Protocol for generating human organoid tumor transplantation and immune-human organoid tumor transplantation models for glioblastoma research

Glioblastoma research requires human models that preserve tumor heterogeneity and microenvironmental interactions. Here, we present a protocol for generating the human organoid tumor transplantation (HOTT) model and its immunocompetent counterpart, the immune-HOTT (iHOTT) model. These platforms use direct-from-patient tumor cells engrafted into organoids to recapitulate the tumor microenvironment and, in the iHOTT system, autologous tumor-immune interactions. We describe steps for organoid generation, patient tumor dissociation, co-culture with autologous peripheral blood mononuclear cells, fluorescence-activated cell sorting, and downstream single-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Ge et al. and Baisiwala et al. 1 1. Baisiwala, S. ∙ Fazzari, E. ∙ Li, M.X. ... A human tumor-immune organoid model of glioblastoma Cell Rep. 2026; 45 , 116790 Full Text Full Text (PDF) Scopus (19) PubMed Google Scholar , 2 2. Ge, W. ∙ Kan, R.L. ∙ Yilgor, C. ... Human organoid tumor transplantation identifies functional glioblastoma-microenvironment communication mediated by PTPRZ1 Cell Rep. 2026; 45 , 116848 Full Text Full Text (PDF) Scopus (6) Google Scholar