A Human Tumor-Immune Organoid Model of Glioblastoma
Shivani BaisiwalaiD, Elisa FazzariiD, Matthew X. LiiD, Antoni Andreu MartijaiD, Daria J. AzizadiD, Lu SuniD, Gilbert Herrera, Trinh Phan, Amber Monteleone, Ryan L. KaniD, David A. NathansoniD, A. Hongjun Wang, Won Kim, Richard G. EversoniD, Kunal S. PateliD, Linda M. LiauiD, Robert Michael PrinsiD, Aparna BhaduriiD
Abstract
A major obstacle to identifying effective therapies for the aggressive brain tumor glioblastoma is the lack of human-specific, immunocompetent models that reflect the human tumor microenvironment. To address this, we developed the immune-Human Organoid Tumor Transplantation (iHOTT) model. This is an autologous co-culture platform that integrates patient-derived tumor cells and matched peripheral blood mononuclear cells (PBMCs) within human cortical organoids, enabling the study of the patient-specific immune response to the tumor and tumor-immune interactions. This platform preserves tumor and immune populations, immune signaling, and cell-cell interactions observed in patient tumors. Treatment of iHOTT with pembrolizumab, a checkpoint inhibitor, mirrored cell type shifts and cell interactions observed in patients. TCR sequencing further revealed pembrolizumab-driven expansion of stem-like CD4-T-cell clonotypes exhibiting patient-specific repertoires. These findings establish iHOTT as a physiologically relevant platform for exploring autologous tumor-immune interactions and underscore the critical need for antigen-targeted strategies to enhance immunotherapy in glioblastoma.
02 /Specification & Classification
Click any tag to browse matching papers- Citations
- 30
- FWCI
- 128.96
- Field percentile
- top 1%
- Organism
- Assay
- Research Focus
- Approach
In-depth summary
Claude Opus 5.5 · xhigh effort · 5 Nov 2026 · from the full text of version 1 (20 Jun 2025) · CC BY-NC 4.0
Autologous co-culture of patient glioblastoma cells and matched peripheral blood mononuclear cells in human cortical organoids (iHOTT) preserves tumor-immune crosstalk and recapitulates patient T cell responses to pembrolizumab.
Glioblastoma immunotherapy models either rely on immunodeficient or murine hosts with non-human cytokine signaling or use tumor-derived organoids containing few exhausted infiltrating immune cells. Integrating freshly isolated patient tumor cells and matched circulating PBMCs into human cortical organoids enables autologous modeling of early immune infiltration and checkpoint blockade.
Key results
ShownCo-culturing patient-derived GFP-labeled glioblastoma cells and matched PBMCs at a 1:1 ratio on 8-12 week human cortical organoids for 7 days preserves major tumor and immune populations and induces tumor-associated cytokines.
In 50-50 Sasai3:RPMI media across 3 patient tumors, scRNA-seq and flow cytometry recover major tumor and immune subsets (from input PBMCs with ~65% CD3+ T, 11% CD14+ myeloid, and 13% CD19+ B cells) with low HAVCR2/LAG3 and high GZMB/CD69 expression, while 37-plex Luminex profiling shows elevated IL-6, IL-8, IL-10, and G-CSF versus tumor-only controls. · iHOTT Enables Tumor-Immune Co-Culture; Cytokines Characteristic of the Tumor…; scRNAseq Reflects Preservation of Tumor and…; Figure legends
ShownTumor-immune co-culture activates antigen presentation and B cell immunity pathways and establishes MHC-I and MIF signaling from tumor oligodendrocyte-like cells to CD8+ T and NK cells.
Gene set enrichment analysis across 3 patients shows significant upregulation (adjusted p < 0.05) of B cell-mediated immunity, complement activation, and antigen presentation in co-cultured PBMCs versus input PBMCs, while CellChat identifies MHC-I and MIF as top upregulated pathways targeting CD8+ T cells, NK cells, and unconventional T cells. · Interactions Between Tumor and Immune Cell Types…; GSEA; Ligand-Receptor Signaling Analysis; Figure legends
ShownPembrolizumab treatment in iHOTT shifts immune composition, cytokine secretion, and intercellular signaling in patterns that mirror recurrent glioblastoma patient tumors from a 42-patient clinical trial.
Over 7 days across 3 patient co-cultures, pembrolizumab increases CD4+ T cells, B cells, and secreted IL-15, IL-17F, and IL-25 (IL-17E) relative to IgG control, and parallels the 42-patient recurrent glioblastoma cohort in upregulating complement, CD226, TIGIT, and CD70 signaling toward unconventional T cells (gamma-delta, MAIT, and double-negative T cells) and ILCs. · Pembrolizumab Treatment in iHOTT Results in…; Pembrolizumab Treatment in iHOTT Alters Cytokine…; iHOTT Mirrors Immune Cell Type Shifts Observed…; iHOTT Mirrors Shifts in Cell-Cell Interactions…
ShownSingle-cell TCR sequencing shows that pembrolizumab drives expansion of novel, patient-private stem-like CD4+ T cell clonotypes in both iHOTT and treated patient tumors.
