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Supplementary figure 1. Comparative spleen size in tumor-free mice and mice engrafted with CT26-GMCSF tumors for 21 days. Photographs taken in parallel upon necropsy of the indicated group of mice.

Purpose: Interleukin-8 (IL8) is a chemokine produced by malignant cells of multiple cancer types. It exerts various functions in shaping protumoral vascularization and inflammation/immunity. We evaluated sequential levels of serum IL8 in preclinical tumor models and in patients to assess its ability to estimate tumor burden.Experimental Design: IL8 levels were monitored by sandwich ELISAs in cultured tumor cells supernatants, tumor-xenografted mice serum, and in samples from 126 patients with cancer. We correlated IL8 serum levels with baseline tumor burden and with treatment-induced changes in tumor burden, as well as with prognosis.Results: IL8 concentrations correlated with the number of IL8-producing tumor cells in culture. In xenografted neoplasms, IL8 serum levels rapidly dropped after surgical excision, indicating an accurate correlation with tumor burden. In patients with melanoma (n = 16), renal cell carcinoma (RCC; n = 23), non–small cell lung cancer (NSCLC; n = 21), or hepatocellular carcinoma (HCC; n = 30), serum IL8 concentrations correlated with tumor burden and stage, survival (melanoma, n = 16; RCC, n = 23; HCC, n = 33), and objective responses to therapy, including those to BRAF inhibitors (melanoma, n = 16) and immunomodulatory monoclonal antibodies (melanoma, n = 8). IL8 concentrations in urine (n = 18) were mainly elevated in tumors with direct contact with the urinary tract.Conclusions: IL8 levels correlate with tumor burden in preclinical models and in patients with cancer. IL8 is a potentially useful biomarker to monitor changes in tumor burden following anticancer therapy, and has prognostic significance. Clin Cancer Res; 20(22); 5697–707. ©2014 AACR.

Supplementary figure 2. IL-8 hydrodynamic gene transfer effects on tumor free mice. Hydrodynamic gene transfer experiments as in figure 2 but performed in tumor-free mice. Results show the content of the indicated cell subsets in the liver and spleen of euthanized mice.

Supplementary Figure 4. Comparison of IL-8 serum concentrations with the serum concentration of IL-6 and c-reactive protein (CRP) in available samples from 14 melanoma and 19 RCC patients from Figure 2A. (**, p

Supplementary Figure 1B. Time course of the serum concentrations of IL-8 (lower panels) and tumor volume (upper panels) in Rag2-/-IL2Rγc-/- mice xenografted with the indicated cell lines. Individual mice are represented and at the time point indicated by the arrow the tumor was surgically removed. Serum IL-8 concentrations are depicted at 2h and 24h after surgery.

Supplementary Figure 2. Serum IL-8 levels before (day -1) and after (day 5-7) disease-reduction surgery of 7 patients: 1 hepatocellular carcinoma (HCC); 2 colorectal carcinoma (CRC); 1 pancreatic cancer (PC); 3 renal cell carcinoma (RCC).

Supplementary figure 3. Number of MDSC in peripheral blood of patients using osmotic lysis of erythrocytes instead of samples from ficoll-gradient centrifugation. Experiments as those shown in figure 3B representing the percentage of positively stained cells analyzed by FACS merely following osmotic lysis of erythrocytes instead of Ficoll gradient enrichment.

Supplementary video 2. IL-8 induces NET extrusion of GrMDSCs to entrap or adhere HT29 tumor cells. GrMDSCs nuclei were pre-stained with DRAQ5, (cyan), HT29 cells with PKH26 cell membrane dye (Red seen intracellular vesicles) and treated or not with IL-8. The non-cell permeant DNA probe (SYTOXgreen/green) is present in the culture media and only stains DNA when it is extruded extracelullarly. Image on the left shows the complete field of view with several GrMDSCs and an inset of an individual GrMDSC interacting with tumor cells but not extruding NETs since no IL-8 was added to the medium. Image on the left shows the complete microscopy field with several GrMDSCs and an inset with three GrMDSC contacting with HT29 cells and entrapping or adhering to them by formation of the NETs induced upon addition of IL-8. Video is a maximum intensity projection of a Z stack, (DiC is shown for contrast). Video is 300x accelerated.

