A Three-Dimensional Organoid Culture System Derived from Human Glioblastomas Recapitulates the Hypoxic Gradients and Cancer Stem Cell Heterogeneity of Tumors Found In Vivo

Abstract

Many cancers feature cellular hierarchies that are driven by tumor-initiating, cancer stem cells (CSCs) and rely on complex interactions with the tumor microenvironment. Standard cell culture conditions fail to recapitulate the original tumor architecture or microenvironmental gradients, and are not designed to retain the cellular heterogeneity of parental tumors. Here, we describe a three-dimensional culture system that supports the long-term growth and expansion of tumor organoids derived directly from glioblastoma specimens, including patient-derived primary cultures, xenografts, genetically engineered glioma models, or patient samples. Organoids derived from multiple regions of patient tumors retain selective tumorigenic potential. Furthermore, organoids could be established directly from brain metastases not typically amenable to in vitro culture. Once formed, tumor organoids grew for months and displayed regional heterogeneity with a rapidly dividing outer region of SOX2+, OLIG2+, and TLX+ cells surrounding a hypoxic core of primarily non-stem senescent cells and diffuse, quiescent CSCs. Notably, non-stem cells within organoids were sensitive to radiation therapy, whereas adjacent CSCs were radioresistant. Orthotopic transplantation of patient-derived organoids resulted in tumors displayed histological features, including single cell invasiveness, that were more representative of the parental tumor compared with those formed from patient-derived sphere cultures. In conclusion, we present a new ex vivo model in which phenotypically diverse stem and non-stem glioblastoma cell populations can be simultaneously cultured to explore new facets of microenvironmental influences and CSC biology.

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“…Both the normal and tumor epithelium can now be readily cultured from biopsies, propagated and expanded in vitro using defined culture conditions. Advances in generating tumoroids from pancreatic cancer [20], glioblastoma [21], prostate cancer [22,23] and colorectal cancer [3] reveal that patient-derived tumoroids recapitulate patient-specific histological features. These tumoroids can also be cryopreserved in a similar manner as continuous cell lines and can subsequently be characterized by genomics, transcriptomics and using high-throughput in vitro drug screening assays, something that has spurred initiatives to develop live patient-derived tumoroid biobanks [1].…”

Biomimetic Strategies for the Glioblastoma Microenvironment

“…Tumor hypoxia is one of the key factors inducing the development of heterogeneous cell subpopulation within the tumor masses, which leads to an aggressive treatment-resistant phenotype, rapid progression, and poor prognosis (Heddleston et al, 2009;Li and Rich, 2010;Cheng et al, 2011). Recent studies have reported that these subcellular populations within the GBM mass are cancer stem cells, which exhibit increased tumorigenicity and stem cell-like capacity (Hubert et al, 2016). Histologically, GBM tissue contains a large hypoxic core, called pseudopalisading necrosis (Rong et al, 2006), which in turn contains a stem-like subpopulation (Mamun et al, 2009).…”

New Advances and Challenges of Targeting Cancer Stem Cells

“…A 3D GBM-CSC organoid culture system has been developed to preserve the cell heterogeneity and microenvironmental gradients in patient tumors and will aid in the development of new therapies to treat the heterogeneity of GBM (Chris Hubert, Cleveland Clinic Foundation; ref. 41). …”