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Tang et al. Neuroimmunol Neuroinflammation 2021;8:64-9 I http://dx.doi.org/10.20517/2347-8659.2020.28 Page 65
[3]
living longer than 5 years . These facts indicate that there is a strong need to develop novel treatments for
glioma.

Recently, our group developed a novel viro-immunotherapy for early-stage murine glioma with 83%
[4]
tumor regression rate . Despite the impressive response that this treatment elicits, such a response is not
representative of what occurs in the clinical setting, since patients are usually diagnosed at an advanced
stage of tumor progression.

Surgical resection has always been the first line of treatment for gliomas. However, surgical resection
[5]
promotes an immunosuppressive tumor microenvironment . It has been reported that interferon-γ
[6]
secretion and cell cytotoxicity of natural killer cells are profoundly suppressed ; the number of cytotoxic
[8,9]
[7]
T cells and T helper cells markedly decreases, while the number of regulatory T cells (Tregs) and low-
[10]
density neutrophils significantly increases in the postoperative period. All these factors could contribute
to the poor prognosis and recurrence of tumor.
As a preliminary step toward testing a novel viro-immunotherapy, we asked whether mice would survive
resection of an advanced murine glioma.

METHODS
Cell culture
Glioma 261 (GL261) is a murine glioma cell line. GL261 cells were purchased from the National Cancer
Institute Division of Cancer Treatment and Diagnosis Repository and tested negative for mycoplasma.
Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; 10013CV, Corning Incorporated)
containing 5 mmol/L HEPES, 1.3 mmol/L L-glutamine, 50 µmol/L 2-ME, penicillin, streptomycin and 10%
fetal bovine serum (FBS) at 37 °C and 5% CO . GL261 neurosphere (GL261 NS) cells, which are cancer
2
stem-like non-adherent cells, were generated by culturing in untreated cell culture flasks (08-757-501,
Corning Incorporated) with DMEM/F12 + GlutaMAX (10565018, Life Technologies) culture medium
containing penicillin/streptomycin (17-602E, Lonza), B27 with vitamin A (17504044, Life Technologies),
20 ng/mL recombinant human epidermal growth factor (EGF; 236-EG-200, R&D Systems), 20 ng/mL
recombinant human fibroblast growth factor (FGF; 233-FB-025/CF, R&D Systems), and 5 µg/mL heparin
(H3149100KU, Sigma-Aldrich) in an incubator at 37 °C with 5% CO 2 [11] .

Animals
C57BL/6J mice were purchased from The Jackson Laboratory and maintained as colonies in animal
facilities at the University of Illinois Urbana-Champaign. Mice of both sexes were used in experiments
when they were 2-3 months old. All animal studies were approved by the Institutional Animal Care and
Use Committee (IACUC) at the University of Illinois Urbana-Champaign.

Intracranial tumor establishment
GL261 NS cells were harvested, washed twice with Hanks’ Balanced Salt Solution (HBSS; 21023CV,
Corning Incorporated) and stereotactically injected into the brain of mice anesthetized with isoflurane
4
(59399-106-01, Akorn). A total of 5 × 10 GL261 NS cells in 0.5 µL HBSS were infused into the ventral
striatum (0.5 mm rostral; 2.25 mm lateral; 3.3 mm ventral). Mice were euthanized at 75% of baseline body
weight or when they exhibited symptoms of neurological impairment, lethargy, or pain, in accordance with
IACUC guidelines.

Intracranial tumor resection
Tumor-bearing mice were anesthetized with isoflurane (59399-106-01, Akorn) and placed in a stereotactic
stage. Mice were treated with a 200-µL subcutaneous injection of carprofen (0.5 mg/mL; Division of