Supplementary video 1. IL-8 induces NET extrusion of GrMDSCs using time-lapse in vivo confocal fluorescence microsocopy. GrMDSCs nuclei were pre-stained with DRAQ5 (Red) and treated with IL-8. The non-cell permeant green DNA probe (SYTOXgreen) is present in the culture media and only stains DNA when is extruded extracelullarly. Video represent over five hours a maximum intensity projection of a Zstack. Video is 600x accelerated

Supplementary figure 6. IL-8 induction of NETs on GrMDSC is dose dependent, similar in intensity to that induced by LPS and can be blocked by IL-8 neutralizing antibodies. (A and B) Experiments as those in figure 6 showing the indicated conditions NET formation induction by LPS and IL-8. (C) GrMDSC induce to extrude NETs by IL-8 or LPS but cocultured with PKH26-labelled (red) HT 29 tumor cells.

Purpose: Myeloid-derived suppressor cells (MDSC) are considered an important T-cell immunosuppressive component in cancer-bearing hosts. The factors that attract these cells to the tumor microenvironment are poorly understood. IL8 (CXCL8) is a potent chemotactic factor for neutrophils and monocytes.Experimental Design: MDSC were characterized and sorted by multicolor flow cytometry on ficoll-gradient isolated blood leucokytes from healthy volunteers (n = 10) and advanced cancer patients (n = 28). In chemotaxis assays, sorted granulocytic and monocytic MDSC were tested in response to recombinant IL8, IL8 derived from cancer cell lines, and patient sera. Neutrophil extracellular traps (NETs) formation was assessed by confocal microscopy, fluorimetry, and time-lapse fluorescence confocal microscopy on short-term MDSC cultures.Results: IL8 chemoattracts both granulocytic (GrMDSC) and monocytic (MoMDSC) human MDSC. Monocytic but not granulocytic MDSC exerted a suppressor activity on the proliferation of autologous T cells isolated from the circulation of cancer patients. IL8 did not modify the T-cell suppressor activity of human MDSC. However, IL8 induced the formation of NETs in the GrMDSC subset.Conclusions: IL8 derived from tumors contributes to the chemotactic recruitment of MDSC and to their functional control. Clin Cancer Res; 22(15); 3924–36. ©2016 AACR.

Supplementary Figure 3. Quantitative RT-PCR analyses of the mRNA encoding IL-8 in malignant cells from the biopsy of three melanoma patients. The serum IL-8 levels at the time of the biopsy from each patient is provided under the graph.

Supplementary Table 2. Surgical patient description 2

Supplemental Legend

Supplementary figure 5. Suppressive activity of MoMDSC on autologous purified CD4 and CD8 T cells. Experiments as in figure 5 but in this case immunomagnetically purified T cells by negative selection were CFSE-labeled co-cultured as indicated. T lymphocytes were differentially analyzed for proliferation in FACS-gated CD4 or CD8 T cell subsets. Representative histograms of one experiment out of two performed are represented.

Supplementary Table 1. Patient description 1

Supplementary figure 4. MDSC Chemotaxis induction by autologous patient serum. chemotactic migration of the indicated MDSC subsets or dextran-purified PMN from cancer patients in response to autologous serum or serum from healthy volunteers. Statistical comparisons were made by U-Mann-Whitney tests.

