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Tumor-Derived Extracellular Vesicles Inhibit Natural Killer Cell Function in Pancreatic Cancer.

ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. Tumor-derived extracellular vesicles (EVs) induce pre-metastatic niche formation to promote metastasis. We isolated EVs from a highly-metastatic pancreatic cancer cell line and patient-derived primary cancer cells by ultracentrifugation. The protein content of EVs was analyzed by mass spectrometry. The effects of PDAC-derived EVs on natural kill (NK) cells were investigated by flow cytometry. The serum EVs' TGF-?1 levels were quantified by ELISA. We found that integrins were enriched in PDAC-derived EVs. The expression of NKG2D, CD107a, TNF-?, and INF-? in NK cells was significantly downregulated after co-culture with EVs. NK cells also exhibited decreased levels of CD71 and CD98, as well as impaired glucose uptake ability. In addition, NK cell cytotoxicity against pancreatic cancer stem cells was attenuated. Moreover, PDAC-derived EVs induced the phosphorylation of Smad2/3 in NK cells. Serum EVs' TGF-?1 was significantly increased in PDAC patients. Our findings emphasize the immunosuppressive role of PDAC-derived EVs and provide new insights into our understanding of NK cell dysfunction regarding pre-metastatic niche formation in PDAC.

SUBMITTER: Zhao J

PROVIDER: S-EPMC6628179 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Tumor-Derived Extracellular Vesicles Inhibit Natural Killer Cell Function in Pancreatic Cancer.

Zhao Jiangang J Schlößer Hans A HA Wang Zhefang Z Qin Jie J Li Jiahui J Popp Felix F Popp Marie Christine MC Alakus Hakan H Chon Seung-Hun SH Hansen Hinrich P HP Neiss Wolfram F WF Jauch Karl-Walter KW Bruns Christiane J CJ Zhao Yue Y

Cancers 20190622 6

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. Tumor-derived extracellular vesicles (EVs) induce pre-metastatic niche formation to promote metastasis. We isolated EVs from a highly-metastatic pancreatic cancer cell line and patient-derived primary cancer cells by ultracentrifugation. The protein content of EVs was analyzed by mass spectrometry. The effects of PDAC-derived EVs on natural kill (NK) cells were investigated by flow cytometry. The serum EVs' TGF-β1 le ...[more]

PMID: 31234517

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Project description:Natural killer (NK) cells contribute to immunosurveillance and first-line defense in the control of tumor growth and metastasis diffusion. NK-cell-derived extracellular vesicles (NKEVs) are constitutively secreted and biologically active. They reflect the protein and genetic repertoire of originating cells, and exert antitumor activity in vitro and in vivo. Cancer can compromise NK cell functions, a status potentially reflected by their extracellular vesicles. Hence, NKEVs could, on the one hand, contribute to improve cancer therapy by interacting with tumor and/or immune cells and on the other hand, sense the actual NK cell status in cancer patients. Here, we investigated the composition of healthy donors' NKEVs, including NK microvesicles and exosomes, and their interaction with uncompromised cells of the immune system. To sense the systemic NK cell status in cancer patients, we developed an immune enzymatic test (NKExoELISA) that measures plasma NK-cell-derived exosomes, captured as tsg101+CD56+ nanovesicles. NKEV mass spectrometry and cytokine analysis showed the expression of NK cell markers, i.e., NKG2D and CD94, perforin, granzymes, CD40L, and other molecules involved in cytotoxicity, homing, cell adhesion, and immune activation, together with EV markers tsg101, CD81, CD63, and CD9 in both NK-derived exosomes and microvesicles. Data are available via Proteome Xchange with identifier PXD014894. Immunomodulation studies revealed that NKEVs displayed main stimulatory functions in peripheral blood mononuclear cells (PBMCs), inducing the expression of human leukocyte antigen DR isotype (HLA-DR) and costimulatory molecules on monocytes and CD25 expression on T cells, which was maintained in the presence of lipopolysaccharide (LPS) and interleukin (IL)-10/transforming growth factor beta (TGF?), respectively. Furthermore, NKEVs increased the CD56+ NK cell fraction, suggesting that effects mediated by NKEVs might be potentially exploited in support of cancer therapy. The measurement of circulating NK exosomes in the plasma of melanoma patients and healthy donors evidenced lower levels of tsg101+CD56+ exosomes in patients with respect to donors. Likewise, we detected lower frequencies of NK cells in PBMCs of these patients. These data highlight the potential of NKExoELISA to sense alterations of the NK cell immune status.

Project description:Extracellular vesicles (EVs) have been the focus of great interest, as they appear to be involved in numerous important cellular processes. They deliver bioactive macromolecules such as proteins, lipids, and nucleic acids, allowing intercellular communication in multicellular organisms. EVs are secreted by all cell types, including immune cells such as natural killer cells (NK), and they may play important roles in the immune system. Currently, a large-scale procedure to obtain functional NK EVs is lacking, limiting their use clinically. In this report, we present a simple, robust, and cost-effective method to isolate a large quantity of NK EVs. After propagating and activating NK cells ex vivo and then incubating them in exosome-free medium for 48 h, EVs were isolated using a polymer precipitation method. The isolated vesicles contain the tetraspanin CD63, an EV marker, and associated proteins (fibronectin), but are devoid of cytochrome C, a cytoplasmic marker. Nanoparticle tracking analysis showed a size distribution between 100 and 200 nm while transmission electron microscopy imaging displayed vesicles with an oval shape and comparable sizes, fulfilling the definition of EV. Importantly, isolated EV fractions were cytotoxic against cancer cells. Furthermore, our results demonstrate for the first time that isolated activated NK (aNK) cell EVs contain the cytotoxic proteins perforin, granulysin, and granzymes A and B, incorporated from the aNK cells. Activation of caspase -3, -7 and -9 was detected in cancer cells incubated with aNK EVs, and caspase inhibitors blocked aNK EV-induced cytotoxicity, suggesting that aNK EVs activate caspase pathways in target cells. The ability to isolate functional aNK EVs on a large scale may lead to new clinical applications. Abbreviations: NK: natural killer cells; activated NK (aNK) cells; EVs: extracellular vesicles; ALL: acute lymphoblastic leukaemia; aAPC: artificial antigen-presenting cell; TEM: transmission electron microscope; PBMC: peripheral blood mononuclear cells; FBS: foetal bovine serum.

Dataset Information

Tumor-Derived Extracellular Vesicles Inhibit Natural Killer Cell Function in Pancreatic Cancer.

Publications

Tumor-Derived Extracellular Vesicles Inhibit Natural Killer Cell Function in Pancreatic Cancer.

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