GL261
Cat.No.: CSC-C9184W
Species: Mus musculus (Mouse)
Source: Brain
Morphology: fibroblastoid
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The use of appropriate animal models in glioma research is essential for the development of new therapeutic approaches. An ideal animal model of human glioma should reproduce the major features of this neoplasm: predictable and reproducible in vitro and in vivo growth patterns; infiltrative, but non-metastatic tumor growth; and poor immunogenicity. Many animal brain tumor models are being used, however, no model currently available simulates human high-grade gliomas exactly. Tumor models vary in their immunogenicity, growth patterns and invasiveness. Therefore, it is important to choose an appropriate animal model depending on the endpoint examined. Most models are derived from rat, and several xenograft models based on the intracerebral transplantation of human brain tumors into immunodeficient nude mice, are also available. Murine models of malignant brain tumors are used much less frequently, mainly because the number of available models is limited.
One of the most commonly used murine brain tumor models is GL261. The GL261 tumor was induced originally by intracranial injection of 3-methylcholantrene into C57BL/6 mice and maintained by serial intracranial and subcutaneous transplantations of small tumor pieces on the syngeneic mouse strain.
The benefits of the GL261 model relate to its widespread use and extensive characterization throughout the literature. GL261 models recapitulate the histology of glioblastoma (GBM) very well. In addition, GL261 cells are easily maintained in culture, allowing them to be investigated in vitro and easily expanded for in vivo tumor injection. When grown in serum-free media, GL261 cells express CD133, a stem cell marker, and have increased tumorgenicity and immunogenicity. Therefore, GL261 cells can be used to investigate glioma stem cells, which contribute to chemo- and radioresistance in human GBM.
Mapping glioblastoma spreading: connexin43 and glial dynamic in mouse and human glioblastoma microenvironment
Background
This preprint investigates glioblastoma (GBM) spreading and glial microenvironment responses using mouse and human organotypic brain slices and patient peritumoral cortex. It shows peritumoral tissue has high astrocytic Connexin43 (Cx43) and CD44, while tumor core has myeloid infiltration and low CD44. Implanted GBM cells infiltrate tissue and induce early glial changes, including increased Cx43 expression and distribution. Blocking Cx43 hemichannels alters human GBM morphology and polarity, suggesting Cx43 is crucial for GBM infiltration and conserved across species.
Key Findings
Human peritumoral high-grade glioma tissue showed elevated astrocytic Cx43 and CD44 and discrete microglial infiltration, whereas tumor core had high myeloid infiltration and reduced CD44. In mouse and human organotypic slice co-cultures, implanted GBM cells infiltrated brain tissue and elicited early glial modifications, notably increased Cx43 expression and redistribution. Cx43 upregulation was consistent in both mouse and human host tissue. Blocking Cx43 hemichannels with GAP19 increased GBM migratory ability and induced morphological changes and polarization of human primary GBM cells, typical of migration. These findings identify Cx43 as a conserved hub in GBM-host interactions and suggest Cx43-targeted approaches.
Contribution
Creative Bioarray supplied the murine glioma GL261 cell line (CSC-C9184W), which the authors used as a well-characterized GBM model for mouse organotypic slice co-cultures and functional assays. This enabled comparative analysis of GBM infiltration, glial Cx43 responses, and GAP19 effects across mouse and human systems.
HDL Nanodiscs Loaded with Liver X Receptor Agonist Decreases Tumor Burden and Mediates Long-term Survival in Mouse Glioma Model
Background
Glioblastoma multiforme relies on astrocyte-derived cholesterol and is immunosuppressive. This study developed synthetic high-density lipoprotein (sHDL) nanodiscs co-loading liver X receptor agonist GW3965 and CpG oligonucleotide to deplete tumor cholesterol and stimulate antitumor immunity. GW-CpG-sHDL upregulated ABCA1, enhanced cholesterol efflux, reduced glioma cell viability, and increased radiosensitivity and caspase-3/7-dependent apoptosis in mouse and patient-derived glioblastoma cells while sparing normal astrocytes. In GL261 tumor-bearing mice, GW-CpG-sHDL plus radiation produced 60% long-term survival, prevented tumor growth upon rechallenge, and showed no major organ toxicity, supporting cholesterol metabolism and immune modulation as a promising GBM strategy.
