Oncolytic Coxsackievirus B3 Strain PD-H Is Effective Against a Broad Spectrum of Pancreatic Cancer Cell Lines and Induces a Growth Delay in Pancreatic KPC Cell Tumors In Vivo

Abstract

Pancreatic cancer is one of the deadliest cancers globally, with limited success from existing therapies, including chemotherapies and immunotherapies like checkpoint inhibitors for patients with advanced pancreatic ductal adenocarcinoma (PDAC). A promising new approach is the use of oncolytic viruses (OV), a form of immunotherapy that has been demonstrated clinical effectiveness in various cancers. Here we investigated the potential of the oncolytic coxsackievirus B3 strain (CVB3) PD-H as a new treatment for pancreatic cancer. In vitro, PD-H exhibited robust replication, as measured by plaque assays, and potent lytic activity, as assessed by XTT assays, in most pancreatic tumor cell lines, outperforming two other coxsackievirus strains tested, H3N-375/1TS and CVA21. Thus, H3N-375/1TS showed efficient replication and lytic efficiency in distinctly fewer tumor cell lines, while most tumor cells were resistant to CVA21. The oncolytic efficiency of the three OV largely correlated with mRNA expression levels of viral receptors and their ability to induce apoptosis, as measured by cleaved caspase 3/7 activity in the tumor cells. In a syngeneic mouse model with subcutaneous pancreatic tumors, intratumoral administration of PD-H significantly inhibited tumor growth but did not completely stop tumor progression. Importantly, no virus-related side effects were observed. Although pancreatic tumors respond to PD-H treatment, its therapeutic efficacy is limited. Combining PD-H with other treatments, such as those aiming at reducing the desmoplastic stroma which impedes viral infection and spread within the tumor, may enhance its efficacy.

Keywords: CVA21; KPC cells; PD-H; coxsackievirus; oncolytic virus; pancreatic cancer.

Figure 1

Oncolytic activity of PD-H, H3N-375/1TS, and CVA21 in pancreatic tumor cells. Cell viability: Pancreatic tumor cell lines (KPC, Beta-TC-3, AsPC-1, MIA Paca-2, Capan-1, Capan-2, and BxPC-3) were seeded in 96-well plates and infected with PD-H, H3N-375/1TS, or CVA21 at the indicated MOI. Cell viability was determined by XTT assay 24 and 48 h post-infection and is set relative to untreated cells (control). Data are shown as mean values ± SD from 2–3 independent experiments with three replicates each. Statistical significance of differences compared to control: * p < 0.05, ** p < 0.01 and *** p < 0.001; n.s., not significant.

Figure 1

Oncolytic activity of PD-H, H3N-375/1TS, and CVA21 in pancreatic tumor cells. Cell viability: Pancreatic tumor cell lines (KPC, Beta-TC-3, AsPC-1, MIA Paca-2, Capan-1, Capan-2, and BxPC-3) were seeded in 96-well plates and infected with PD-H, H3N-375/1TS, or CVA21 at the indicated MOI. Cell viability was determined by XTT assay 24 and 48 h post-infection and is set relative to untreated cells (control). Data are shown as mean values ± SD from 2–3 independent experiments with three replicates each. Statistical significance of differences compared to control: * p < 0.05, ** p < 0.01 and *** p < 0.001; n.s., not significant.

Figure 2

Virus growth curves kinetics of PD-H, H3N-375/1TS, and CVA21 in pancreatic tumor cells. The indicated pancreatic tumor cell lines were seeded in 96-well plates and infected with 1 MOI of PD-H, H3N-375/1TS, or CVA21. The virus was isolated at the indicated time points through three freeze/thaw cycles, and the virus titer was determined by plaque assay on HeLa cells. Data are shown as mean values ± SD from 2–3 independent experiments with two replicates each.

Figure 3

Viral receptor expression in pancreatic tumor cells. (A) Relative expression levels of CAR, ICAM-1, and DAF: The expression levels of CAR, ICAM-1, and DAF were determined by qRT-PCR. Each receptor’s expression level was normalized to the endogenous HPRT expression level and is set relative to the corresponding level in HeLa cells, which was set to 1. Data are shown as mean values ± SD from 2 independent samples with two replicates each. (B) Effect of heparin on PD-H infection in pancreatic tumor cell lines: PD-H at MOI 1 (MOI 10 for BxPC-3) was incubated with DMEM containing heparin (5000 µg/mL) or without heparin for 1 h before being applied to the cells. Cell viability was measured 48 h post-infection using the XTT assay. Data are shown as mean values ± SD from 3 independent experiments with three replicates each. Statistical significance as indicated: ** p < 0.01 and *** p < 0.001; n.s., not significant.

Figure 4

Relative cleaved caspase 3/7 activity in pancreatic tumor cells upon infection with PD-H, H3N-375/1TS, and CVA21. Pancreatic tumor cells were seeded in 96-well plates and infected with PD-H, H3N-375/1TS, or CVA21 at MOI 1. Cleaved caspase 3/7 activity was measured 24 h post-infection and normalized to the activity in untreated cells (control). Data are shown as mean values ± SD from 3 independent experiments with three replicates each. Statistical significance compared to control as indicated: * p < 0.05, ** p < 0.01 and *** p < 0.001; n.s., not significant.

Figure 5

Oncolytic efficiency of PD-H in a syngeneic KPC tumor mouse model. Subcutaneous KPC tumors were established on both flanks of C57BL/6J mice. When the tumors reached a volume of 60–100 mm³, one of the tumors was injected with 3 × 10⁶ pfu of PD-H (n = 5) or 0.9% NaCl solution (n = 6), while the contralateral tumor remained untreated. The same dose of PD-H was administered on Days 2, 6, 10 and 14 post-initial injection. (A) Growth of the injected tumor: Shown are the mean values ± SD for each group (upper panel) and for each individual animal (lower panel). Statistical significance between PD-H-injected vs. NaCl 0.9%-injected as indicated: * p < 0.05, ** p < 0.01; n.s., not significant. Red arrows indicate the time points of PD-H/NaCl 0.9% injection. (B) Growth of the non-injected contralateral tumor: Shown are the mean values ± SD for each group (upper panel) and for each individual animal (lower panel). Statistical significance between untreated PD-H and untreated NaCl 0.9% tumors as indicated: * p < 0.05; n.s., not significant. (C) Kaplan–Meier survival curve; n.s., not significant. (D) Development of animal body weight. Shown are the mean values ± SD for each group. (E) Histological examination of the KPC tumor. Representative tumor slides stained with hematoxylin and eosin (H&E), Trichrome, and Sirius Red from a PD-H-injected tumor and a 0.9% NaCl-treated tumor on Day 28 post-tumor inoculation. Scale bars = 2 mm. Magnification scale bars = 200 µm. (F) Histological examination of murine tissues. Representative slides of murine organs (heart, pancreas, spleen, lung, liver) stained with H&E are shown on Day 26 (0.9% NaCl-treated) and Day 28 (PD-H-treated) after tumor inoculation. Scale bars = 2 mm (heart) and 300 µm (magnification heart, pancreas, spleen, lung, liver).