Origin and tumor characteristics
The Glioma 261 (GL261) orthotopic model for murine glioma was established in 1970 via chemical induction with methylcholanthrene. Ausman
et al. transplanted tumor fragments subcutaneously and intracranially into C57BL/6 mice, with the latter resulting in a median survival of 24–25 days when implanted with 1×10
5 tumor cells/10 μl[
64,
65]. Stable GL261 cell lines for transplantation were constituted in the mid-1990s[
65]. Although the GL261 tumors most resemble ependymoblastomas on histology, GL261 tumors closely mimic GBM phenotypes[
64]. They stain positively for the GBM marker vimentin[
66] and harbor activating mutations of the K-ras oncogene as well as mutations of the p53 tumor suppressor gene, resulting in high expression of c-myc[
65]. Similar genetic derangements have been reported in human gliomas[
67‐
69]. GL261 tumors are partially immunogenic, as they express high levels of MHC I. However, GL261 expression of MHC II, B7-1, and B7-2, the latter two of which are co-stimulatory molecules required for T cell activation, is limited[
65]. Reduced MHC and B7 expression is also characteristic of GBM cell lines and CD133
+ BTSCs, which contributes to their escape from immune surveillance[
60,
70,
71].
Tumors established from GL261 cells recapitulate many characteristics of GBM. Tumor formation proceeds through four stages, over a four-week period, following implantation: perivascular organization, proliferation near vasculature, hypoxia through blood vessel degeneration, and neovascularization towards necrotic regions[
72]. Histologic analysis reveals pleomorphism, pseudopalisading necrosis, and angiogenesis[
72]. While invasive, GL261 tumors are not known to be metastatic[
65] and, importantly, these tumors do not spontaneously regress as other murine tumors are known to do on occasion[
73].
Applications in immunotherapy research for GBM
The GL261 murine model has perhaps been the most extensively used for preclinical testing of immunotherapeutic approaches for GBM[
74]. Among these studies are: the use of adoptive T cell transfer to restore and induce long-term immunity[
75]; the use of antibodies to improve antitumor T cell activity via augmentation of costimulatory signaling[
76]; and the abrogation of the survival advantage of T
regs[
77]. Gene therapy studies, involving tumor modification for production of inflammatory cytokines (e.g. IL-2) to enhance tumor immunogenicity[
78] as well as with IL-12-expressing DNA plasmids to slow tumor growth and stimulate a robust CTL response,[
79] have also utilized the GL261 model.
The GL261 model has also been widely used in support of vaccine-based studies. GL261 cells express unique tumor antigens, including HMP/AN2,[
80] EphA-2,[
81] and GARC-1,[
82] and these induce a GL261-specific CTL response. GL261 vaccines, used for pulsing DCs, have been curative and, at times, even preventative of tumor engraftment[
83‐
85]. DC vaccines have also been augmented using adjuvants such as plasmid vectors for IFN-γ-inducible protein-10 (IP-10)[
86] or by antibody-mediated depletion of T
regs.[
87] The results of these studies have helped validate GL261 as the model of choice when investigating immunotherapeutic treatment modalities.
The GL261 model has also been used to test experimental methods for mitigating GBM-induced immunosuppression. For instance, in a study by Ueda
et al., mice were treated with peptide vaccinations using GL 261-specific antigens and a TGF-β neutralizing antibody (1D11). Mice receiving both treatments showed 60% 100-day survival, in contrast to the 0-20% survival rates for mice receiving treatments independently. Analysis of animal subjects from this study revealed significantly elevated CTL activity within the lymph nodes and spleens, as well as greater immune cell infiltration, with concurrent reduction of T
regs, in the brains of the mouse hosts. Overall, treatment was associated with promoting the Th
1 phenotype[
88].
The GL261 model has also been used for studying the immunosuppressive effects of TGF-β, which promotes T
reg activity, on B and T cell function[
36,
89,
90]. Additional inhibitory mechanisms beyond TGF-β can be studied using this model. Given GL261 deficiency in PTEN, GL261 tumors accurately model PI3K pathway dysregulation, which is known to promote glial tumor development[
91]. Importantly, PTEN mutations up-regulate expression of PD-L1, a cell surface protein that can be expressed in GBM tumors but not in normal physiologic states[
10]. PD-L1 promotes immunosuppression by inducing T lymphocyte apoptosis,[
92] and monocytes exposed to PD-L1
+ gliomas adopt similar expression patterns of PD-L1, leading to increased T cell apoptosis and tumor resistance to immunotherapy[
93,
94]. Devising methods to target and reverse PD-L1-mediated immunosuppression is thus an important objective for optimizing immunotherapies[
95].
Since GL261 cells express stem cell markers such as CD133 when grown in serum-free media, these cell subpopulations can be isolated and propagated for experimental use. Importantly, when grown in serum-positive media, GL261 cells differentiate and do not express CD133,[
96] as has been similarly reported with primary human GBM cell lines[
45]. Isolation of CD133
+ GL261 cells appears to increase tumorigenicity, as even IC implantation of CD133
+ GL261 cells at small volumes (~100 cells) leads to tumor formation and GL261 neurosphere formation is also greater when culturing CD133
+ cells in serum-free media[
96]. Thus, as is the case for the CT-2A model, GL261 tumors may find use for studying BTSCs and immunosuppression. However, one potentially important consideration to this is that GL261 stem cells appear to enhance tumor immunogenicity. As demonstrated by Pellegatta
et al., DCs pulsed with tumor lysates from GL261 neurospheres, as opposed to normal GL261 cells, generated a more robust T cell and antitumor immune response[
97]. More recently, Xu
et al. lent further evidence to this by showing that immunotherapy with DCs pulsed with GL261 stem cell lysates or DCs pulsed with GL261 lysates were able to prevent tumor formation in 37.5% and 0% of mice, respectively, and induced a significant CTL response[
98].
Despite the extensive information yield from using the GL261 cells, an inherent disadvantage of this model is its moderate immunogenicity which may complicate interpretation of experimental data[
65]. Reduced immunogenicity can confound evaluation of responses to immunotherapy, and extrapolation of results to the clinical setting, as GBM is known to be an immune-privileged tumor that evades host immune recognition[
99]. Nevertheless, the GL261 system presents one of the best characterized syngeneic, immunocompetent models, and it is likely that this model will continue to see extensive use in immunotherapy preclinical research.