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. 2019 May 15:14:107-120.
doi: 10.1016/j.omto.2019.05.001. eCollection 2019 Sep 27.

An Oncolytic Adenovirus Vector Expressing p14 FAST Protein Induces Widespread Syncytium Formation and Reduces Tumor Growth Rate In Vivo

Josh Del Papa  1   2   3 , Julia Petryk  4 , John C Bell  2   3   4 , Robin J Parks  1   2   3   5
Affiliations

An Oncolytic Adenovirus Vector Expressing p14 FAST Protein Induces Widespread Syncytium Formation and Reduces Tumor Growth Rate In Vivo

Josh Del Papa et al. Mol Ther Oncolytics. .

Abstract

Intratumoral injection of oncolytic viruses provides a direct means of tumor cell destruction for inoperable tumors. Unfortunately, oncolytic vectors based on human adenovirus (HAdV) typically do not spread efficiently throughout the tumor mass, reducing the efficacy of treatment. In this study, we explore the efficacy of a conditionally replicating HAdV vector expressing the p14 Fusion-Associated Small Transmembrane (FAST) protein (CRAdFAST) in both immunocompetent and immunodeficient mouse models of cancer. The p14 FAST protein mediates cell-cell fusion, which may enhance spread of the virus-mediated, tumor cell-killing effect. In the murine 4T1 model of cancer, treatment with CRAdFAST resulted in enhanced cell death compared to vector lacking the p14 FAST gene, but it did not reduce the tumor growth rate in vivo. In the human A549 lung adenocarcinoma model of cancer, CRAdFAST showed significantly improved oncolytic efficacy in vitro and in vivo. In an A549 xenograft tumor model in vivo, CRAdFAST induced tumor cell fusion, which led to the formation of large acellular regions within the tumor and significantly reduced the tumor growth rate compared to control vector. Our results indicate that expression of p14 FAST from an oncolytic HAdV can improve vector efficacy for the treatment of cancer.

Keywords: FAST; adenovirus; cancer; cell fusion; oncolytic.

