This study comprises two complementary investigations that leverage innovative human model systems and cutting-edge molecular profiling to dissect glioblastoma (GBM) heterogeneity and tumor-microenvironment interactions. The first study uses a novel Human Organoid Tumor Transplantation (HOTT) model, in which freshly dissociated GBM tumor cells from five patients were transplanted into human embryonic stem cell-derived cortical organoids. This system enables the interrogation of tumor and microenvironment interactions in a human-specific, developmentally relevant context. Using single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA-seq (snRNA-seq), the authors profiled over 216,000 cells, analyzing tumor heterogeneity across multiple media conditions. Ligand-receptor analysis (CellChat), immunofluorescence, and RNAScope were used to validate findings. The study revealed that the HOTT model faithfully recapitulates primary GBM cell types and microenvironmental components, and uncovered a novel bidirectional signaling axis mediated by <a href="https://www.ncbi.nlm.nih.gov/gene/5803">PTPRZ1</a> that influences both tumor migration and cell fate in a context-dependent manner. <a href="https://www.ncbi.nlm.nih.gov/gene/5803">PTPRZ1</a> is identified to be important for intercellular communication between tumor and microenvironmental cells, while <a href="https://www.ncbi.nlm.nih.gov/gene/5159">PDGFRB</a>, <a href="https://www.ncbi.nlm.nih.gov/gene/4854">NOTCH3</a>, <a href="https://www.ncbi.nlm.nih.gov/gene/84525">HOPX</a>, <a href="https://www.ncbi.nlm.nih.gov/gene/11170">FAM107A</a>, <a href="https://www.ncbi.nlm.nih.gov/gene/7122">CLDN5</a>, <a href="https://www.ncbi.nlm.nih.gov/gene/8490">RGS5</a>, <a href="https://www.ncbi.nlm.nih.gov/gene/2296">FOXC1</a>, <a href="https://www.ncbi.nlm.nih.gov/gene/54345">SOX18</a> are key markers of the neurovascular progenitor that we identify to be a multipotent, restricted progenitor in GBM. Related datasets include the inclusion of immune cells in the HOTT system, termed “iHOTT”. The second study focuses on identifying and characterizing a rare but functionally potent neurovascular progenitor (NVP) population within GBM tumors. By generating a meta-atlas from seven publicly available GBM scRNA-seq datasets, the authors identified a unique tumor-derived cell population co-expressing neural progenitor and vascular markers. This population was validated in situ and in vivo through immunostaining and lineage tracing via DNA barcoding in the HOTT system. Functional knockdown of NVPs in a murine GBM model resulted in reduced cycling cells, altered tumor composition, and increased survival. Clonal analysis showed NVPs have dual potency to give rise to both neuronal and vascular-like tumor cells, contributing significantly to tumor cell diversity. Molecular technologies used across the studies include: <ul><li>Single-cell and single-nucleus RNA sequencing </li><li>DNA barcoding and clonal lineage tracing (CellTag) </li></ul>Population information: <ul><li>Tumor samples were obtained directly from multiple GBM patients (all IDH1 wild-type, adult), including core and peripheral tumor regions. Human cortical organoids derived from human embryonic stem cells (hESCs) served as the microenvironment in the HOTT model. </li></ul>Principal findings include: <ul><li>HOTT recapitulates the heterogeneity and cell type fidelity of GBM, while enabling the study of tumor–microenvironment signaling. </li><li>PTPRZ1 has opposing roles in tumor and microenvironment, influencing GBM cell migration and fate via non-catalytic mechanisms. </li><li>A novel GBM neurovascular progenitor population (NVP) was identified, validated, and shown to contribute clonally to distinct tumor cell types. </li><li>Elimination of NVPs in vivo reduces tumor aggressiveness and improves survival. </li></ul>Data to be available through dbGaP: <ul><li>Raw and processed single-cell and single-nucleus RNA-seq data (HOTT model and NVP studies) </li><li>CellTag barcode-labeled scRNA-seq clonal lineage data </li><li>Associated metadata including patient of origin, experimental condition, and sample annotations.</li></ul>

Studying Glioblastoma in a Human Organoid Tumor Transplantation Model

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Studying Glioblastoma in a Human Organoid Tumor Transplantation Model

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