Purpose: Myeloid-derived suppressor cells (MDSC) are considered an important T-cell immunosuppressive component in cancer-bearing hosts. The factors that attract these cells to the tumor microenvironment are poorly understood. IL8 (CXCL8) is a potent chemotactic factor for neutrophils and monocytes. Experimental Design: MDSC were characterized and sorted by multicolor flow cytometry on ficoll-gradient isolated blood leucokytes from healthy volunteers (n = 10) and advanced cancer patients (n = 28). In chemotaxis assays, sorted granulocytic and monocytic MDSC were tested in response to recombinant IL8, IL8 derived from cancer cell lines, and patient sera. Neutrophil extracellular traps (NETs) formation was assessed by confocal microscopy, fluorimetry, and time-lapse fluorescence confocal microscopy on short-term MDSC cultures. Results: IL8 chemoattracts both granulocytic (GrMDSC) and monocytic (MoMDSC) human MDSC. Monocytic but not granulocytic MDSC exerted a suppressor activity on the proliferation of autologous T cells isolated from the circulation of cancer patients. IL8 did not modify the T-cell suppressor activity of human MDSC. However, IL8 induced the formation of NETs in the GrMDSC subset. Conclusions: IL8 derived from tumors contributes to the chemotactic recruitment of MDSC and to their functional control. Clin Cancer Res; 22(15); 3924–36. ©2016 AACR.

Purpose: Interleukin-8 (IL8) is a chemokine produced by malignant cells of multiple cancer types. It exerts various functions in shaping protumoral vascularization and inflammation/immunity. We evaluated sequential levels of serum IL8 in preclinical tumor models and in patients to assess its ability to estimate tumor burden. Experimental Design: IL8 levelsweremonitored by sandwich ELISAs incultured tumor cells supernatants, tumor-xenograftedmice serum, and in samples from126 patients with cancer. Wecorrelated IL8 serumlevels with baseline tumor burden and with treatment-induced changes in tumor burden, as well as with prognosis. Results: IL8 concentrations correlated with the number of IL8-producing tumor cells in culture. In xenografted neoplasms, IL8 serum levels rapidly dropped after surgical excision, indicating an accurate correlation with tumor burden. In patients with melanoma (n = 16), renal cell carcinoma (RCC; n = 23), non-small cell lung cancer (NSCLC; n = 21), or hepatocellular carcinoma (HCC; n = 30), serum IL8 concentrations correlated with tumor burden and stage, survival (melanoma, n = 16; RCC, n = 23; HCC, n = 33), and objective responses to therapy, including those to BRAF inhibitors (melanoma, n = 16) and immunomodulatory monoclonal antibodies (melanoma, n = 8). IL8 concentrations in urine (n = 18) were mainly elevated in tumors with direct contact with the urinary tract. Conclusions: IL8 levels correlate with tumor burden in preclinical models and in patients with cancer. IL8 is a potentially useful biomarker to monitor changes in tumor burden following anticancer therapy, and has prognostic significance. Fil: Sanmamed, Miguel F.. Universidad de Navarra; España Fil: Carranza Rua, Omar. Universidad de Navarra; España Fil: Alfaro, Carlos. Universidad de Navarra; España Fil: Oñate, Carmen. Universidad de Navarra; España Fil: Martín Algarra, Salvador. Universidad de Navarra; España Fil: Perez, Guiomar. Universidad de Navarra; España Fil: Landazuri, Sara F.. Universidad de Navarra; España Fil: Gonzalez, Alvaro. Universidad de Navarra; España Fil: Gross, Stefanie. University Hospital Erlangen; Alemania Fil: Rodriguez, Inmaculada. Universidad de Navarra; España Fil: Muñoz Calleja, Cecilia. Universidad Autonoma de Madrid. Hospital Universitario de la Princesa; España Fil: Rodríguez Ruiz, María. Universidad de Navarra; España Fil: Sangro, Bruno. Universidad de Navarra; España Fil: López Picazo, José M.. Universidad de Navarra; España Fil: Rizzo, Manglio Miguel. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mazzolini Rizzo, Guillermo Daniel. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pascual, Juan I.. Universidad de Navarra; España Fil: Andueza, Maria Pilar. Universidad de Navarra; España Fil: Perez Gracia, Jose Luis. Universidad de Navarra; España Fil: Melero, Ignacio. Universidad de Navarra; España