Published OnlineFirst March 8, 2016; DOI: 10.1158/1078-0432.CCR-15-2463 Biology of Human Tumors Clinical Cancer Research Tumor-Produced Interleukin-8 Attracts Human Myeloid-Derived Suppressor Cells and Elicits Extrusion of Neutrophil Extracellular Traps (NETs) Carlos Alfaro1,2,3, Alvaro Teijeira1,2,3, Carmen Onate~ 1,2,3, Guiomar Perez 1,2,3, Miguel F. Sanmamed2,3, Maria Pilar Andueza2,3, Diego Alignani4, Sara Labiano1, Arantza Azpilikueta1, Alfonso Rodriguez-Paulete1, Saray Garasa1, Juan P. Fusco2,3, Angela Aznar1, Susana Inoges 2,3, Maria De Pizzol5, Marcello Allegretti5, Jose Medina-Echeverz1, Pedro Berraondo1, Jose L. Perez-Gracia2,3, and Ignacio Melero1,2,3 Abstract Purpose: Myeloid-derived suppressor cells (MDSC) are con- microscopy, fluorimetry, and time-lapse fluorescence confocal sidered an important T-cell immunosuppressive component in microscopy on short-term MDSC cultures. cancer-bearing hosts. The factors that attract these cells to the Results: IL8 chemoattracts both granulocytic (GrMDSC) and tumor microenvironment are poorly understood. IL8 (CXCL8) is monocytic (MoMDSC) human MDSC. Monocytic but not gran- a potent chemotactic factor for neutrophils and monocytes. ulocytic MDSC exerted a suppressor activity on the proliferation Experimental Design: MDSC were characterized and sorted by of autologous T cells isolated from the circulation of cancer multicolor flow cytometry on ficoll-gradient isolated blood leu- patients. IL8 did not modify the T-cell suppressor activity of cokytes from healthy volunteers (n ¼ 10) and advanced cancer human MDSC. However, IL8 induced the formation of NETs in patients (n ¼ 28). In chemotaxis assays, sorted granulocytic and the GrMDSC subset. monocytic MDSC were tested in response to recombinant IL8, IL8 Conclusion: IL8 derived from tumors contributes to the che- derived from cancer cell lines, and patient sera. Neutrophil motactic recruitment of MDSC and to their functional control. extracellular traps (NETs) formation was assessed by confocal Clin Cancer Res; 1–13. Ó2016 AACR. Introduction genic activities by means of providing growth factors (12, 13), upregulating angiogenesis (2, 4, 7, 14), and suppressing and IL8 (CXCL8) is a chemokine originally discovered for its biasing antitumor immune responses (13, 15). In mice, a subset ability to attract neutrophils to inflammatory foci (1). In cancer of myeloid cells suppressing T-cell proliferation and activation its role in promoting angiogenesis is well characterized. IL8 accumulates in the spleen and tumor microenvironment of does not only attract neutrophilsbutalsomonocytesto tumor-bearing hosts (16). This population-termed myeloid- inflamed tissues (2). Tumor cells constitutively produce IL8 derived suppressor cells (MDSC) is characterized by surface (2–9) and hence plasma concentrations of IL8 reflect tumor coexpression of CD11b and Ly6 (16). Its functions cause sup- burden (2) and determine prognosis (2–9). IL8 is functional on pression of T-cell immunity by a variety of metabolic actions (17), CXCR1 and CXCR2 where it induces chemotactic responses. which ultimately repress the proliferation of T lymphocytes. There is no IL8 analogue in the mouse genome (10, 11), but IL8 Therefore, reductions in the numbers of such myeloid cells is functional on murine receptors (11). enhance therapeutic immune responses (18, 19) and have yielded Little is known about the function of IL8 to attract leukocytes in a therapeutic benefit in several tumor models. Two subsets of cancer. However, myeloid leukocytes are involved in protumoro- MDSC have been described based on selective expression of Ly6C þ and Ly6G (16, 20). Morphologically, Ly6C MDSC resemble þ 1Division of Gene Therapy and Hepatology, Centre for Applied Medical monocytes, whereas Ly6G MDSC share morphological features Research (CIMA), Pamplona, Spain. 2Department of Oncology, Uni- with polymorphonuclear (PMN) leukocytes. versity Clinic of Navarra, Pamplona, Spain. 3Department of Immunol- In the peripheral blood of human cancer patients, several ogy, University Clinic of Navarra, Pamplona, Spain. 4Cytometry Plat- fi form, Centre for Applied Medical Research (CIMA), Pamplona, Spain. investigators have de ned myeloid subsets that now also receive 5Dompé Farmaceutici SpA, L'Aquila, Italy. the name of MDSCs (21–25) even if their suppressive activity on Note: Supplementary data for this article are available at Clinical Cancer the activation and proliferation of T lymphocytes is not well fl Research Online (http://clincancerres.aacrjournals.org/). studied. Using ow cytometry multiple staining on peripheral blood leukocytes recovered from ficoll gradients permits the J.L. Perez-Gracia and I. Melero share equal credit for senior authorship. identification of human monocytic MDSC that are defined as þ þ À þ Corresponding Author: Ignacio Melero, Avda.Pio XII, 55, Pamplona 31008, CD33 CD11b HLA-DR /low CD14 , whereas granulocytic Spain. Phone: 349-4819-4700; Fax: 349-4819-4717; E-mail: [email protected] þ þ À þ MDSC are defined as CD33 CD11b HLA-DR /low CD15 (25). doi: 10.1158/1078-0432.CCR-15-2463 The numbers of both populations rise in advanced cancer Ó2016 American Association for Cancer Research. patients and several articles have reported a correlation www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst March 8, 2016; DOI: 10.1158/1078-0432.CCR-15-2463 Alfaro et al. clinics. Patients provided informed consent under a protocol Translational Relevance approved by the institutional ethics committee. The cell suspen- IL8 is a chemokine produced by cancer cells and whose sion was laid over 15 mL of Ficoll-Hypaque (GE Healthcare serum concentration correlates with poor prognosis. In this Europe GMBH) gradients in 50 mL tubes and centrifuged at study, we demonstrate that IL8 chemotactically attracts 2,200 rpm for 20 minutes at 20C. The leukocyte layer was myeloid-derived suppressor cells (MDSC) sorted from the resuspended in 5 mL ice-cold PBS. Subsequently, cells were peripheral blood of advanced cancer patients and that this incubated with PBS/human IgG (50 mg/mL; Beriglobina P; Behr- chemotactic activity can be pharmacologically disrupted in ing) for 10 minutes on ice, to block Fc receptors and then aliquots vivo. Surprisingly, we also found that IL8 activates granulo- of cells (106) were washed in ice-cold PBS and incubated 15 min- cytic MDSC to extrude DNA-forming neutrophil extracellu- utes at 4C with specific labeled mAb: Lin [CD3 PE (Biolegend), lar traps (NET). These IL8-mediated mechanisms are likely CD16 PE (Biolegend), CD19 PE (BD Biosciences), CD20 PE to be relevant in shaping a protumoral leukocyte microen- (Biolegend), CD56 PE (BD Biosciences)], HLA-DR APC (Biole- vironment in cancer. gend), CD11b PeCy5 (IoTest), CD33 FITC (Biolegend), CD14 BV421 (Biolegend), and CD15 BV510 (Biolegend). Immediately after, FACS sorting was performed using a BD FACSAria III with the gating strategy shown in Fig. 1A. For surface expression of CXCR1 and CXCR2 in human and mouse MDSC, the following between higher levels and worse prognosis (21, 26–28). We antibodies were used: Lin PE, CD11b PeCy5 (IoTest), CD33 have previously reported that IL8 attracts dendritic cells and BV421 (Biolegend), HLA-DR APC Cy7 (Biolegend), CD14 PeCy7 discovered that tumors producing IL8 were chemotactically (Biolegend), CD15 BV510 (Biolegend), human CXCR1 FITC retaining dendritic cells injected intratumorally, thereby avoid- (Biolegend), human CXCR2 AF647 (Biolegend), mouse CXCR1 ing their outmigration to draining lymph nodes (10, 29). In FITC (Biorbyt), and mouse CXCR2 APC (R&D Systems). For this study, we explored if IL8 could attract and functionally characterization the mouse MDSC, obtained from the spleen, the modulate MDSC. To this end, human MoMDSC and GrMDSC following antibodies were used: CD45.2 Pacific Blue (Biolegend), were identified and FACS-sorted from the peripheral blood of a CD11b APC (BD Biosciences), CD124 PE (BD Biosciences), Ly6G series of advanced cancer patients. It was found that IL8 readily BV510 (Biolegend), and Ly6C PerCP Cy5.5 (Biolegend). Samples attracts all these cell subsets, although it does not regulate their were analyzed using a FACSCanto flow cytometer (BD Bios- T-cell suppressive activity. However, IL8 induces the formation ciences) and data were analyzed with the FlowJo (FlowJo, TreeStar of neutrophil extracellular traps (NET) with projected DNA as Inc.) software. seen in human and mouse PMNs (30–32). Such NETs made of For neutrophil enrichment, 10 mL of peripheral blood DNA and DNA-binding proteins are known to have important drawn in heparin-containing tubes were mixed with 10 mL of bacteriostatic and bactericidal functions (32–35) and have cold PBS and 10 mL of 6% dextran/0.9% NaCl solution. After recently been discovered to play a role in autoimmunity being inverted 18 to 20 times to ensure adequate mixing, the (36) and in organizing metastatic emboli in cancer (37). mixture was pipetted into a 50 mL tube and the tube was placed Collectively, our experiments demonstrate that tumor-pro- upright at room temperature for 1 to 1.5 hours, or until duced IL8 chemoattracts and modulates human and mouse MDSC separation was completed. The yellowish supernatant was in a fashion that can be interfered with in vivo using the CXCR1/2 recovered into a 50 mL tube and spun at 1,200 rpm for 12 blocking agent reparixin, that are under clinical development in minutes at 4C with low brake. The supernatant was then oncology (38, 39). Given the protumorogenic immunosuppres- discarded and the pellet resuspended in 5 mL ACK buffer, sive function of these myeloid subsets, the IL8–MDSC axis offers 5 minutes at room temperature. After that incubation, the cells an attractive target for cancer immunotherapy strategies. were washed with 45 mL of cold PBS. The supernatant was discarded and the pellet was resuspended in 2.5 mL of PBS. The Materials and Methods cell suspension was laid over 3 mL of Ficoll-Hypaque (GE Healthcare Europe GMBH) gradients in a 15 mL tube and spun Cell lines and tumor grafting at 1500 rpm for 30 minutes at 4C.