Efficacy of a novel double-controlled oncolytic adenovirus driven by the Ki67 core promoter and armed with IL-15 against glioblastoma cells

Abstract

Background: Glioblastoma (GBM) is an immunosuppressive, highly vascular and devastating malignant brain tumor. Even with progressive combination treatment that includes surgery, radiotherapy, and chemotherapy, the prognosis for GBM patients is still extremely poor. Oncolytic adenovirus (OAd) can specifically replicate in GBM cells, permitting the rapid copy of the therapeutic genes it carries. Moreover, E1A is an essential gene in adenoviral replication and is the first gene expressed upon viral infection. E1A expression can be regulated by the Ki67 promoter, while the CMV promoter drives therapeutic gene expression. However, the efficacy of a double-controlled OAd driven by the Ki67 core promoter and armed with IL-15 against GBM cells has not been investigated.

Methods: Fluorescence microscopy was performed to evaluate infection ability in the viruses. Cell viability was detected by CCK-8 assay. Levels of cytokines in different supernatants were determined by ELISA, and IL-15 gene expression was measured by RT-PCR. Angiogenic capacity was analyzed by tube formation assay.

Results: We successfully constructed a double-controlled oncolytic adenovirus driven by the Ki67 core promoter and armed with IL-15 that selectively infected and killed GBM cells while sparing normal cells. The adenoviruses prime IL-15 gene expression to significantly enhance anti-GBM efficacy through effective activation of microglial cells. Moreover, OAd not only directly inhibits angiogenesis but exhibits potent antiangiogenic capacity mediated by the reduction of VEGF secretion.

Conclusions: These results provide new insight into the effects of a novel double-controlled OAd driven by the Ki67 core promoter and armed with IL-15 in glioblastoma treatment, which may help in the development of novel therapies in solid tumors.

Keywords: Angiogenesis; Glioblastoma; IL-15; Immunotherapy; Ki-67 promoter; Oncolytic adenovirus.

Fig. 1

Expression of IL-15 in GBM cells treated with Ad5-Ki67/IL-15. a–d ELISA was used to measure IL-15 levels in conditioned media that different glioma cells were treated with Ad5-Ki67/IL-15 (MOI = 40) for 24, 48, 72, and 96 h. Untreated conditioned medium was used as a control. e–h IL-15 gene expression in GL261, U251, U87 and primary cells BT-01 treated with Ad5-Ki67/IL-15 for 72 h by qPCR. Untreated GBM cells were used as a control

Fig. 2

Characteristics of novel constructed oncolytic adenovirus. a, b Recombinant oncolytic adenovirus (MOI = 40) does not infect microglial HMC3 and BV2 cells (×50, scale bars = 1000 µm). c The capacity of recombinant oncolytic adenovirus to infect GBM cells. Representative photomicrographs were obtained from GL261, U251, U87, and primary cells BT-01 that treated with Ad5-GFP and Ad5-Ki67/GFP at an MOI of 40 for 72 h (×50, scale bars = 1000 µm). d Quantification of the average intensity difference in GBM cells treated with Ad5-GFP and Ad5-Ki67/GFP for 72 h using Image Pro Plus. (n ≥ 3) *P < 0.05 and **P < 0.01. ***P < 0.001 and ****P < 0.0001

Fig. 3

The novel double-controlled OAd driven by the Ki67 core promoter selectively kills GBM cells. a, b CCK-8 assay was performed to evaluate cell viability of HMC3 and BV2 cells that treated with Ad5-GFP, Ad5-Ki67/GFP, and Ad5-Ki67/IL15 at different MOI. c–f CCK-8 assay demonstrated that recombinant oncolytic adenovirus inhibited proliferation of GL261, U251, U87, and primary cells BT-01 at different MOI. Inhibition levels were significantly higher in Ad5-Ki67/GFP and Ad5-Ki67/IL15 compared to Ad5. Results are expressed as a percentage of untreated controls

Fig. 4

The OAd driven by the Ki67 core promoter and armed with IL-15 enhanced GBM eradication. a–d CCK-8 assay was performed to evaluate cell viability of GL261, U251, U87 and primary cells BT-01 that treated with Ad5-GFP, Ad5-Ki67/GFP, and Ad5-Ki67/IL15 (MOI = 40). The inhibition ability of Ad5-Ki67/GFP and Ad5-Ki67/IL15 was more potent than Ad5-GFP at different time points. e GL261 cell proliferation in response to the conditioned medium from virus-treated BV2 microglial cells (BV2-Ad5-GFP-CM, BV2-Ad5-Ki67/GFP-CM, and BV2-Ad5-Ki67/IL15-CM, MOI = 40) at different time points was determined by CCK8. f–h U251, U87 and primary cells BT-01 in response to the conditioned medium from virus-treated HMC3 microglial cells (HMC3-Ad5-GFP-CM, HMC3-Ad5-Ki67/GFP-CM, and HMC3-Ad5-Ki67/IL15-CM, MOI = 40) at different time points was determined by CCK8. All results are expressed as a percentage of untreated controls. Data are presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

Tube formation capacity of HUVECs incubated in different U251 conditioned media. a Glioma cells U251 were respectively treated with Ad5-GFP, Ad5-Ki67/GFP, Ad5-Ki67/IL15 (MOI = 40) for 72 h, and at which time different U251 conditioned media (Ad5-GFP-CM, Ad5-Ki67/GFP-CM and Ad5-Ki67/IL15-CM) were collected. Angiogenic capacity of HUVECs cultured by CM, Ad5-GFP-CM, Ad5-Ki67/GFP-CM and Ad5-Ki67/IL15-CM (MOI = 40) for 6 h on Matrigel (×100, scale bars = 500 µm). CM represented the untreated U251 conditioned medium. b, c Total segments length and quantification of number of tubes generated by HUVECs incubated with different conditioned media. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 6

Tube formation capacity of HUVECs incubated in different U251 conditioned media. a Angiogenic capacity of HUVECs cultured in Ad5-GFP-CM, Ad5-Ki67/GFP-CM, Ad5-Ki67/IL15-CM, Ad5-GFP-CM + VEGF, Ad5-Ki67/GFP-CM + VEGF and Ad5-Ki67/IL15-CM + VEGF (MOI = 40) for 6 h on Matrigel (×100, scale bars = 500 µm). b, c Total segments length and quantification of number of tubes generated by HUVECs incubated with different culture conditions. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 7

VEGF levels in different glioma conditioned media measured by ELISA. a VEGF levels were significantly lower in Ad5-GFP-CM, Ad5-Ki67/GFP-CM, and Ad5-Ki67/IL15-CM (MOI = 40) than in untreated conditioned medium in GL261 cells. b U251. c U87. d Primary cells BT-01. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001