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Figures

Figure 1
Figure 1
Adenovirus Constructs Schematic diagrams of HAdV vectors used in this study: AdFAST, an E1- and E3-deleted non-replicating HAdV vector expressing p14 FAST from the cytomegalovirus immediate early enhancer-promoter in the E1; CRAd, an E1Δ24, E3-deleted conditionally replicating vector; and CRAdFAST, an E1Δ24, E3-deleted conditionally replicating vector expressing p14 FAST from within the E3 region through the inclusion of a major late promoter splice acceptor (SA).
Figure 2
Figure 2
Replication of HAdV-5 Oncolytic Vectors in 4T1 Cells Is Non-productive (A) 4T1 cells were infected with either AdFAST or CRAdFAST at an MOI of 10. At 4, 24, 48, and 72 hpi, media were removed and the cells were harvested in SDS-Proteinase K buffer for DNA extraction. qPCR was performed on 200 ng total isolated DNA for the Ad hexon genome region. Error bars represent mean ± SD. (B) 4T1 cells were infected with either CRAd or CRAdFAST at an MOI of 10. Cells and medium were collected at 4, 24, and 72 hpi. Viral titers were determined by plaque assay on 293 cells. Error bars represent mean ± SD. (C) 4T1 cells were infected with either AdFAST or CRAdFAST at an MOI of 3 and harvested at 24, 48, and 72 hpi. Immunoblot analysis was performed to examine HAdV fiber and HA (p14 FAST) protein levels, and actin was used as a loading control. (D) 4T1 cells were infected at an MOI of 50 with CRAdFAST for 1 h, and they were overlaid with medium lacking (−) or containing (+) 20 μg/mL cytosine arabinoside (Ara-C) to inhibit DNA replication. Crude protein samples were harvested at 24, 48, and 72 hpi, and they were assayed by immunoblot for the expressions of fiber protein, p14 FAST protein, and actin (loading control).
Figure 3
Figure 3
CRAdFAST Induces Cellular Fusion and Cell Death in 4T1 Cells More Efficiently Than Non-replicating AdFAST (A) 4T1 cells were mock infected or infected with AdFAST, CRAd, or CRAdFAST at an MOI of 100. Phase-contrast images were obtained at 24, 48, and 72 hpi. (B) 4T1 cells were mock infected or infected with AdFAST, CRAd, or CRAdFAST at an MOI of 1,000 in triplicate wells (n = 6 technical replicates, n = 2 independent experimental replicates). At 48 hpi, metabolic activity was assessed by MTS assay, and the values are reported relative to metabolic activity in mock-infected cells. Mean values reported ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (C) 4T1 cells were mock infected or infected with CRAd or CRAdFAST at MOIs of 10 and 1,000, and protein samples were harvested at 48 hpi. Protein lysates from infected cells were subjected to immunoblot for HAdV fiber, full-length caspase-3, and cleaved caspase-3, with actin used as a loading control.
Figure 4
Figure 4
Expression of p14 FAST Protein Does Not Improve Oncolytic Efficacy of CRAd in the Murine 4T1 Model of Cancer Tumor-bearing mice were injected intratumorally with PBS or 7 × 109 PFU CRAd or CRAdFAST, and tumor volumes were measured for eight mice per group three times weekly. (A) Symbols represent mean calculated tumor volumes over time. Lines represent non-linear regression model of tumor growth rates. At day 11, PBS-treated tumors were significantly larger than CRAdFAST-treated tumors (*p = 0.03). Error bars represent mean ± SEM; * p = 0.033 for CRAdFAST compared to PBS. (B) Tumor-specific growth rates were calculated for each tumor and averaged; error bars represent SD. (C) Sections of tumor and liver from mice sacrificed at day 5 post-injection were probed with anti-HA antibody (p14 FAST) and counterstained with hematoxylin to assess p14 FAST expression. At 1×, 20×, and 100× magnifications, scale bars represent 2,000, 100, and 20 μm, respectively.
Figure 5
Figure 5
Analysis of Protein Expression and Virus Yield of CRAdFAST in A549 Cells In Vitro (A) A549 and 4T1 cells were infected at an MOI of 10 and harvested at 48 hpi. A549 samples were diluted to 1/100, 1/400, 1/800, and 1/1,000 to compare protein levels to those obtained in 4T1 cells. (B) A549 cells were infected at an MOI of 3 with CRAd, AdRFP, or CRAdFAST or mock infected, and they were overlaid with medium lacking (−) or containing (+) 20 μg/mL Ara-C to inhibit viral DNA replication. Cells were harvested at 24 hpi, and immunoblot analysis was performed to examine HAdV fiber, RFP, and HA (p14 FAST) protein levels, with actin used as a loading control. (C) A549 cells were infected at an MOI of 10 with either CRAd or CRAdFAST. Cells and media were harvested at 4, 24, and 72 hpi. Viral titers were determined by plaque assay. Error bars represent mean ± SD.
Figure 6
Figure 6
CRAdFAST Mediates Enhanced Cell Fusion and Cell Death in A549 Cells In Vitro (A) Phase-contrast images taken at 24 and 48 hpi of A549 cells infected at MOIs of 0.01, 0.1, and 1 with AdFAST, CRAd, or CRAdFAST. (B) A549 cells were mock infected or infected with AdFAST, CRAd, or CRAdFAST at an MOI of 1 in triplicate (n = 6 technical replicates, n = 2 independent experimental replicates). At 24, 48, and 72 hpi, metabolic activity was assessed by MTS assay; values reported are relative to mock-infected cells and represent mean ± SD (****p < 0.0001). (C) A549 cells were mock infected or infected at MOIs of 0.0625, 0.125, 0.25, 0.5, and 1 with CRAd or CRAdFAST, and they were harvested for immunoblot analysis at 48 hpi. Blots were probed for HAdV fiber, full-length caspase-3, or cleaved caspase-3, and actin was used as a loading control.
Figure 7
Figure 7
Expression of p14 FAST Protein Significantly Improves Oncolytic Efficacy of CRAdFAST following Intratumoral Injection in a Human A549 Xenograft Model of Cancer Mice bearing A549 tumors were injected intratumorally with PBS, CRAd, or CRAdFAST. Tumor volumes were measured for five mice per group, three times weekly. (A) Symbols represent calculated tumor volumes over time. Lines represent linear regression model of tumor growth rates. Beyond day 29, PBS-treated tumors are significantly larger than both CRAd- and CRAdFAST-treated tumors. Error bars represent mean ± SEM; *p <0.05 for CRAdFAST compared to PBS; Y represents p < 0.05 for CRAd compared to PBS. (B) Tumor growth rates were calculated for each tumor and averaged; error bars represent SD (**p = 0.0013, ****p < 0.0001).
Figure 8
Figure 8
Treatment with CRAdFAST Induces Syncytium Formation in A549 Tumors In Vivo (A) Sections of A549 xenograft tumors from mice treated with CRAd or CRAdFAST and sacrificed at day 5 post-injection were probed with anti-HAdV-5 antibody and counterstained with hematoxylin to assess HAdV replication. Images were taken at 1× and 20× magnifications. (B) Tumor sections from mice treated with CRAd or CRAdFAST and sacrificed at day 5 and day 50 post-injection were probed with anti-HA (p14 FAST) antibody and counterstained with hematoxylin to assess p14 FAST expression. Images were taken at 1×, 20×, and 100× magnifications. (C) Liver sections from mice treated with CRAd or CRAdFAST and sacrificed at day 5 and day 50 post-injection were probed with anti-HA (p14 FAST) and counterstained with hematoxylin to assess p14 FAST expression. Images were taken at 1× and 20× magnifications. (D) Liver sections from mice treated with CRAd or CRAdFAST and sacrificed at day 5 post-injection were probed with anti-HAdV-5 and counterstained with hematoxylin to assess HAdV replication. Images were taken at 5× and 20× magnifications. At 1×, 5×, 20×, and 100×, scale bars represent 2,000, 500, 100, and 20 μm, respectively.
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