The first and perhaps the most impactful decision the investigator has to make is the origin of the tumor. They can be from the same species as the model animal (allogeneic) or patient-derived (xenograft). Allogeneic tumors can be generated from spontaneously occurring tumors, carcinogen mutagenesis, genetic engineering, and transposon mutagenesis. Xenograft tumors are patient-derived cell lines and cancer stem cells (CSCs). Allogeneic tumors can be implanted on immunocompetent mice whereas xenograft models can only be implanted in immunocompromised or humanized mice.

Therapeutic approaches such as CAR-T and tumor vaccines require that animal models express some of the same tumor neo-antigens as the human tumor. In this respect, genetically engineered mouse models (GEMMs) of gliomas are not always the best choice. GEMM does a good job at recapitulating driver mutations, however, these tumors poorly express tumor neoantigens that are expressed by gliomas, limiting the usefulness of this model when evaluating immune therapies.

For some tumors residing outside the CNS, it has been observed that tumors having a higher mutational burden are better candidates for immunotherapy. This higher mutational burden often leads to the production of more tumor neoantigens which can be targeted by the immune system. This observation has been made in colorectal cancer, as well as other cancers outside the CNS (11, 12). However, the opposite has been observed in gliomas, where a higher tumor mutational burden is often associated with worse survival (12, 13). These findings highlight the need for paying close attention to tumor mutational burden when choosing a pre-clinical model for glioma immunotherapy studies as models having very high tumor mutational burden may not be best suited for immunotherapy studies.

Gliomas are known as aggressive malignancies that are known to grow quickly. It has been reported that GBM specifically has a median specific growth rate of 1.4% per day, with an equivalent volume doubling time of 49.6 days (14). Choosing a pre-clinical model that has a high growth rate is crucial for immunotherapy studies as these tumors grow quickly in patients. Additionally, GBM as well as other gliomas, grow in an infiltrative manner unlike most CNS tumors (15). Given these findings, it is crucial for investigators performing glioma immunotherapy studies to choose pre-clinical models that possess high growth rates and closely parallel glioma growth patterns as this will best replicate what is observed in patients.

GL261 is an allogeneic tumor cell line that was originally created by intracranially injecting C57BL/6 mice with a known carcinogen, that being, 3-methylcholantrene (16). Small pieces of the tumor were taken and subjected to serial passaging over time which is believed to be one of the reasons that GL261 lacks important glial differentiation markers (17). The growth of intracranial GL261 tumors has been described in the literature as rapid with a slightly invasive growth pattern. Additionally, it has been noted that lymphocyte infiltration is extremely low in these tumors. Szatmári and colleagues found that after intracranially implanting 1 × 10⁵, 1 × 10⁴, 1 × 10³ and 1 × 10² GL261 cells into immunocompetent mice, the mean survival time was 25, 27, 36 and 55 days respectively (16). It has been noted that a higher level of MHC1 antigens can be detected in wildtype GL261 tumors when compared to healthy brain, and it has been noted that MHC1 is upregulated in cells exposed to interferon-gamma (16). Compared to other tumor lines, GL261 has a higher mutational burden as whole exome sequencing has shown in vitro GL261 to have 212 frameshift and 4766 missense mutations (18). In the same study, it was shown that in vitro SB28 had 67 frameshift and 41 missense mutations (18). Commonly, GL261 cells are administered to mice via intracranial injection, but these tumors can also be grown in the subcutaneous space by injecting mice with GL261 cells in the flank.

After H. Fraser and colleagues observed the first incidences of mice developing spontaneous gliomas, Serano and colleagues developed the SMA-560 cell line after performing a serial transplantation of spontaneous murine astrocytoma (19). Specifically, tumor tissue underwent homogenization, in vitro culturing, and subsequent transplantation into VM/Dk mice (19). The median survival for animals bearing SMA-560 tumors following injection with 1×10⁴ tumor cells/5 μl has been reported to be approximately 26 days (20). Notably, SMA-560 has high expression of glial fibrillary acid protein (GFAP) and the astrocyte marker glutamine synthetase, and low expression of S-100 proteins (21, 22). Additionally, it has been noted that while MHC1 expression is low in SMA-560 at baseline, it is upregulated in cells exposed to interferon-gamma (23). In a study by Johanns and colleagues, it was observed that SMA-560 had 2171 non-synonymous exome mutations as compared to 4,932 for GL261 (24). SMA-560 cells can be administered to mice via intracranial injection, but these tumors can also be grown in the subcutaneous space by injecting mice with SMA-560 cells in the flank.

CT-2A is an allogeneic cell line that was generated from a malignant astrocytoma that was formed in C57BL/6J mice that were injected in the cerebrum with a known carcinogen, that being, 20-methylcholanthrene (25). These cells have a high tumorigenicity as mice have a median survival of 20 days after intracranial injection with 1x10⁴ cells (23). This tumor is known to have high levels of complex gangliosides and very low distribution of GM3 (monosialodihexosylganglioside) which has been classified as an anti-angiogenic ganglioside (26, 27). Additionally, CT-2A cells are known to be deficient in the tumor suppressor PTEN and are wild-type for p53. These tumors have a high mitotic index and unlike many other tumors, demonstrate high levels of microvascular proliferation (25, 28). Commonly, CT-2A cells are administered to mice via intracranial injection, but these tumors can also be grown in the subcutaneous space by injecting mice with CT-2A cells in the flank.

