lysates were procured from Biochain, Newark, CA, USA.

Gelatin zymography and ELISA The concentrated serum-free media from a2MF was mixed with sample buffer and applied directly, without prior heating or reduction, to 10% zymogram gels containing 1 mg/ml gelatin. After electrophoresis, the SDS was removed from the gel by incubating in 2.5% (v/v) Triton X-100 (Sigma) for 30 min. The gels were then incubated at 37 1C overnight in development buffer (50 mmol/l Tris-HCl, pH 7.6, containing 0.2 M NaCl, 5 mmol/l CaCl2) for 24 h at 37 1C. Gels were then stained with 0.2% Coomassie blue. White bands representing gelatinase activity were photographed. The same serum-free supernatants were subjected to ELISA for IL-10, VEGF-A and MMP-9 (R&D Systems) in accordance with the manufacturer’s instructions.

Arginase activity assay Arginase activity was measured in untreated or a2MF cell lysates by using arginase assay kit, Bioassay (US Biologicals, Swampscott, MA, USA). Briefly, cells were lysed with lysis buffer (0.1% Triton X-100, 100 mg/ml pepstatin, 100 mg/ml aprotinin and 100 mg/ml antipain). A 10-ml sample volume was subjected to arginase assay in accordance with the manufacturer’s instructions. Data are given as milliunits of arginase/106 cells, where 1 U of arginase is defined as the amount of enzyme that catalyzes the formation of 1 mg of urea/min. & 2014 Macmillan Publishers Limited


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Figure 4. Cocultivation of a2NTD-polarized macrophages with tumor cells enhances the invasive capacity and angiogenesis of tumor cells and suppresses T-cell activation. PMF harvested from a2NTD- or PBS-injected mice were seeded (2 105) into the upper insets (0.4 mm) and cocultured with B-16F0 melanoma cells (2 105 cells per well) in a 12-well plate for 48 h. After coculture, MF were discarded and F0 cells were washed and used for cell invasion assay or wound-healing assay as described in Materials and Methods. (a) Wound-healing assay: B-16F0 cells were subjected to in vitro scratch assay with images captured at 0, 12 and 24 h after incubation using phase-contrast microscope ( 40). (b) Tumor-cell invasion assay: The cocultured F0 cells (2 105 cells per well) were seeded into Matrigel-coated invasion chambers (24-well, 8 mm pore size) and allowed to invade towards FBS for 24 h. Invasive cells on the bottom of the membrane were stained and quantified by florescent intensity. Data are reported as mean±s.d. *Po0.05, as compared with non-cocultured B-16F0 cells. (c, d) In vitro angiogenesis assay: Conditioned media from B-16F0 cells non-cocultured or cocultured with control or a2NTD-treated macrophages was concentrated 10-fold and added to C166 endothelial cells seeded into a Matrigel-coated 48-well plate for 48 h at 37 1C. Tube-like structures of C166 formed at 48 h were counted under a microscope. Data are reported as mean±s.d. *Po0.05, as compared with non-cocultured B-16F0 cells. (e–g) Macrophage-mediated suppression of T-cell activation: Splenocytes were cocultured with unstimulated or a2NTD- or IFN-g- or IL-4stimulated macrophages. After 48 h anti-CD3e and anti-CD28 were added to the cultures and incubated for 24 h. Expression of CD25 activation marker or T-cell proliferation was analyzed. (e) Representative CD25 expression on Tcells cocultured with unstimulated or a2NTDstimulated macrophages examined by flow cytometry. (f ), CD25 þ cell numbers in the CD3-positive fraction of all experimental test and control subsets are shown. The results are representative of four individual experiments. (g) T-cell proliferation assay: The same set of T cells was used for T-cell proliferation and assessed by MTT assay. *Po0.05, compared with the control cells. Error bars shows mean±s.e.

In vitro invasion assay

In vitro angiogenesis assay

The invasion assay was conducted using the CytoSelect 24-well cell invasion assay kit (Cell Biolabs, Inc, San Diego, CA, USA). Briefly, B-16F0 cells (1 105) were resuspended in 200 ml of serum-free DMEM and added to the upper inserts. DMEM (500 ml) with 10% FCS was added to the lower chamber. After 24 h, invaded cells were used for fluorometric analysis as per the manufacturer’s instructions.

C166 (1 104 cells in 100 ml/well) were seeded into a BD Matrigel (BD Biosciences, San Jose, CA, USA) coated (50 ml/well) 48-well plate and 100 ml of 10-fold concentrated conditioned media from B-16F0 cells, previously cocultured or non-cocultured with a2MF, was added and incubated for 48 h at 37 1C. Tube-like structures of C166 formed at 48 h were counted under a microscope.


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5654 Suppression of T-cell activation Mouse peritoneal macrophages were isolated and stimulated with a2NTD (500 ng/ml) or interferon-g (20 ng/ml) or IL-4 (20 ng/ml). After 48 h, an equal number of splenocytes was added and cocultured for 48 h in flatbottom 48-well plates. These cocultures were stimulated with anti-CD3F and anti-CD28 for 24 h. Expression of CD25 (eBiosciences, San Diego, CA, USA) (a T-cell activation marker) was analyzed on CD3 þ cells by flow cytometry.24 A similar set of cells was used to analyze T-cell proliferation by methylthiazol tetrazolium assay (Promega, Madison, WI, USA) as described before.25

RNA preparation and real-time PCR See Supplementary Materials and methods.

Immunohistochemistry and immunofluorescence See Supplementary Materials and methods.

Immunoblotting See Supplementary Materials and methods.

Flow cytometry See Supplementary Materials and methods.

Statistical analysis Statistical analysis was performed with Mann–Whitney test/unpaired t-test using Graph Pad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA).

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank Robert Dickinson for technical assistance and the Rosalind Franklin University of Medicine and Science Flow Cytometry Core Facility. This work was supported by grants from the Clinical Immunology Laboratory, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA.

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2014) 5649 – 5654
