Immunostimulatory Profile of Cancer Cell Death by the AdV-Lumc007-Derived Oncolytic Virus 'GoraVir' in Cultured Pancreatic Cancer Cells

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy which shows unparalleled therapeutic resistance. Oncolytic viruses have emerged as a new treatment approach and convey their antitumor activity through lysis of cancer cells. The therapeutic efficacy of oncolytic viruses is largely dependent on the induction of immunogenic cell death (ICD) and the subsequent antitumor immune responses. However, the concurrent generation of antiviral immune responses may also limit the a virus' therapeutic window. GoraVir is a new oncolytic adenovirus derived from the Human Adenovirus B (HAdV-B) isolate AdV-lumc007 which was isolated from a gorilla and has demonstrated excellent lytic activity in both in vitro and in vivo models of PDAC. In this study, we characterized the immunostimulatory profile of cancer cell death induced by GoraVir and the concerted cellular antiviral responses in three conventional pancreatic cancer cell lines. While GoraVir was shown to induce late apoptotic/necrotic cell death at earlier time points post infection than the human adenovirus type 5 (HAdV-C5), similar levels of ICD markers were expressed. Moreover, GoraVir was shown to induce ICD not dependent on STING expression and regardless of subsequent antiviral responses. Together, these data demonstrate that GoraVir is an excellent candidate for use in oncolytic virotherapy.

Keywords: STING; immunogenic cell death; non-human primate adenovirus; oncolytic virus; pancreatic ductal adenocarcinoma.

Figure 1

Virus-induced cell death of pancreatic cancer cell lines by GoraVir. Cells were infected with GoraVir or HAdV-C5 at MOI 10 and stained with Annexin V and propidium iodide at 48 and 72 hrs post infection (hpi). Depicted are representative figures out of n = 3 for (A) BxPC-3, (D) HPAF-II, and (G) MIA PaCa-2. Annexin V + PI- cells were classified as early apoptotic (EA) cells and Annexin V-PI+ and Annexin V + PI+ together as late apoptotic/necrotic (LA/N) cells. Percentage of single cell population in EA or LA/N was determined at 48 and 72 hpi for BxPC-3 (B,C), HPAF-II (E,F), and MIA PaCa-2 (H,I), respectively. Depicted are means of n = 3 and comparisons were performed using one-way ANOVA with Tukey correction for multiple testing. Significant differences are indicated by asterisks, with p values < 0.05 shown as *, <0.01 as **, and <0.001 as ***.

Figure 2

Protein expression of immunogenic cell death (ICD) markers in pancreatic cancer cell lines upon infection with GoraVir and HAdV-C5. (A) Light microscopy images of pancreatic cancer cells infected with GoraVir or HAdV-C5 at 72 hpi. Scale bar represents 500 µm; (B) BxPC-3, (C) HPAF-II, and (D) MIA PaCa-2 cells were infected with GoraVir or HAdV-C5 at MOI 10 and 72 hpi. The percentage of calreticulin (CTR)-positive, HSP70-positive, or double-positive cells was determined by flow cytometry. Means are depicted of n = 3 biologically independent replicates. Means were compared using one-way ANOVA with Tukey correction for multiple testing. Significant differences are indicated by asterisks, with p values < 0.05 shown as *, <0.01 shown as **, and <0.001 shown as ***.

Figure 3

Replication kinetics of GoraVir and HAdV-C5 in pancreatic cancer cell lines. (A) BxPC-3, (B) HPAF-II, and (C) MIA PaCa-2 cells were infected with either GoraVir or HAdV-C5 at MOI 2 and viral genome copies were determined at 24, 48, and 72 hpi. Means are depicted of n = 3 biologically independent replicates. Statistical analyses were performed using multiple unpaired t tests and the Holm-Šídák correction. Significant differences are indicated by asterisks, with p values < 0.001 shown as ***.

Figure 4

ISG response upon infection with GoraVir or HAdV-C5 through cGAS/STING signaling. (A) BxPC-3, (B) HPAF-II, and (C) MIA PaCa-2 cells were infected with GoraVir or HAdV-C5 at MOI 10 and mRNA expression of ISG15 and ISG54 was measured at 24 h post infection. Fold change expression was calculated relative to GAPDH. Means are depicted of n = 3 biologically independent replicates; (D) Protein expression of cGAS, STING, and Vinculin in pancreatic cancer cells.; (E) Quantification of cGAS and STING protein levels normalized to Vinculin. Means were compared using multiple unpaired t tests and the Holm-Šídák correction. Significant differences are indicated by asterisks, with p values < 0.05 shown as *, <0.01 shown as **, and <0.001 shown as ***.