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Normoxic Tumour Extracellular Vesicles Modulate the Response of Hypoxic Cancer and Stromal Cells to Doxorubicin In Vitro.

ABSTRACT: Extracellular vesicles (EV) secreted in the tumour microenvironment (TME) are emerging as major antagonists of anticancer therapies by orchestrating the therapeutic outcome through altering the behaviour of recipient cells. Recent evidence suggested that chemotherapeutic drugs could be responsible for the EV-mediated tumour-stroma crosstalk associated with cancer cell drug resistance. Here, we investigated the capacity of tumour EV (TEV) secreted by normoxic and hypoxic (1% oxygen) C26 cancer cells after doxorubicin (DOX) treatment to alter the response of naïve C26 cells and RAW 264.7 macrophages to DOX. We observed that C26 cells were less responsive to DOX treatment under normoxia compared to hypoxia, and a minimally cytotoxic DOX concentration that mounted distinct effects on cell viability was selected for TEV harvesting. Homotypic and heterotypic pretreatment of naïve hypoxic cancer and macrophage-like cells with normoxic DOX-elicited TEV rendered these cells slightly less responsive to DOX treatment. The observed effects were associated with strong hypoxia-inducible factor 1-alpha (HIF-1?) induction and B-cell lymphoma-extra-large anti-apoptotic protein (Bcl-xL)-mediated anti-apoptotic response in normoxic DOX-treated TEV donor cells, being also tightly connected to the DOX-TEV-mediated HIF-1? induction, as well as Bcl-xL levels increasing in recipient cells. Altogether, our results could open new perspectives for investigating the role of chemotherapy-elicited TEV in the colorectal cancer TME and their modulatory actions on promoting drug resistance.

SUBMITTER: Patras L

PROVIDER: S-EPMC7503554 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Normoxic Tumour Extracellular Vesicles Modulate the Response of Hypoxic Cancer and Stromal Cells to Doxorubicin In Vitro.

Patras Laura L Fens Marcel H A M MHAM Vader Pieter P Barendrecht Arjan A Sesarman Alina A Banciu Manuela M Schiffelers Raymond R

International journal of molecular sciences 20200819 17

Extracellular vesicles (EV) secreted in the tumour microenvironment (TME) are emerging as major antagonists of anticancer therapies by orchestrating the therapeutic outcome through altering the behaviour of recipient cells. Recent evidence suggested that chemotherapeutic drugs could be responsible for the EV-mediated tumour-stroma crosstalk associated with cancer cell drug resistance. Here, we investigated the capacity of tumour EV (TEV) secreted by normoxic and hypoxic (1% oxygen) C26 cancer ce ...[more]

PMID: 32824972

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Project description:BackgroundHuman mesenchymal stromal cell (MSC)-derived extracellular vesicles (EV) revealed neuroprotective potentials in various brain injury models, including neonatal encephalopathy caused by hypoxia-ischemia (HI). However, for clinical translation of an MSC-EV therapy, scaled manufacturing strategies are required, which is challenging with primary MSCs due to inter- and intra-donor heterogeneities. Therefore, we established a clonally expanded and immortalized human MSC line (ciMSC) and compared the neuroprotective potential of their EVs with EVs from primary MSCs in a murine model of HI-induced brain injury. In vivo activities of ciMSC-EVs were comprehensively characterized according to their proposed multimodal mechanisms of action.MethodsNine-day-old C57BL/6 mice were exposed to HI followed by repetitive intranasal delivery of primary MSC-EVs or ciMSC-EVs 1, 3, and 5 days after HI. Sham-operated animals served as healthy controls. To compare neuroprotective effects of both EV preparations, total and regional brain atrophy was assessed by cresyl-violet-staining 7 days after HI. Immunohistochemistry, western blot, and real-time PCR were performed to investigate neuroinflammatory and regenerative processes. The amount of peripheral inflammatory mediators was evaluated by multiplex analyses in serum samples.ResultsIntranasal delivery of ciMSC-EVs and primary MSC-EVs comparably protected neonatal mice from HI-induced brain tissue atrophy. Mechanistically, ciMSC-EV application reduced microglia activation and astrogliosis, endothelial activation, and leukocyte infiltration. These effects were associated with a downregulation of the pro-inflammatory cytokine IL-1 beta and an elevated expression of the anti-inflammatory cytokines IL-4 and TGF-beta in the brain, while concentrations of cytokines in the peripheral blood were not affected. ciMSC-EV-mediated anti-inflammatory effects in the brain were accompanied by an increased neural progenitor and endothelial cell proliferation, oligodendrocyte maturation, and neurotrophic growth factor expression.ConclusionOur data demonstrate that ciMSC-EVs conserve neuroprotective effects of primary MSC-EVs via inhibition of neuroinflammation and promotion of neuroregeneration. Since ciMSCs can overcome challenges associated with MSC heterogeneity, they appear as an ideal cell source for the scaled manufacturing of EV-based therapeutics to treat neonatal and possibly also adult brain injury.