Key Findings
Synthetic HDL nanodiscs (8–12 nm) co-encapsulated LXR agonists and cholesterol-CpG with >90% efficiency. GW-CpG-sHDL increased ABCA1 mRNA ~20-fold and cholesterol efflux to ~14%, exceeding blank sHDL (~7%). It dose-dependently reduced viability of murine GL26 EGFRvIII and human HF2303/MGG8 glioblastoma cells, enhanced radiation-induced cytotoxicity, increased sub-G1/apoptotic populations, and activated caspase-3/7, with minimal effects on normal human astrocytes. In GL261 glioma-bearing mice, GW-CpG-sHDL plus irradiation significantly extended survival versus either alone; 60% survived long-term, and rechallenged survivors showed immunological memory with 2/3 tumor-free. Treated tumors averaged 2.56 mm³ versus 23.65 mm³ for saline. Combination therapy reduced microglial/macrophage infiltration, increased CD4+/CD8+ T-cell infiltration in tumors, preserved brain architecture, and caused no liver/kidney toxicity.
Contribution
Creative Bioarray provided GL261 cells (CSC-C9184W) used for in vitro characterization and as the basis for the syngeneic GL261 mouse glioma model. These cells enabled evaluation of LXR agonist/CpG-loaded sHDL nanodiscs on cholesterol efflux, cytotoxicity, and in vivo survival, supporting translational testing.
mLumiOpto Is a Mitochondrial-Targeted Gene Therapy for Treating Cancer
Background
mLumiOpto is a mitochondrial-targeted luminoptogenetics gene therapy designed to disrupt the inner mitochondrial membrane potential and kill cancer cells. It coexpresses a blue light-gated cationic channelrhodopsin (CoChR) in the inner mitochondrial membrane and a bioluminescent nanoluciferase (NLuc) in the cytosol of the same cells. Delivered via AAV or mAb-tagged exosome-associated AAV, luciferin-triggered endogenous bioluminescence activates CoChR, causing mitochondrial depolarization, apoptosis, and DNA damage. In glioblastoma and triple-negative breast cancer models, mLumiOpto reduced tumor burden, extended survival, and boosted antitumor immunity with minimal toxicity.
Key Findings
The authors developed mLumiOpto, a next-generation optogenetic therapy that selectively delivers genes to cancer cells to trigger mitochondrial depolarization and cell death. CoChR and NLuc were linked via a cleavable 2A linker, with CoChR targeted to mitochondria using the ABCB10 leading sequence. ViviRen-induced bioluminescence activated CoChR, producing dose-dependent ΔΨm depolarization and cancer cell death primarily through intrinsic apoptosis and DNA damage, independent of mPTP opening and excessive ROS. AAV-DJ/8 carrying a cancer-selective cfos promoter, or anti-EGFR mAb-Exo-AAV, enabled cancer-specific gene delivery. In glioblastoma (U87, PDX) and triple-negative breast cancer (MDA-MB-231, 4T1) mouse models, mLumiOpto significantly reduced tumor burden, extended survival, and increased dendritic cell and CD8+ T-cell infiltration without organ toxicity. It also killed drug-resistant U251-TMZ cells, suggesting potential to overcome drug resistance.
Contribution
Creative Bioarray provided the mouse glioblastoma GL261 cell line, which was used to validate mLumiOpto-mediated cytotoxicity. Treatment killed 90%–99% of GL261 cells along with other GBM lines, supporting the broad anticancer efficacy of the mitochondrial-targeted luminoptogenetic therapy.
Progesterone boosts abiraterone-driven target and NK cell therapies against glioblastoma
Background
This study investigates combining progesterone (Prog) with abiraterone (Abi) to treat temozolomide-resistant glioblastoma (GBM). Using in vitro and orthotopic mouse models, the authors examine cell viability, apoptosis, metabolism, MGMT expression, and immune microenvironment changes. Prog enhanced Abi cytotoxicity, reduced MGMT via promoter methylation and protein stability, suppressed mitochondrial respiration and glycolysis, and increased NK cell infiltration. The combination prolonged survival and boosted NK-92 cell-mediated killing, suggesting a promising immunometabolic strategy for GBM.
