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. 2023 Sep 27;15(19):4738.
doi: 10.3390/cancers15194738.

Oncolytic Viral Therapy for Glioma by Recombinant Sindbis Virus

Kangyixin Sun  1   2   3 , Xiangwei Shi  2   3   4 , Li Li  2 , Xiupeng Nie  5 , Lin Xu  5 , Fan Jia  2   4 , Fuqiang Xu  1   2   3   4   6
Affiliations

Oncolytic Viral Therapy for Glioma by Recombinant Sindbis Virus

Kangyixin Sun et al. Cancers (Basel). .

Abstract

Background: The characteristics of glioblastoma, such as drug resistance during treatment, short patient survival, and high recurrence rates, have made patients with glioblastoma more likely to benefit from oncolytic therapy.

Methods: In this study, we investigated the safety of the sindbis virus by injecting virus intravenously and intracranially in mice and evaluated the therapeutic effect of the virus carrying different combinations of IL-12, IL-7, and GM-CSF on glioma in a glioma-bearing mouse model.

Results: SINV was autologously eliminated from the serum and organs as well as from neural networks after entering mice. Furthermore, SINV was restricted to the injection site in the tree shrew brain and did not spread throughout the whole brain. In addition, we found that SINV-induced apoptosis in conjunction with the stimulation of the immune system by tumor-killing cytokines substantially suppressed tumor development. It is worth mentioning that SINV carrying IL-7 and IL-12 had the most notable glioma-killing effect. Furthermore, in an intracranial glioma model, SINV containing IL-7 and IL-12 effectively prolonged the survival time of mice and inhibited glioma progression.

Conclusions: These results suggest that SINV has a significant safety profile as an oncolytic virus and that combining SINV with cytokines is an efficient treatment option for malignant gliomas.

Keywords: cytokines; glioblastoma; killing glioma; oncolytic virus; sindbis virus.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Figure 1
Figure 1
Safety assessment of SINV. (A) The 100 µL blood samples of mice from 1 to 5 h after tail vein injection of SINV-EGFP were collected and used to infect BHK-21 cells, areas with viral expression were selected for recording in an inverted microscope at 24 h post-infection. Scale bars, 100 µm; The mean value of the number of viral particles per 1 mL of blood samples is shown in (B). (C) The distribution of viral RNA extracted from the heart, liver, spleen, kidney, brain, and blood of mice after 1, 5, and 10 days (n = 3 at each time point) of intraperitoneal injection of SINV-EGFP. (D) H&E staining results of the organs of the mice at 60 days post-injection. As shown by the red arrow in the figure, the number of lymphocytes was reduced in the spleen, and more multinucleated giant cell infiltration was seen in the tissue, as shown by the black arrows in the Figure. In addition, a small number of loose and edematous renal tubular epithelial cells were seen in the kidney, as shown by the red arrow, and a small number of inflammatory cells were seen in the tissue, as shown by the black arrow. The rest of the tissue is normal and unlabeled. Scale bars, 100 µm. (E) The signal of the virus in different brain regions at 6 days as well as at 16 days after SINV intracranial injection (magnification, 10×). Scale bars, 20 µm. (F) The SINV could infect the neurons of tree shrews. The superficial gray layer, optic nerve layer, and intermediate gray layer of the superior colliculus were detected with green positive signals. Scale bars: upper left image, 1 mm; top right image, 200 µm; bottom image, 20 µm.
Figure 2
Figure 2
SINV-EGFP effectively induces apoptosis in some GBM cells in vitro. (A) Glioma cells and glial cells infected with SINV-EGFP (MOI = 0.1); representative images were obtained under an inverted microscope at 24 h post-infection (magnification, 20×). Scale bars, 100 µm. (B) All cells were infected with SINV-EGFP (MOI = 1, 0.1 or 0.01) and cell viability was assessed at 48 h post-infection. Cell viability was considered to be 100% in mock-infected cells. Data are presented as the mean and s.d. of 6 independent experiments.
Figure 3
Figure 3
Tumor-killing effect of SINV on subcutaneous gliomas. (A) Treatment schema. After subcutaneous injection of U-87MG cells with PBS into the right thigh of nude mice, SINV-EGFP was injected into glioma tumors on days 7, 9, and 11 after cell implantation. (B) Tumor length and width were measured using calipers and tumor volume was calculated using the formula (n = 5 for each group), and the weight of the mice was also recorded continuously using an electronic scale. (C) H&E staining results of subcutaneous tumor in mice. The SINV group showed a large area of tumor cell necrosis, as shown by the red arrow, and a small amount of inflammatory cell infiltration, as shown by the black arrow. Scale bars, 50 µm.
Figure 4
Figure 4
Cytokine arming improves therapeutic efficacy. The tumor-killing effect of 6 engineered SINVs carrying cytokines on U-87MG. (A) The schematic diagram of recombinant viral vectors (B) BHK-21 and U-87MG cells were infected with recombinant SINVs (MOI = 1, 0.1, 0.01) and at 48 h post-injection, the effect of each virus on the viability of both cells was counted (n = 6). (C) Treatment strategy. A total of 5 × 106 U-87MG-Luc cells with 100 µL Matrigel were implanted into the thigh of mice. Recombinant SINVs were intratumorally injected into glioma tumors on days 7, 9, and 11 after cell implantation. (D) Representative tumor photo at 21 days post-treatment. (E) Volume–time plot of mouse tumors during treatment and comparison of tumor weights at 21 days after treatment (n = 5). (F) Comparison of NK cells within the tumor after 4 days of treatment. Error bars indicate S.E.M.; results are representatives of 3 independent experiments.
Figure 5
Figure 5
The performance of SINV IL-12/IL-7 in the U-87MG intracranial model. (A) Experimental timeline for SINV IL-12/IL-7 treatment in GBM model. (B) Luciferase imaging of U-87MG tumors 21 days after tumor implantation (n = 3). (C) The quantitative result of luciferase in (B) plot. Mean ± s.e.m., n = 3 mice per group, two-tailed unpaired t-test with Welch correction. (D) Survival curves of U-87MG-loaded mice treated with PBS, SINV IL-12/IL-7, respectively (n = 6). (E) H&E-stained coronal sections of U-87MG tumor-bearing mice brains treated at 21 days and 45 days post-cell implantation, respectively. Representative images of each group are presented. Scale bar, 1000 µm.
Figure 6
Figure 6
SINV carrying IL-12 and IL-7 can attack distal lesions. (A)Treatment options. 100 µL of SINV (1 × 107 PFU) was injected into the tail vein of mice on days 7, 9, and 11 after cell transplantation. Tumors expressing luciferase were observed by IVIS on the day of administration and after administration. (B) Luciferase imaging of U-87MG tumors before and after treatment (n = 4). (C) Corresponding quantification of luciferase expression in (B). Mean ± s.e.m., n = 4 per group, two-tailed unpaired t-test, Welch’s correction. Quantitative results 21 days after treatment were statistically significant and are labeled with “**” in (C). (D) Comparison of tumors removed after treatment. (E) Volume–time plot of mouse tumors during treatment.
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