Project description:Extracellular vesicles (EVs) mediate cell-to-cell communication by horizontally transferring biological materials from host cells to target cells. During exposure to pathogens, pathogen-associated molecular patterns (e.g., lipopolysaccharide, LPS) get in contact with endothelial cells and stimulate the secretion of endothelial cell-derived EVs (E-EVs). The triggered EVs secretion is known to have a modulating influence on the EVs-receiving cells. Macrophages, a major component of innate immunity, are polarized upon receiving external inflammatory stimuli, in which toll-like receptor4 (TLR4)-nuclear factor kappa B (NFκB) pathway plays a key role. However, the functions of LPS-induced E-EVs (ELPS-EVs) in modulating macrophage phenotype and activation remain elusive. We collected the EVs from quiescent endothelial cells (ENor-EVs) and ELPS-EVs to detect their stimulatory role on NR8383 macrophages. Isolated EVs were characterized by transmission electron microscopy (TEM), western blot assay, and nanoparticle tracking analysis (NTA). NR8383 macrophages were stimulated with ELPS-EVs, ENor-EVs, or PBS for 24 h. Hereafter, the uptake of EVs by the macrophages was investigated. Upon EVs stimulation, cellular viability was determined by MTT assay, while macrophage phenotype was analyzed by flow cytometry and immunofluorescence analysis. Furthermore, a western blot assay was conducted to evaluate the potentially involved TLR4-NFκB pathway. Interestingly, upon exposure to LPS, endothelial cells secreted significantly higher amounts of EVs (i.e., ELPS-EVs) when compared to quiescent cells or cells in PBS. The ELPS-EVs were also better internalized by NR8383 macrophages than ENor-EVs. The cellular viability of ELPS-EVs-treated macrophages was 1.2 times higher than those in the ENor-EVs and PBS groups. In addition, ELPS-EVs modulated NR8383 macrophages towards a proinflammatory macrophage M1-like phenotype. This was indicated by the significantly upregulated expressions of proinflammatory macrophage biomarkers CD86 and inducible nitric oxide synthase (iNOS) observed in ELPS-EVs-treated macrophages. The TLR4-NFκB signaling pathway was substantially activated in ELPS-EVs-treated macrophages, indicated by the elevated expressions of makers TLR4 and phosphorylated form of nuclear factor kappa B p65 subunit (p-NFκBp65). Overall, our results indicate that E-EVs play a crucial role in macrophage phenotype modulation under inflammatory conditions.

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Normoxic Tumour Extracellular Vesicles Modulate the Response of Hypoxic Cancer and Stromal Cells to Doxorubicin In Vitro.

Publications

Normoxic Tumour Extracellular Vesicles Modulate the Response of Hypoxic Cancer and Stromal Cells to Doxorubicin In Vitro.

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