GL261 luciferase-expressing cells elicit an anti-tumor immune response: an evaluation of murine glioma models

Abstract

Preclinical models that reliably recapitulate the immunosuppressive properties of human gliomas are essential to assess immune-based therapies. GL261 murine glioma cells are widely used as a syngeneic animal model of glioma, however, it has become common practice to transfect these cells with luciferase for fluorescent tumor tracking. The aim of this study was to compare the survival of mice injected with fluorescent or non-fluorescent GL261 cells and characterize the differences in their tumor microenvironment. Mice were intracranially implanted with GL261, GL261 Red-FLuc or GL261-Luc2 cells at varying doses. Cytokine profiles were evaluated by proteome microarray and Kaplan-Meier survival analysis was used to determine survival differences. Median survival for mice implanted with 5 × 10 4 GL261 cells was 18 to 21 days. The GL261 Red-FLuc implanted mice cells did not reach median survival at any tumor dose. Mice injected with 3 × 10 5 GL261-Luc2 cells reached median survival at 23 days. However, median survival was significantly prolonged to 37 days in mice implanted with 5 × 10 4 GL261-Luc2 cells. Additionally, proteomic analyses revealed significantly elevated inflammatory cytokines in the supernatants of the GL261 Red-FLuc cells and GL261-Luc2 cells. Our data suggest that GL261 Red-FLuc and GL261-Luc2 murine models elicit an anti-tumor immune response by increasing pro-inflammatory modulators. Glioblastoma (GBM) is the most common malignant primary brain tumor in adults 1. Despite ongoing studies and numerous clinical trials, the prognosis remains poor 2. There is an urgent need to provide these patients with new therapies, as the standard of care treatment has gone unchanged for more than a decade 3. In light of the promising advances made in the area of immune therapy as a treatment for various solid cancers 4-7 , the field of neuro-oncology has embraced immunotherapies for gliomas as a promising area of preclinical and clinical investigation 8. However, recent trials like the Phase III CheckMate-143 9 and Phase III CheckMate-498 10 show poor efficacy despite promising results from pre-clinical studies 11-15. The lack of translation from preclinical to clinical studies illustrates the need for a more rigorous characterization of the pre-clinical models currently being used in the scientific community. The development of novel immune therapeutics is not possible without a reliable preclinical animal model that accurately mimics the complex immune landscape of GBM. Of the various classes of preclinical mouse models, syngeneic models have been indispensable for evaluating immune therapies in GBM 16. Syngeneic murine models are models that rely on allografts of immortalized cancer cells from the same mouse strain from which the model originates.

Mouse models of glioblastoma for the evaluation of novel therapeutic strategies

“… 90 However, a more recent study by Sanchez et al highlighted improved survival in mice injected with GL261 tumors expressing luciferase with or without an additional fluorescent protein marker, relative to wild-type tumor cells. 91 They also showed increased inflammatory immune cells in tumors expressing luciferase and a fluorescent protein marker as well as increased secretion of inflammatory cytokines by these cells. 91 Given the mixed literature on the subject, attention should be paid to the use of luciferase or other nonendogenous tumor tracking proteins, such as GFP, especially if investigating immunotherapies.…”

Anti-PD-1 checkpoint blockade monotherapy in the orthotopic GL261 glioma model: the devil is in the detail

“…Just as increased knowledge about the mutational burden of GL261 tumors has led increasingly to them being viewed as a model of hypermutated glioblastoma, 26 , 82 increased knowledge of other experimental variables should change our practices moving forward. For instance, knowing that modification of the GL261 cell line with bioluminescent reporters enhances its antigenicity 52 should temper the enthusiasm of successful immunotherapeutic strategies vetted in that model and perhaps encourage the utilization of a second model system like CT-2A, SB28, or wild-type GL261 for verification. Other sets of experimental factors should inform our practices as well.…”

“…While an older study failed to observe any gross changes in overall growth kinetics or T-cell infiltration between GL261 and GL261-Luc tumors, 51 a more recent study found that wild-type GL261-bearing mice had shorter survival than mice bearing either of 2 types of Luciferase-expressing GL261. 52 The ability of each of these 3 cell lines to replicate in vitro was comparable and the difference in survival was instead linked to a more inflammatory state within the Luciferase-expressing tumors as shown by a cytokine microarray revealing elevated IFN-y and IL-1α levels. 52 Whether the differential cytokine levels in the tumor microenvironment are sufficient to dictate a differential response to anti-PD-1 therapy is yet unknown, but the equivalence of GL261 and GL261-Luciferase cell lines should not be taken for granted without further study.…”

“… 52 The ability of each of these 3 cell lines to replicate in vitro was comparable and the difference in survival was instead linked to a more inflammatory state within the Luciferase-expressing tumors as shown by a cytokine microarray revealing elevated IFN-y and IL-1α levels. 52 Whether the differential cytokine levels in the tumor microenvironment are sufficient to dictate a differential response to anti-PD-1 therapy is yet unknown, but the equivalence of GL261 and GL261-Luciferase cell lines should not be taken for granted without further study. Another minimally explored, but potentially significant, factor in influencing murine survival between studies is the location of tumor inoculation.…”

Genetically Engineered Mouse Model of Brainstem High-Grade Glioma

“…A recent study reports that the implantation of adult mice with cells expressing luciferase may elicit an increased pro-inflammatory immune response, when compared with the implantation of Wild type glioma cells ( Sanchez et al., 2020 ). In this study, luciferase expressing cells were implanted in adult animals, which have a fully developed immune system.…”