American Association for Cancer Research
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supplementary video 2 from Tumor-Produced Interleukin-8 Attracts Human Myeloid-Derived Suppressor Cells and Elicits Extrusion of Neutrophil Extracellular Traps (NETs)

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posted on 2023-03-31, 18:27 authored by Carlos Alfaro, Alvaro Teijeira, Carmen Oñate, Guiomar Pérez, Miguel F. Sanmamed, Maria Pilar Andueza, Diego Alignani, Sara Labiano, Arantza Azpilikueta, Alfonso Rodriguez-Paulete, Saray Garasa, Juan P. Fusco, Angela Aznar, Susana Inogés, Maria De Pizzol, Marcello Allegretti, Jose Medina-Echeverz, Pedro Berraondo, Jose L. Perez-Gracia, Ignacio Melero

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.



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.

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