1a98708ace8abf30f5398e351130d333 ijms-24-17628-v2_2_.pdf a8ee266cbe0539bdf890bcbf8b6e74cfb868df67 ijms-24-17628-v2_2_.pdf 1c83fb43a385f42557ea673b98c7f9f10bbab61da76346c5d8ece81297ce6a82 ijms-24-17628-v2_2_.pdf Title: Correlation of MR-Based Metabolomics and Molecular Profiling in the Tumor Microenvironment of Temozolomide-Treated Orthotopic GL261 Glioblastoma in Mice Subject: The tumor microenvironment in glioblastoma (GB) is considered to be “cold”, i.e., the fraction of cytotoxic T cells, for instance, is low. Instead, macrophages are the major immune cell population in GB, which stem either from tissue response (resident microglia) or recruitment of macrophages from the periphery, thereby undergoing tumor-dependent “imprinting” mechanisms by which macrophages can adapt a tumor-supportive phenotype. In this regard, it is important to describe the nature of macrophages associated with GB, in particular under therapy conditions using the gold standard chemotherapy drug temozolomide (TMZ). Here, we explored the suitability of combining information from in vivo magnetic resonance spectroscopic (MRS) approaches (metabolomics) with in vitro molecular analyses to assess therapy response and characterize macrophage populations in mouse GB using an isogenic GL261 model. For macrophage profiling, expression levels of matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs) were determined, since their gene products affect macrophage–tumor cell communication by extensive cleavage of immunomodulatory membrane proteins, such as PD-L1. In tumor mice with an overall therapy response, expression of genes encoding the proteases ADAM8, ADAM10, and ADAM17 was increased and might contribute to the immunosuppressive phenotype of GB and immune cells. In tumors responding to therapy, expression levels of ADAM8 were upregulated by TMZ, and higher levels of PD-L1 were correlated significantly. Using a CRISPR/Cas9 knockout of ADAM8 in GL261 cells, we demonstrated that soluble PD-L1 (sPD-L1) is only generated in the presence of ADAM8. Moreover, primary macrophages from WT and ADAM8-deficient mice showed ADAM8-dependent release of sPD-L1, independent of the macrophage polarization state. Since ADAM8 expression is induced in responding tumors and PD-L1 shedding is likely to decrease the anti-tumor activities of T-cells, we conclude that immunotherapy resistance is caused, at least in part, by the increased presence of proteases, such as ADAM8. Keywords: glioblastoma; MR spectroscopic imaging; temozolomide; therapy; metalloproteases; PD-L1; shedding; macrophages Author: Kai Zhao, Pilar Calero-Pérez, Miriam H. A. Bopp, Vincent Möschl, Axel Pagenstecher, Marta Mulero-Acevedo, Mario Vázquez, Carlos Barcia, Carles Arús, Christopher Nimsky, Tillmann Rusch, Jörg W. 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1.12.4 (2023-03-16) LastModified: 2024-05-16 11:51:37 CEST Size: 4900496 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 896 FreeObjects: 37 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Item: Title: Discussion Destination: section.3 Children: Item: Title: Microglia/Macrophage Populations Present Changes in Their Infiltration Behavior and Phenotypic Characteristics during Successful Response to IMS-TMZ Treatment Destination: subsection.3.1 Item: Title: Carbonic Anhydrase Results Suggest That Unresponsive Tumors Could Overexpress This Gene Destination: subsection.3.2 Item: Title: Changes in the Expression of Metalloproteases Can Shape TME, and Relevant Differences Are Found during Response to IMS-TMZ Destination: subsection.3.3 Item: Title: Correlation between the Expression Levels of Different Genes Showed Unexpected Links Destination: subsection.3.4 Item: Title: Microenvironment Changes during Sustained Response to IMS-TMZ Were Translated into Metabolomic Changes Noninvasively Assessed with MRSI Destination: subsection.3.5 Item: Title: A New Therapeutic Target Emerges from the ADAM8/PD-L1 Relationship Destination: subsection.3.6 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: GL261 Cell Culture Destination: subsection.4.1 Item: Title: Preclinical GL261 GB Model and IMS-TMZ Treatment Destination: subsection.4.2 Item: Title: MR Data Acquisition (MRI and MRSI), Processing, and Postprocessing, and Murine Tumor Sample Collection Destination: subsection.4.3 Item: Title: RNA Isolation, cDNA Synthesis, and RT-PCR from Mouse Brain Tissue Destination: subsection.4.4 Item: Title: Construction of Stable GL261 ADAM8 Knockout Cells Destination: subsection.4.5 Item: Title: RNA Extraction, cDNA Transcription, and RT-PCR from GL261 Cells Destination: subsection.4.6 Item: Title: ELISA Measurements Destination: subsection.4.7 Item: Title: In Vitro Polarization of Macrophages Destination: subsection.4.8 Item: Title: Immunofluorescence Staining Destination: subsection.4.9 Item: Title: Statistics, Algorithms, and Tools Used for Data Analyses Destination: subsection.4.10 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.A. Info: Title: Correlation of MR-Based Metabolomics and Molecular Profiling in the Tumor Microenvironment of Temozolomide-Treated Orthotopic GL261 Glioblastoma in Mice Author: Kai Zhao, Pilar Calero-Pérez, Miriam H. A. Bopp, Vincent Möschl, Axel Pagenstecher, Marta Mulero-Acevedo, Mario Vázquez, Carlos Barcia, Carles Arús, Christopher Nimsky, Tillmann Rusch, Jörg W. Bartsch and Ana Paula Candiota Subject: The tumor microenvironment in glioblastoma (GB) is considered to be “cold”, i.e., the fraction of cytotoxic T cells, for instance, is low. Instead, macrophages are the major immune cell population in GB, which stem either from tissue response (resident microglia) or recruitment of macrophages from the periphery, thereby undergoing tumor-dependent “imprinting” mechanisms by which macrophages can adapt a tumor-supportive phenotype. In this regard, it is important to describe the nature of macrophages associated with GB, in particular under therapy conditions using the gold standard chemotherapy drug temozolomide (TMZ). Here, we explored the suitability of combining information from in vivo magnetic resonance spectroscopic (MRS) approaches (metabolomics) with in vitro molecular analyses to assess therapy response and characterize macrophage populations in mouse GB using an isogenic GL261 model. For macrophage profiling, expression levels of matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs) were determined, since their gene products affect macrophage tumor cell communication by extensive cleavage of immunomodulatory membrane proteins, such as PD-L1. In tumor mice with an overall therapy response, expression of genes encoding the proteases ADAM8, ADAM10, and ADAM17 was increased and might contribute to the immunosuppressive phenotype of GB and immune cells. In tumors responding to therapy, expression levels of ADAM8 were upregulated by TMZ, and higher levels of PD-L1 were correlated significantly. Using a CRISPR/Cas9 knockout of ADAM8 in GL261 cells, we demonstrated that soluble PD-L1 (sPD-L1) is only generated in the presence of ADAM8. Moreover, primary macrophages from WT and ADAM8-deficient mice showed ADAM8-dependent release of sPD-L1, independent of the macrophage polarization state. Since ADAM8 expression is induced in responding tumors and PD-L1 shedding is likely to decrease the anti-tumor activities of T-cells, we conclude that immunotherapy resistance is caused, at least in part, by the increased presence of proteases, such as ADAM8. 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