512e4ffaf6865ca5fb24f9a17c57bf0f ijmolsci_a2021v22n7p3453.pdf 90d9c02e6199abff19103bab6f85b74199a95df1 ijmolsci_a2021v22n7p3453.pdf c4e8c19856e8c70d06e85cd5fc50c606ff23d80cc20e5830ed49e1eb144dade0 ijmolsci_a2021v22n7p3453.pdf Title: Successful Partnerships: Exploring the Potential of Immunogenic Signals Triggered by TMZ, CX-4945, and Combined Treatment in GL261 Glioblastoma Cells Subject: Background: The relevance of the cancer immune cycle in therapy response implies that successful treatment may trigger the exposure or the release of immunogenic signals. Previous results with the preclinical GL261 glioblastoma (GB) showed that combination treatment of temozolomide (TMZ) + CX-4945 (protein kinase CK2 inhibitor) outperformed single treatments, provided an immune-friendly schedule was followed. Our purpose was to study possible immunogenic signals released in vitro by GB cells. Methods: GL261 GB cells were treated with TMZ and CX-4945 at different concentrations (25 M–4 mM) and time frames (12–72 h). Cell viability was measured with Trypan Blue and propidium iodide. Calreticulin exposure was assessed with immunofluorescence, and ATP release was measured with bioluminescence. Results: TMZ showed cytostatic rather than cytotoxic effects, while CX-4945 showed remarkable cytotoxic effects already at low concentrations. Calreticulin exposure after 24 h was detected with TMZ treatment, as well as TMZ/CX-4945 low concentration combined treatment. ATP release was significantly higher with CX-4945, especially at high concentrations, as well as with TMZ/CX-4945. Conclusions: combined treatment may produce the simultaneous release of two potent immunogenic signals, which can explain the outperformance over single treatments in vivo. A word of caution may be raised since in vitro conditions are not able to mimic pharmacokinetics observed in vivo fully. Keywords: preclinical glioblastoma; cancer immune cycle; immunogenic signals; calreticulin; ATP; protein kinase CK2; temozolomide Author: Lucía Villamañan, Laura Martínez-Escardó, Carles Arús, Victor J. Yuste and Ana P. 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SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: GL261 Cell Culture Destination: subsection.2.1 Item: Title: Cell Treatments Destination: subsection.2.2 Item: Title: Cell Viability Assays Destination: subsection.2.3 Children: Item: Title: Trypan Blue Exclusion Assay Destination: subsubsection.2.3.1 Item: Title: Flow Cytometry Analysis with Propidium Iodide Destination: subsubsection.2.3.2 Item: Title: DAMPs Release/Exposure Assessment Destination: subsection.2.4 Children: Item: Title: Immunofluorescence for CRT Detection Destination: subsubsection.2.4.1 Item: Title: Bioluminescent Assay for ATP Measurement Destination: subsubsection.2.4.2 Item: Title: Statistical Analysis Destination: subsection.2.5 Item: Title: Results Destination: section.3 Children: Item: Title: Effects over GL261 Cell Viability Destination: subsection.3.1 Children: Item: Title: TMZ Single Treatment Destination: subsubsection.3.1.1 Item: Title: CX-4945 Single Treatment Destination: subsubsection.3.1.2 Item: Title: CX-4945 and TMZ Combined Treatment Destination: subsubsection.3.1.3 Item: Title: Immunogenic Cell Death Signals Triggered In Vitro under TMZ and CX-4945 Treatment Alone and in Combination Destination: subsection.3.2 Children: Item: Title: Calreticulin Exposure Destination: subsubsection.3.2.1 Item: Title: Assessment of ATP Release Destination: subsubsection.3.2.2 Item: Title: Discussion Destination: section.4 Children: Item: Title: TMZ Is Not Enough: Pursuing Better Combinations/Schedules Destination: subsection.4.1 Item: Title: Immunogenic Potential of Single and Combined Treatments and Possible Pitfalls While Interpreting Results Destination: subsection.4.2 Item: Title: Word of Caution While Interpreting the Additive Effect of Immunogenic Signals In Vitro Destination: subsection.4.3 Item: Title: In Vitro Studies Do Not Properly Mimic the In Vivo Environment Destination: subsection.4.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: Successful Partnerships: Exploring the Potential of Immunogenic Signals Triggered by TMZ, CX-4945, and Combined Treatment in GL261 Glioblastoma Cells Author: Lucía Villamañan, Laura Martínez-Escardó, Carles Arús, Victor J. Yuste and Ana P. Candiota Subject: Background: The relevance of the cancer immune cycle in therapy response implies that successful treatment may trigger the exposure or the release of immunogenic signals. Previous results with the preclinical GL261 glioblastoma (GB) showed that combination treatment of temozolomide (TMZ) + CX-4945 (protein kinase CK2 inhibitor) outperformed single treatments, provided an immune-friendly schedule was followed. Our purpose was to study possible immunogenic signals released in vitro by GB cells. Methods: GL261 GB cells were treated with TMZ and CX-4945 at different concentrations (25 M 4 mM) and time frames (12 72 h). Cell viability was measured with Trypan Blue and propidium iodide. Calreticulin exposure was assessed with immunofluorescence, and ATP release was measured with bioluminescence. Results: TMZ showed cytostatic rather than cytotoxic effects, while CX-4945 showed remarkable cytotoxic effects already at low concentrations. Calreticulin exposure after 24 h was detected with TMZ treatment, as well as TMZ/CX-4945 low concentration combined treatment. ATP release was significantly higher with CX-4945, especially at high concentrations, as well as with TMZ/CX-4945. Conclusions: combined treatment may produce the simultaneous release of two potent immunogenic signals, which can explain the outperformance over single treatments in vivo. A word of caution may be raised since in vitro conditions are not able to mimic pharmacokinetics observed in vivo fully. 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