RESUMO
BACKGROUND AND PURPOSE: Hypoxia is a common feature of tumours, associated with poor prognosis due to increased resistance to radio- and chemotherapy and enhanced metastasis development. Previously we demonstrated that GABARAPL1 is required for the secretion of extracellular vesicles (EV) with pro-angiogenic properties during hypoxia. Here, we explored the role of GABARAPL1+ EV in the metastatic cascade. MATERIALS AND METHODS: GABARAPL1 deficient or control MDA-MB-231 cells were injected in murine mammary fat pads. Lungs were dissected and analysed for human cytokeratin 18. EV from control and GABARAPL1 deficient cells exposed to normoxia (21% O2) or hypoxia (O2 < 0.02%) were isolated and analysed by immunoblot, nanoparticle tracking analysis, high resolution flow cytometry, mass spectrometry and next-generation sequencing. Cellular migration and invasion were analysed using scratch assays and transwell-invasion assays, respectively. RESULTS: The number of pulmonary metastases derived from GABARAPL1 deficient tumours decreased by 84%. GABARAPL1 deficient cells migrate slower but display a comparable invasive capacity. Both normoxic and hypoxic EV contain proteins and miRNAs associated with metastasis development and, in line, increase cancer cell invasiveness. Although GABARAPL1 deficiency alters EV content, it does not alter the EV-induced increase in cancer cell invasiveness. CONCLUSION: GABARAPL1 is essential for metastasis development. This is unrelated to changes in migration and invasion and suggests that GABARAPL1 or GABARAPL1+ EV are essential in other processes related to the metastatic cascade.
Assuntos
Vesículas Extracelulares , MicroRNAs , Neoplasias , Humanos , Animais , Camundongos , Hipóxia/metabolismo , Hipóxia Celular , Vesículas Extracelulares/metabolismo , Proteínas Associadas aos Microtúbulos , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status.
Assuntos
Proteínas Quinases Ativadas por AMP , Vesículas Extracelulares , Glicólise , Neoplasias , Biossíntese de Proteínas , Serina-Treonina Quinases TOR , Humanos , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Vesículas Extracelulares/metabolismo , Neoplasias/metabolismo , Células Endoteliais/metabolismo , Glucose/metabolismo , Linhagem Celular Tumoral , Microambiente Tumoral , Células Endoteliais da Veia Umbilical Humana/metabolismoRESUMO
The second extracellular loop (LFWQYFVGKRTVPPGECFIQFLSEPTITFGTAI, aa 205-237) of muscarinic acetylcholine 3 receptor (M3R) has been reported to be an epitope for autoantibodies generated during certain autoimmune disorders, including Sjögren's syndrome (SS). Autoantibodies against M3R(228-237) have been shown to interfere with the function of M3R. However, few studies have been performed on the M3R(205-227) peptide of the second extracellular loop. In the current study, we sought to investigate the effect of M3R(208-227) peptide immunization on autoimmune response in NOD/LtJ mice. We synthesized the M3R(208-227) peptide and immunized NOD/LtJ mice to investigate whether peptide-specific antibodies could be generated and whether immunization would lead to changes in autoimmune response in NOD/LtJ mice. Our results demonstrate that the secretions of Th-1, Th-2, and Th-17 cytokines are downregulated and lymphocytic infiltration is improved in the salivary glands and lacrimal glands following immunization with M3R(208-227) peptide in NOD/LtJ mice, suggesting that peptide immunotherapy using the M3R(208-227) peptide may represent a potential therapeutic alternative.
Assuntos
Autoimunidade , Epitopos/imunologia , Peptídeos/imunologia , Receptor Muscarínico M3/imunologia , Animais , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Citocinas/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Aparelho Lacrimal/imunologia , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/patologia , Linfócitos/imunologia , Linfócitos/patologia , Camundongos , Camundongos Endogâmicos NOD , Receptor Muscarínico M3/química , Glândulas Salivares/imunologia , Glândulas Salivares/metabolismo , Glândulas Salivares/patologiaRESUMO
AIM: To evaluate whether or not the changes in the secretions of IL-17 and IFN-γ can be induced by the immunization with 2nd extracellular loop peptide of muscarinic acetylcholine 3 receptor (M3R) in NOD-scid (nonobese diabetic-severe combined immunodeficiency) mice. METHODS: We synthesized the 2nd extracellular loop peptide of M3R and immunized NOD-scid mice subcutaneously with the 1:1 mixture of the peptide and the incomplete Freund's adjuvant (IFA). At day 1, 7, 14, 21 after immunization, tail blood samples were taken to determine the antibody titer and evaluate the secretions of IL-17 and IFN-γ in sera. Meanwhile, we recorded the fluid intake amount per mouse every week. At day 21, all of the NOD-scid mice were killed to measure the concentrations of IL-17 and IFN-γ in cell supernatants. Immunofluorescence staining of lacrimal glands was performed to observe the changes in the secretions of IL-17 and IFN-γ. RESULTS: Compared with the control group, the sera titers of anti-2nd extracellular loop peptide antibodies were significantly higher in 2nd extracellular loop peptide immunized NOD-scid mice at day 14 (P<0.05). The concentrations of IL-17 and IFN-γ increased significantly in sera of the 2nd extracellular loop peptide immunized NOD-scid mice at day 7 and 14 (P<0.01). The concentration of IL-17 maintained at a certain level in the supernatants of spleen cells co-cultured with 2nd extracellular loop peptide, while it decreased significantly in the control groups (P<0.01). Immunofluorescence staining demonstrated that the production of IL-17 and IFN-γ increased in the lacrimal glands of NOD-scid mice immunized with the 2nd extracellular loop peptide. However, no changes in fluid intake was observed in NOD-scid mice immunized with the 2nd extracellular loop peptide(P>0.05). CONCLUSION: Immunization with 2nd extracellular loop peptide of M3R can induce the production of anti-2nd extracellular loop peptide antibodies and the secretions of IL-17 and IFN-γ in NOD-scid mice.