SB28 is an allogeneic cell line that was generated by using sleeping beauty transposons to insert constructs capable of targeting the P53, RAS and PDGF pathways. These sleeping beauty transposon flanked pT2/CAG-NRasV12 and pT2/shp53/mPDGF constructs were then injected into the right ventricle of C57L/6 mice (23). It has been noted that SB28 bearing mice have extremely low MHC1 expression and limited CD8 T cell infiltration posing a challenge for immunotherapy-based studies using this cell line (18). The median overall survival for mice injected with 1x10⁴ SB28 cells is 19 days and whole exome sequencing has demonstrated these cells have just 108 mutations as compared to the over 4900 mutations present in GL261 (18, 24). This cell line can be injected intracranially, but these tumors can also be grown in the subcutaneous space by injecting mice with SB28 cells in the flank.

U251 is a xenograft cell line that was derived from a glioblastoma multiforme using explant technique (29). These cells must be injected into immunocompromised mice and the median overall survival for tumor bearing mice is 22 days (30). It has been noted in previous studies that B7-H4 expression is upregulated in U251 glioma stem-like cells and while U251 cells do not carry an IDH1 mutation, these cells do carry mutations in hTERT, PTEN and p53 (31). Additionally, these cells have a methylated MGMT status (31). These cells must be injected into immunocompromised mice, limiting their usability in immunotherapy-based studies.

U87 is a xenograft cell line that was derived from a GBM in a female patient. Immunocompromised mice bearing U87 tumors have a median survival of 28.6 days following tumor implantation (30). Interestingly, it has been observed that U87 and U251 tumors only grow to kill their hosts when 1,000,000 or 1,500,000 cells, respectively, are injected in the striatum of nude mice (30). It has been shown that injecting less cells leads to a lack of tumor growth and avoidance of death of the host. It has been noted that U87 cells possess hTERT, ATRX and PTEN mutations, however, these cells do not carry p53 or IDH1 mutations (31). Additionally, these cells have a methylated MGMT status (32). These cells must be injected into immunocompromised mice and can be injected intracranially or into the flank region.

QPP is an immunocompetent murine spontaneous GBM model, in which three common patient-relevant tumor suppressor genes, Quaking (Qk in mouse and QKI in human), Trp53, and PTEN, were deleted (33). The tumors that were derived from this model displayed histopathological and transcriptomic heterogeneity, which can manifest the subtypes of GBM (33). The cell line QPP7, isolated from this model, was used to establish the syngeneic orthotopic glioma in C57L/6 mice with genetic manipulations in previous research (34). Importantly, this syngeneic mouse glioma demonstrated the landscape of the tumor immune microenvironment, including M1/M2-like macrophages, T cells, NK cells, and myeloid-derived suppressor cells (MDSCs) (34). Based on immune profiling and single-cell sequencing analyses, a most recent study reported that both implanted and spontaneous QPP models recapitulate the immunosuppressive myeloid dominant nature of the tumor microenvironment of human gliomas (35).

The Cre-LoxP system allows for the targeting of tumor genes in mouse brain tissue of interest which provides for insight into the genetic drivers of GBM and the differences in genetic drivers between primary and secondary GBMs (36). To create this model, a mouse strain known as the “Cre driver strain” which has Cre recombinase with a promoter, and a mouse strain known as the “LoxP floxed strain” that has LoxP floxed exons of the target gene, were bred together (36). By breeding these strains together this would result in a deletion of the floxed region and a subsequent inactivation of the gene in the desired brain tissue of interest, leaving the target gene active in tissues outside this region (36). Specifically, this model has been used to test the role of p53 and PTEN function in GFAP positive GBM. In a study by Zheng and colleagues, the research team created a p53 and PTEN double knockout mouse where this knockout was targeted to astrocytes specifically (37). The research team found that a loss of both p53 and PTEN would regulate Myc levels and subsequently control NSC self-renewal and differentiation (37, 38). The Cre-LoxP mouse model is extremely valuable for testing immunotherapy applications as this model can activate or inactivate genes that can impact the tumor microenvironment, and this system has also been used to control the cytotoxic potential of CAR-T cells (39).

The HGMM model was developed by Huang and colleagues to better understand the role of CCL18 which is expressed in humans but not in rodents (40). Specifically, the research team developed this model to study the interaction of human glioma cells with human microglia. To create this model, the research team depleted intrinsic microglia from murine organotypic brain slices and then injected either human glioma cells into these slices, or injected both human glioma cells and human stem cell derived microglial cells into these slices (40). Interestingly, the research team found that in slices injected with both human glioma cells and human stem cell derived microglial cells, human stem cell derived microglial cells showed higher levels of sphericity and cell body volume highlighting how the tumor microenvironment impacts the morphology of these cells (40). Additionally, the research team assessed whether the presence of human stem cell derived microglial cells with human glioma cells created an increase in the expression level of genes known to be upregulated by the glioma environment. The research team found that there was an upregulation in IL-10, Osteopontin, MMP14, VEGF, TGF β, and CCL18 in samples containing both human stem cell derived microglial cells with human glioma cells, as opposed to samples containing human glioma cells alone (40).

Ultimately, this model highlights that the presence of human stem cell derived microglial cells with human glioma cells results in not only larger tumors but also an upregulation of genes similar to those known to be upregulated in certain gliomas (40–45). Recent findings have begun to shed light on how GBM can use microglia to induce immunosuppression within the tumor microenvironment. This model will be valuable in developing a deeper understanding of this hijacking and potentially enable therapeutic exploitation of this mechanism (46).