Blood Outgrowth Endothelial Cells as a Cellular Carrier for Oncolytic Vesicular Stomatitis Virus Expressing Interferon-β in Preclinical Models of Non-Small Cell Lung Cancer

Abstract

Oncolytic viruses have demonstrated efficacy in numerous tumor models including non-small cell lung cancer (NSCLC). One limitation of viral therapy for metastatic lung cancer is that systemic administration can be hindered by complement and antiviral immunity. Thus, we investigated whether ex vivo-infected blood outgrowth endothelial cells (BOECs) could be used to deliver VSV-IFNβ in preclinical models of NSCLC. BOECs were obtained from human donors or C57/Bl6 mice. VSV was engineered to produce GFP or IFNβ. Human and murine BOECs could be infected by VSV-GFP and VSV-IFNβ. Infected BOECs resulted in killing of NSCLC cells in vitro and shielded VSV-IFNβ from antibody neutralization. Mouse BOECs localized to lungs of mice bearing syngeneic LM2 lung tumors, and infected murine BOECs reduced tumor burden in this model. In an immune-deficient A549 xenograft model, mice treated with VSV-IFNβ-infected human BOECs exhibited superior antitumor activity and survival of mice (n = 10, P < .05 compared to VSV-IFNβ alone). We conclude that BOECs can be used as a carrier for delivery of oncolytic VSV-IFNβ. This may be an effective strategy for clinical translation of oncolytic virotherapy for patients with metastatic NSCLC.

Figure 1

Susceptibility of BOECs to VSV-IFNβ infection. Human BOEC (hBOECs) were infected with VSV-GFP or VSV-IFNβ at MOI of 1. A) Cell viability was assayed by trypan blue exclusion from time of infection to 96 hours post-infection. B) Viral titers were determined from media employing viral plaque assay after hBOEC infection daily up to 72 hours post-infection. C-D) Murine BOECs (mBOECs) derived from C57/Bl6 mice were infected with VSV-GFP and VSV-IFNβ as in A-B. *denotes P < .02 compared to PBS control. # denotes P < .05 between VSV-GFP and VSV-IFNβ. E) hBOECs were infected at the indicated MOI with both VSV-GFP and VSV-IFNβ and assayed for cell viability at indicated time points.

Figure 2

VSV can infect BOECs and transfer infection to NSCLC cells in vitro. A) An equal number of VSV-infected BOECs were co-cultured with H2009 cells and cell viability measured after 72 hours by trypan blue exclusion (Note: BOECs are not adherent to wells in absence of collagen-coating and are washed away prior to counting). Cell viability is normalized to control (PBS-treated BOECs co-cultured with H2009 cells). *indicates statistical significance (P < .002) compared to control. B) Co-culture of H2009 cells to VSV-hIFNβ-infected hBOECs (MOI =10) was done at varying ratios of H2009:hBOECs as indicated. Cell viability was normalized to H2009 cells treated with uninfected hBOEC cells. *indicates statistical significance (P < .03) compared to control. C) Viral titer was determined from medium harvested from a ratio of H2009 cells to VSV-hIFNβ infected hBOECs. Viral titers were determined using viral plaque assay.

Figure 3

Virus from infected BOECs were protected from neutralizing antibodies. A) H2009 and H2030 cells were treated with VSV-GFP or VSV-GFP infected BOECS in the presence of decreasing concentration of anti-VSV neutralizing antibodies (NAB) and assayed using fluorescence microscopy 24 hours after infection. B) VSV titer from cell medium of co-cultured VSV infected BOECs or naked VSV-GFP with H2009 cells. TCID₅₀ is tissue culture infective dose 50%.

Figure 4

Trafficking of mBOEC cells to lung tumor growth in mice and preliminary efficacy in immune competent mice. A) A/J mice were IV injected with lung adenocarcinoma cells, and 34 days later were IV injected with mBOEC-fLuc cells. Luminescent imaging of mice along with resected lungs and livers are shown at the indicated times. The control mouse did not receive mBOEC-fLuc cells as a negative control. B) A single A/J mouse was IV injected with LM2 cells, and 34 d later was IV injected with mBOEC-fLuc cells. A second mouse without tumors was IV injected with mBOEC-fLuc cells at the same time as the first mouse. Luminescent imaging of mice along with resected lungs and livers are shown at the indicated times. C) 2 × 10⁵ LM2 cells were IV injected into A/J mice and were treated with mBOECs (n = 5), VSV-mIFNβ (n = 5), or VSV-mIFNβ infected mBOECs (n = 5) by tail vein injection on days 20, 22, 24, 41, 43, and 45. On day 48 mice were sacrificed and their lungs removed and weighed. Lung weights were normalized to the lung weight of untreated normal mice (n = 5). Relative tumor burden, normalized to untreated normal lung weight, at necropsy is shown for each experimental group. Results are expressed as the mean +/− standard deviation. # P = .3791 for mBOEC cells alone compared to VSV-mIFNβ alone and * P = .09 for mBOEC cells alone compared to VSV-mIFNβ infected mBOEC cells. D) RNA from lungs of treated mice was extracted on day 27 and subjected to RT-PCR for VSV-N RNA to estimate the copy number in lung tissue compared between naked VSV-IFNβ and BOEC-infected with VSV-IFNβ (n = 4 for naked VSV-IFNβ and 5 for BOEC-VSV-IFNβ).

Figure 5

Systemic delivery of VSV infection by infected mBOECs to orthotopically implanted lung tumors. A) Luc-A549 tumor bearing SCID Beige mice were given either PBS, VSV-mIFNβ, mBOECs, or VSV-mIFNβ infected mBOECs. Tumor burden was estimated using levels of luciferase activity measured in radiance. *P < .02 for mBOEC infected with VSV-mIFNβ compared to mBOEC cells alone. # P < .03 for mBOEC infected with VSV-mIFNβ compared to PBS control. B) Bioluminescent imaging to detect the Luc-A549 cell signal in mice was performed at the indicated times. C) Survival of mice was determined using Kaplan–Meier method. Systemic delivery of VSV-mIFNβ infected mBOECs significantly prolonged the life of mice with lung cancer compared to PBS, mBOECs, or VSV-mIFNβ treatments. # P < .001 for PBS or mBOEC cells alone compared to VSV-mIFNβ infected mBOEC cells and * P < .05 for VSV-mIFNβ alone compared to VSV-mIFNβ infected mBOEC cells.

Supplemental Figure 1

VSV-hIFNβ from infected BOECS is shielded from neutralizing antibodies. H2030 was treated in vitro with VSV-hIFNβ or BOECS infected with VSV-hIFNβ in the presence of decreasing concentration of anti-VSV neutralizing antibodies (NAB). A) Photomicrographs were taken 24 hours after infection to assess cytopathic effect. B) VSV-hIFNβ titer from cell medium of co-cultured VSV-hIFNβ infected BOECs or naked VSV-hIFNβ with H2030 cells.