-------------------------- GENERAL INFORMATION -------------------------- 1. Title of dataset:Datos de investigación Proyecto PID2023-149111OB-I00 - Estudio de la Biología del Pericito Dependiente de Autofagia Mediada por Chaperonas Como Diana Clave para el Desarrrollo de Terapias en Glioblastoma 2. Author information: Name: Maria Dolores Salinas Institution: Biochemistry, Molecular Biology B and Immunology Department, University of Murcia (UMU), 30120 Murcia, Spain Email: mdsh@um.es ORCID: 0000-0002-5859-0969 Name: Isabel María Martínez Institution: Biochemistry, Molecular Biology B and Immunology Department, University of Murcia (UMU), 30120 Murcia, Spain Email: isabelmaria.martinezg@um.es ORCID: Name: Elena Naranjo Institution: Biochemistry, Molecular Biology B and Immunology Department, University of Murcia (UMU), 30120 Murcia, Spain Email: elena.naranjo1@um.es ORCID: Name: Rut Valdor Institution: Biochemistry, Molecular Biology B and Immunology Department, University of Murcia (UMU), 30120 Murcia, Spain Email: rut.valdor@um.es ORCID: 0000-0002-2681-0779 3. Date of data collection (single date or date range): [2024-09-01] - [2027-12-31] 4. Deposit date: [2026-05-15] 5. Language: Spanish English -------------------------- METHODOLOGICAL INFORMATION -------------------------- ### 1. Description of the methodology used to generate the dataset The dataset was generated from a combination of *in vitro* glioblastoma (GB)–pericyte (PC) coculture systems, molecular and functional assays, *in vivo* murine GB xenograft models, immunological analyses, fluorescence microscopy, and histopathological studies designed to investigate the role of chaperone-mediated autophagy (CMA) in GB progression and tumor–microenvironment interactions. #### Cell culture and coculture assays Primary murine brain pericytes (PCs), including wild-type (WT), CMA-deficient (Lamp2a knockout), and genetically modified PCs, were isolated and expanded under standardized culture conditions. Human glioblastoma stem cells (GSCs) and glioblastoma cell lines (U87, U251, U373) were maintained under defined conditions and used for coculture experiments. Pericytes were pretreated with a CMA-modulating peptide or control peptide prior to coculture with GB cells or GSCs. Functional assays included: * Cell proliferation assays using cumulative population doubling (CPD) measurements * Tumor cell adhesion assays using fluorescent tracers (DiI/DiD) * Cell survival and death analyses by flow cytometry * Quantification of stemness-associated markers (SOX2, SOX9) * Evaluation of CMA activity using KFERQ-based fluorescent CMA reporters * Macroautophagy flux monitoring using LC3 fluorescent reporters * Analysis of lysosomal/endosomal transfer between GB cells and PCs * Lipophagy and lipid droplet degradation analyses using fluorescent BODIPY labeling Additional analyses included quantification of: * Lamp2a mRNA expression by RT-qPCR * LAMP-2A and autophagy-related proteins by immunofluorescence and Western blot * Nitric oxide synthase (iNOS)-dependent signaling * Peroxynitrite and nitrosylation-related pathways * Endoplasmic reticulum stress markers (CHOP, GRP78/BiP) * Inflammasome activation markers (NLRP3, IL1B, IL18, Caspase-1) Secretome studies were performed using concentrated supernatants from GSCs or modified PCs to evaluate paracrine effects on tumor progression, autophagy, and immune modulation. #### Animal models Immunocompetent C57BL/6 mice and genetically modified mouse strains (Lamp2a knockout and NLRP3 knockout) were xenografted intracranially with patient-derived glioma stem cells or GB cell lines using stereotaxic surgery. Three weeks after grafting, mice received: * Intravenous administration of modified PCs * CMA-modulating peptide therapy * Control peptide treatments Animals were sacrificed at multiple time points (1–5 weeks post-treatment). Brain tissues were collected for histological and immunofluorescence analyses. Quantitative assessments included: * Tumor size and infiltration * Perivascular pericyte coverage * Brain inflammatory markers (IFNγ, CD68, Iba1) * Immune cell recruitment and activation * T-cell phenotype characterization from lymph nodes (FoxP3, CD4/CD8, CD44, CD62L, CTLA4, PD-1) #### Zebrafish models Transgenic zebrafish glioma models expressing fluorescent autophagy reporters (eGFP-map1lc3b; mCherry-lamp1) under pericyte-specific promoters were used to monitor autophagy flux and organelle transfer in vivo. Human glioma stem cells and modified human pericytes were xenografted into zebrafish embryos, followed by live imaging and convolutional neural network–based analysis. #### Histological and imaging analyses Brain sections from murine models and archived paraffin-embedded human GB samples were analyzed by: * Chromogenic immunohistochemistry * Immunofluorescence microscopy * Confocal microscopy * Western blot densitometry Markers analyzed included: * LAMP-2A * αSMA and PDGFRβ (pericyte markers) * IFNγ, CD68, Iba1 (inflammatory markers) * SOX2/SOX9 * LC3 and LAMP1 * NLRP3 inflammasome markers Raw and processed datasets include fluorescence intensity values, puncta counts, Western blot densitometry, qPCR Ct values, flow cytometry percentages, ELISA absorbance measurements, tumor morphometric analyses, and cell proliferation/survival quantifications. --- ### 2. Software or tools needed to interpret the data The following software and analytical tools were used for data acquisition, processing, quantification, and statistical analysis: * **ImageJ (NIH), version 1.54** – fluorescence quantification, puncta analysis, morphometric measurements, densitometry * **FlowJo**, version 10.0.7 (TreeStar) – flow cytometry analysis * **GraphPad Prism**, version 8.3.0 – statistical analysis and graphical representation * **LAS X**, version 5.2.2 (Leica Microsystems) – confocal microscopy image processing * **NIS-Elements AR (Nikon)** – fluorescence microscopy acquisition * **Western blot image acquisition software** associated with chemiluminescence imaging systems * **Convolutional neural network–based image analysis tools** for zebrafish live imaging datasets * **qPCR acquisition software** associated with real-time PCR systems Some acquisition software packages (FlowJo, GraphPad Prism, LAS X, NIS-Elements) require institutional or commercial licenses and can be obtained from the corresponding vendors. However, all datasets deposited in DIGITUM are provided in reusable and open formats (CSV, XLSX, TXT, TIFF), allowing data interpretation without requiring proprietary software. Image-derived quantitative data were exported into tabular formats suitable for reuse and secondary analysis. --- ### 3. Procedures followed for data quality assurance Several experimental and analytical quality assurance procedures were implemented to ensure reproducibility and data reliability. * All animal procedures were conducted under approved ethical protocols and institutional animal care regulations. * Biological replicates and independent experimental repeats were performed for all major assays. * Coculture experiments included appropriate controls: * untreated cells * control peptide–treated cells * CMA-deficient pericytes * conditioned media controls * lysosomal inhibitor controls * Flow cytometry analyses included: * viability dyes * fluorescence-minus-one controls * isotype controls * compensation controls * Quantitative microscopy analyses were performed using standardized acquisition parameters and predefined thresholds across all experimental groups. * Western blot analyses included housekeeping/loading controls and repeated measurements. * RT-qPCR assays were performed with technical replicates and normalized using housekeeping genes. * Functional assays (cell proliferation, survival, adhesion, cytokine secretion) were conducted in multiple independent experiments. * Histological analyses were performed on multiple tissue sections and fields per sample. * Zebrafish imaging experiments were standardized for developmental stage, imaging timing, and acquisition conditions. * Statistical analyses were conducted using appropriate parametric tests (Student’s t-test, one-way ANOVA with post hoc correction), with statistical significance defined as p < 0.05. Human samples used for validation studies were anonymized and derived from previously approved biobanked collections. Prior to repository deposition, datasets were curated, checked for consistency, and converted into interoperable formats compatible with FAIR data principles. -------------------------- FILES -------------------------- 1. File name: [List all the files included in the dataset, with the name and extension of each file (.csv, .pdf, etc.)]. 2. File format: -------------------------- KEYWORDS -------------------------- 1. keywords: autophagy, pericytes, glioblastoma, therapy, biomarkers ------------------------------------- SPONSORSHIP INFORMATION AND GRANT IDs ------------------------------------- 1. Grant Information: Título: "Estudio de la biología del pericito dependiente de autofagia mediada por chaperonas como diana clave para el desarrollo de terapias en glioblastoma". Investigadora Principal: Rut Valdor Alonso. Institución: Universidad de Murcia (Facultad de Medicina). Temática: Biomedicina - Cáncer (Glioblastoma). Objetivo: Investigar la biología de los pericitos en relación con la autofagia mediada por chaperonas para mejorar el tratamiento de los glioblastomas, que son tumores cerebrales agresivos con una tasa de supervivencia baja. Financiación: 200.000 €. Marco: Proyectos de Generación de Conocimiento 2023. -------------------------- RELATED PUBLICATIONS -------------------------- 1. Related publication: 2. Related dataset: -------------------------- LICENSES AND PRIVACITY -------------------------- 1. Licenses: 2. Privacity: -------------------------- MORE INFO -------------------------- [Include any other information about the data set that is not reflected in this template and that is considered relevant.]