Key Findings
Prog significantly lowered Abi IC50 in TMZ-resistant GBM cells without affecting proliferation alone, indicating synergy (CI<1). Combined Abi+Prog induced apoptosis, suppressed tumor growth, and extended survival versus Abi alone in U87MG-R and GL261-R orthotopic models (Scid and C57BL/6). Prog reduced MGMT expression by increasing promoter methylation and decreasing protein stability. Abi inhibited mitochondrial respiration via CDK1 downregulation, while Prog suppressed glycolysis and TCA metabolites, also involving CDK1. RNA-seq showed Prog upregulated NK-activating genes (ULBP1, IL15) and downregulated inhibitory genes (IL1B, TGFB2); Abi increased NK recognition ligands. Combination increased CD3−/CD19−/CD56+/NK1.1+ NK infiltration in tumors. In co-culture and in vivo, Abi+Prog enhanced NK-92-mediated GBM killing and reduced tumor burden. These results support an immunometabolic combination strategy overcoming TMZ resistance.
Contribution
Creative Bioarray provided GL-261 murine glioma cells used to establish GL261-R tumors in immunocompetent C57BL/6 mice. These cells enabled in vivo evaluation of abiraterone plus progesterone on tumor growth, survival, and NK cell infiltration, supporting the study's key combination efficacy and immune microenvironment findings.
Comparison of methods for in vivo and ex vivo quantification of iron in murine brain tumors
Background
This study compares four methods for quantifying iron oxide nanoparticles in murine brain tumors in vivo and ex vivo: T2* MRI, portable X-ray fluorescence (XRF), UV-Vis spectroscopy, and ICP-MS. Using ferumoxytol-treated GL-261 glioblastoma models and cell cultures, the authors assess dose-response correlations, detection limits, and agreement with the ICP-MS gold standard. The work provides practical guidance for selecting an iron quantification method based on sample type, equipment, cost, and whether spatial information or noninvasive monitoring is required.
Key Findings
In vivo MRI T2* and portable XRF measurements correlated with administered ferumoxytol dose and ex vivo ICP-MS results, but XRF showed stronger, more significant correlations (dose: ρ=0.97; ICP-MS: ρ=0.91) than MRI T2* (dose: ρ=-0.64; ICP-MS: ρ=-0.69). XRF also quantified iron across the full 0–300 mg/kg/day dose range, whereas MRI T2* became unreliable above 100 mg/kg/day due to image distortion and signal dropout, and unlike MRI, XRF cannot provide spatial iron distribution. UV-Vis spectroscopy performed well in cell culture samples but failed in complex brain tumor tissue due to incomplete dissolution and interfering absorbance. ICP-MS remained the gold standard and confirmed pXRF and MRI results, with pXRF typically yielding higher regional iron concentrations because it sampled the tumor region while ICP-MS analyzed whole brain. The study concludes that method choice should depend on sample type, iron concentration, equipment, budget, and whether noninvasive or spatially resolved measurement is required.
Contribution
Creative Bioarray supplied the GL-261 glioma cell line (Shirley, NY) used in this study. This cell line enabled the in vitro ferumoxytol uptake assays and orthotopic murine brain tumor experiments, which were central to comparing MRI, XRF, UV-Vis, and ICP-MS iron quantification.
Implanted Luciferase-Expressing GL261 Models Prolonged Survival in C57BL/6 Mice
In order to validate controls for use in future preclinical work, we first implanted 5 × 104 cells/5 μL of GL261 or GL261 Red-FLuc into the brains of C57BL/6 mice (Fig. 1a), expecting to find equivalent survival rates, as assessed by the Kaplan–Meier method. However, we found a significant difference in overall survival between these two groups (n = 20 per group, P < 0.0001). The GL261 implanted mice reached median survival at 18 days post-implantation with 100% of animals succumbing to disease after showing clear signs of clinical decline. Conversely, the GL261 Red-FLuc implanted mice did not reach median survival, and 60% of animals were described as long-term (≥ 100 days) survivors without neurological morbidity.
The significant differences found in overall survival prompted us to investigate if tumor dose or the type of luciferase expressed affect results found. We therefore evaluated an escalating tumor dose (Fig. 1b) of 3 × 105 cells/5 μL of luciferase-expressing GL261 Red-FLuc or GL261-Luc2 cells against our starting dose of 5 × 104 cells/5 μL. In this subsequent experiment, we found that mice implanted with 5 × 104 cells of GL261 reached median survival at 21 days with 100% tumor penetrance (n = 20), similar to previous experiments. For GL261-Luc2 cells, the higher tumor dose of 3 × 105 GL261-Luc2 cells (n = 10) approached the median survival of the GL261 mice dosed with 5 × 104 cells, and 100% symptomatic tumor penetrance. However, mice implanted with 5 × 104 GL261-Luc2 cells (n = 10) demonstrated a significantly prolonged median survival of 37 days in comparison to GL261 implanted mice (P = 0.0011). Of note, 40% of animals implanted with 5 × 104 GL261-Luc2 cells reached long term survival (> 100 days), without symptomatic tumor burden. In contrast, the mice implanted with either dose of GL261 Red-FLuc cells did not reach median survival, regardless of dose. Specifically, 60% of those implanted with 5 × 104 cells (n = 10) achieved long-term survival and 70% of those intracranially injected with a tumor dose of 3 × 105 cells (n = 20) were termed long-term survivors.