In iHOTT (~45% T cell TCR coverage; top 5 clonotypes of 4 to 54 cells) and patient tumors (~16% paired TCR coverage in text, ~30% in Fig. 4D; top 5 clonotypes of 10 to 33 cells), pembrolizumab enriches novel CD4+ T cell clones with elevated TCF7 and LEF1 and reduced TOX, while GLIPH2 clusters are predominantly private to individual patients. · T Cell Receptor Sequencing (TCRseq) in iHOTT…; Integration of TCR Clonotypes with…; Checkpoint Inhibition Responses in Patients…; Figure legends
Methods, data and reproducibility
Human embryonic stem cell (UCLA6) cortical organoids matured for 8-12 weeks were engrafted with 500,000 GFP-labeled fresh patient glioblastoma cells and 500,000 matched PBMCs (1:1) and cultured for 7 days in 50-50 Sasai3:RPMI media with IgG or pembrolizumab. Co-cultures and 42 clinical trial tumor samples were profiled by flow cytometry, 37-plex Luminex arrays, 10x Genomics scRNA-seq, and single-cell TCR sequencing.
- Fresh primary glioblastoma surgical resections and matched intraoperative peripheral blood mononuclear cells (PBMCs) co-cultured in iHOTT · human · n = 3 patients for scRNA-seq/TCR-seq co-cultures; 4 tumors with PBMC-only controls · https://cells-test.gi.ucsc.edu/?ds=ihott
- Pembrolizumab-treated and IgG control iHOTT organoid co-cultures · human · n = 3 patient-matched tumor and PBMC co-cultures · https://cells-test.gi.ucsc.edu/?ds=ihott+treated
- Untreated iHOTT organoid dataset on UCSC Cell Browser · human · n = 3 patients · https://cells-test.gi.ucsc.edu/?ds=ihott+untreated
- Recurrent glioblastoma patient tumor immune fraction from randomized trial of pembrolizumab vs no treatment on first or second recurrence · human · n = 42 patients
- Human embryonic stem cells (UCLA6 line) used to generate cortical organoids · human
- Methods & tools
- Cell Ranger v8.0.0, Seurat v5.0, SCTransform, clusterProfiler v4.6.2, CellChat v1.6.1, scRepertoire v1.7.1, immunarch, GLIPH2, FlowJo, 10x Genomics Chromium, Luminex 37-plex panel
- Data & accessions
- https://cells-test.gi.ucsc.edu/?ds=ihotthttps://cells-test.gi.ucsc.edu/?ds=ihott+untreatedhttps://cells-test.gi.ucsc.edu/?ds=ihott+treated
- Reagents & environment
- Cell Ranger v8.0.0, R/RStudio, Seurat v5.0, clusterProfiler v4.6.2, CellChat v1.6.1, scRepertoire v1.7.1, immunarch, GLIPH2, FlowJo, GRCh38 reference genome
- Reproducibility gaps
- Raw sequencing data are being deposited in dbGAP (accession number not yet provided), and no custom analysis code repository is linked.
Limitations (3)
- Myeloid cells (CD14+) are underrepresented in iHOTT after 7 days compared to fresh patient tumors, likely due to limited ex vivo monocyte and dendritic cell survival and absence of stromal or vascular cues. · stated by the authors
- Using freshly isolated circulating PBMCs over a 7-day co-culture models early immune infiltration and activation rather than the chronic exhaustion state of long-term tumor-infiltrating lymphocytes. · stated by the authors
- PBMC and cortical organoid viability decline after day 7 in time-course optimization experiments, restricting the duration of co-culture. · stated by the authors
Who should read:Read this if you study glioblastoma immunotherapy or tumor-immune organoid models and want an autologous human cortical organoid platform that tracks scRNA-seq and TCR clonal dynamics under PD-1 blockade.
Reproducibility ledger
Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.
Sign in to file a reproducibility report.
Related papers
- Humanized glioblastoma patient-derived orthotopic xenografts recreate a locally immunosuppressed human immune ecosystem amenable to immunotherapeutic modulation
Pilar M. Moreno‐Sanchez, Anaïs Oudin, … Anna Golebiewska·bioRxiv·Nov 20, 2025
- Development of a human glioblastoma model using humanized DRAG mice for immunotherapy
R. Srivastava, A. Labani-Motlagh, … G. Yi·bioRxiv·Feb 15, 2023
- Human Organoid Tumor Transplantation Identifies Functional Glioblastoma - Microenvironmental Communication Mediated by PTPRZ1
Weihong Ge, Ryan L. Kan, … Aparna Bhaduri·Cell ReportsPeer-reviewed·Jan 1, 2026
- Patient-Derived Organoids Recapitulate Intrinsic Immune Landscapes and Progenitor Populations of Glioblastoma.
Fumihiro Watanabe, Ethan W. Hollingsworth, … Jaime Imitola·PNAS NexusPeer-reviewed·Feb 1, 2024
- Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR-T cell testing
F. Jacob, G.-l. Ming, H. Song·bioRxiv·Mar 19, 2020
- Exploring the immune environment of glioblastoma through single-cell RNA sequencing in humanized mouse models
Jun Takei, Ken Furudate, … Satoru Osuka·bioRxiv·May 17, 2025