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Archive > Volume 29 Issue 8 > 2022,29(8):714-723. DOI:10.3872/j.issn.1007-385X.2022.08.003 Prev Next
Prompt Report
A preliminary study of ATF6-mediated regulation of the immunogenicity of MCA205 osteosarcoma cells and its underlying mechanisms
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Abstract:
Objective: To investigate the impact of activating transcription factor 6 (ATF6) on the immunogenicity of osteosarcoma cells MCA205, and to preliminarily explore the underlying regulatory mechanisms. Methods: The CRISPR-Cas9 technology was utilized to knock out Atf6 gene in MCA205 cells. By using CCK-8 assays, cell energy metabolism assays, flow cytometry, ATP detection kits, interferon-sensitive response element (ISRE) -luciferase reporter cells and qPCR, we analyzed cell viability,mitochondrial oxygen consumption rate (OCR), extracellular acidification rate (ECAR), phosphatidylserine exposure and permeabilization of cell membranes, intracellular calcium mobilization, intracellular and extracellular ATP concentration, IFN-a/b secretion, and the expression of interferon-stimulated genes (ISG) in wild-type (WT) and Atf6-/- MCA205 cells after PBS or tunicamycin (Tm) treatment, respectively. WT or Atf6-/- MCA205 cells were subcutaneously inoculated in immunocompetent mice. Tumor growth kinetics, gene transcription profiles in tumor tissues and activation of local anti-tumor effector T cells of the two groups were compared.WT and Atf6-/- MCA205 cells were simultaneously inoculated on two sides of nu/nu mice. Alternatively, Atf6-/- MCA205 cells were inoculated subcutaneously in immunocompetent mice and Ifnar-/- mice. Tumor growth curves were recorded. Tm-pretreated WT and Atf6-/- MCA205 cells were used to stimulate na?ve mice (without any previous immunostimulation) and prime tumor antigen-specific T cells, respectively. Cells from draining lymph nodes were then collected and boosted in vitro. Activation of antigen-specific T cells of the two groups were compared. At different effector-target ratios, NK cells were mixed with fluorescent dye-prelabeled WT and Atf6-/-MCA205 cells. NK cell-based killing was detected by flow cytometry. Results: Upon PBS or Tm treatment, we haven’t observed any significant differences between WT and Atf6-/- tumor cells in their viability and proliferation, oxidative phosphorylation and glycolysis,ionomycin-triggered intracellular calcium mobilization, intracellular and extracellular ATP content and IFN-a/b secretion. Upon Tm treatment, the death ratio of Atf6-/- tumor cells was significantly lower than that of WT tumor cells (P<0.01). In immunocompetent mice,the growth of Atf6-/- tumors was significantly slower than that of WT tumors (P<0.05). However, their differences in growth kinetics were largely diminished in nu/nu mice. The growth of Atf6-/- tumors in Ifnar-/- mice was slightly faster than that in immunocompetent mice (P<0.05). The expression of immune response-related genes and the activation of effector T cells in Atf6-/- tumors were significantly higher than those in WT tumors (P<0.05). Compared with that of WT MCA205 cells, Atf6-/- MCA205 cells were more sensitive to NK cell-mediated cytolysis. Upon Tm preconditioning, Atf6-/- MCA205 cells expressed more Irf3 and Irf7 and can stimulate more IFN-g production from T cells in the prime-boost setting than WT cells. Conclusion: Blocking ATF6 signaling pathway can significantly enhance the immunogenicity of MCA205 cells, promote immune surveillance and delay tumor progression.
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