GL261 and GL261 Luciferase-Expressing Cells Have Similar Proliferation Rates In Vitro
To explain the underlying differences found in overall survival, we next examined these cells for differences in cell growth and proliferation. First, we assessed in vitro proliferation of both cell lines via spectrophotometric CCK-8 assay and cell counting 48 h after seeding cells. We observed no differences for in vitro proliferation between the cell lines GL261 (3.503 ± 0.178, n = 6), GL261 Red-FLuc (3.395 ± 0.106, n = 6) and GL261-Luc2 (3.57 ± 0.144, n = 6) (Fig. 1c). In addition, we verified luciferase reporter gene and protein expression by ONE-Glo EX Luciferase Assay System (Fig. 1d). Here, we demonstrate that the original GL261 cells do not express luciferase, whereas the GL261 Red-FLuc and GL261-Luc2 cells are strongly positive for luciferase expression at (2.48 ± 0.08)105 RLU (relative light unit) (n = 6) and (1.29 ± 0.2)106 RLU (n = 6), respectively (P < 0.0001).
Fig. 6. Outcomes for C57BL/6 mice intracranially injected with glioma cells. (a) Kaplan–Meier survival analysis showing median survival for GL261 and GL261 Red-FLuc implanted mice. (b) Displays median survival at increasing tumor doses. (c) Measurements of proliferation rate for all three cell lines. (d) Luciferase expression verified reporter gene expression in GL261 Red-FLuc and GL261-Luc2 cells. (Sanchez, Victoria E., et al., 2020)
GL261-Red Luciferase-Expressing Tumors Recruit More Pro-inflammatory Cells to the Brain Microenvironment
Flow cytometry experiments were used to analyze the immune cell frequency in dissociated brain tissue cell suspensions from mice injected with 5 × 104 cells/5 μL of GL261 or GL261 Red-FLuc tumor cell lines sacrificed 10 days following tumor implantation (n = 5 per group) (Fig. 2a). Day 10 was chosen to capture the immune infiltration into the GL261 tumors before growth caused significant morbidity but before luciferase-expressing tumors regressed.
First, we compared the frequency of phenotypically sub-gated T-cells of mice injected with either GL261 or GL261 Red-FLuc (Fig. 2b). We observed a significant increase in the frequency of CD4+T cells of the GL261-injected mice (mean 0.8896%) compared to that of the GL261 Red-FLuc injected mice (mean 2.731%). Conversely, there was a significant decrease in CD4+Tregs (from mean 45.28% to 16.82%) in GL261 compared to the GL261 Red-FLuc. The CD8+T-cell population frequency showed a significant increase in the GL261 Red-FLuc (mean 1.478%) compared to GL261 (mean 0.324%). There was an increase in the frequency of CD8+Tregs in the GL261 group that did not reach significance (p = 0.08). When comparing macrophage populations, there was a significant increase in activated macrophages (F4/80+CD11b+cells) in GL261 Red-FLuc (mean 15.33%) compared to GL261 (mean 5.6%) tumors.
We repeated this flow cytometry analysis on mice injected with 5 × 104 cells/5 μL of GL261 or GL261-Luc2 to compare immune cell frequencies between these two tumor cell lines (Fig. 2c). There was a slight observed increase in activated macrophages, however, there were no significant differences observed between brains of mice implanted with these two cell lines.
Fig. 7. (a) Displays the various cell populations gated for F4/80+ macrophages and CD3+ T cells. CD3+ T-cell subsets were then fractioned by CD4 and CD8 positivity. (b) Shows the frequencies of immune cells observed in the GL261 and GL261 Red-FLuc implanted brains dissociated into a single cell suspension 10 days post-implantation. (c) Displays a panel of immune cell frequencies observed in GL261 and GL261-Luc2 implanted brains (Sanchez, Victoria E., et al., 2020).
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