Efficacy of Different Oncolytic Vaccinia Virus Strains for the Treatment of Murine Peritoneal Mesothelioma

Abstract

Effective treatment options for peritoneal surface malignancies (PSMs) are scarce. Oncolytic virotherapy with recombinant vaccinia viruses might constitute a novel treatment option for PSM. We aimed to identify the most effective oncolytic vaccinia virus strain in two murine mesothelioma cell lines and the oncolytic potential in a murine model of peritoneal mesothelioma. Cell lines AB12 and AC29 were infected in vitro with vaccinia virus strains Lister (GLV-1h254), Western Reserve (GLV-0b347), and Copenhagen (GLV-4h463). The virus strain GLV-0b347 was shown most effective in vitro and was further investigated by intraperitoneal (i.p.) application to AB12 and AC29 mesothelioma-bearing mice. Feasibility, safety, and effectiveness of virotherapy were assessed by evaluating the peritoneal cancer index (PCI), virus detection in tumor tissues and ascites, virus growth curves, and comparison of overall survival. After i.p. injection of GLV-0b347, virus was detected in both tumor cells and ascites. In comparison to mock-treated mice, overall survival was significantly prolonged, ascites was less frequent and PCI values declined. However, effective treatment was only observed in animals with limited tumor burden at the time point of virus application. Nonetheless, intraperitoneal virotherapy with GLV-0b347 might constitute a novel therapeutic option for the treatment of peritoneal mesothelioma. Additional treatment modifications and combinational regimes will be investigated to further enhance treatment efficacy.

Keywords: immunovirotherapy; intraperitoneal therapy; oncolytic virotherapy; peritoneal carcinomatosis; syngeneic murine tumor model.

Figure 1

Different strains of oncolytic vaccinia virus; (top): GLV-1h254 (Lister), (middle): GLV-4h463 (Copenhagen), and (bottom): GLV-0b347 (Western Reserve).

Figure 2

Schematic illustration of the timing of treatment procedures in the in vivo experiments. Infection of tumors by intraperitoneal virus application was assessed by experiments that were terminated 24 h and 48 h post infection, respectively. Overall survival was investigated over 14 days after intraperitoneal virus application.

Figure 3

Viability of murine peritoneal mesothelioma cell lines after treatment with different vaccinia virus strains. AB12 (A) and AC29 (B) tumor cells were infected with GLV-0b347 (Western Reserve), GLV-1h254 (Lister) or GLV-4h463 (Copenhagen) at various multiplicities of infection (MOIs) ranging from 0.0001 to 1 or remained uninfected (mock). At 96 h post infection (hpi), the remaining tumor cell masses were determined by SRB viability assay. Vaccinia virus mediated oncolysis was calculated relative to mock control. The mean ± SD of at least two independent experiments performed in quadruplicates is shown. For clarity reasons, only the lowest MOI that first reached statistical significance is annotated by asterisks. p ≤ 0.01 (**), p ≤ 0.0001 (****).

Figure 4

Viral replication of different vaccinia virus strains in murine peritoneal mesothelioma cell lines. AB12 (A) and AC29 (B) tumor cells were infected with GLV-0b347 (Western Reserve), GLV-1h254 (Lister) or GLV-4h463 (Copenhagen) at indicated MOIs and viral replication was analyzed via plaque assay at 1, 24, 48, 72, and 96 hpi. The mean ± SD of at least two independent experiments performed in duplicates is shown. hpi: hours post infection; MOI: multiplicity of infection; pfu: plaque-forming units.

Figure 5

Intraperitoneal treatment of peritoneal mesothelioma in a murine tumor model of AC29 tumor cells in CBA/j mice with GLV-0b347. (A) Open implantation of AC29 cells into the peritoneal cavity of anesthetized CBA/j mouse. (B) Two-layered abdominal wall closure with braided sutures. (C) Exploration of the abdominal cavity after euthanasia 48 h after intraabdominal virus application showing multiple indured tumor formations. The tweezers point to a subphrenic mass of peritoneal mesothelioma beneath the left diaphragm.

Figure 6

Detection of infectious GLV-0b347 viral particles (pfu/mL) in tumor lysates 24 and 48 h after treatment of i.p. tumors in AB12 tumor cell-bearing BALB/c mice (n = 5) (A) and AC29 tumor cells in CBA/j mice (n = 5) (B), respectively, and extent of peritoneal tumors quantified by PCI value according to Ottow et al. [28]. hpi: hours post infection; i.p.: intraperitoneal; pfu: plaque-forming unit; PCI: peritoneal-cancer index.

Figure 7

Survival of AB12 tumor-bearing BALB/c mice (n = 10) (A) and AC29 tumor-bearing CBA/j mice (n = 10) (B) in the course of 14 days after virus treatment with GLV-0b347 (green) and in control (mock; black), respectively. There was no statistically significant (ns) difference regarding survival between treatment groups. dpi: days post infection; ns: not significant.

Figure 8

Kaplan–Meier survival curves of AB12 (n = 10) (A) or AC29 (n = 10) (B) tumor-bearing mice treated with modified experimental conditions including surgical implantation of a reduced tumor cell number and earlier onset of i.p. virotherapy with GLV-0b347 (green) compared to control animals (mock; black) until 14 dpi. dpi: days post infection. p < 0.01 (**), p < 0.001 (***).

Figure 9

Intra-abdominal tumor burden given as peritoneal cancer index (PCI) quantified according to Ottow et al. [28] at individual end of experiment based on either signs of strain or maximum 14 days after virus application in AB12 (n = 10) (A) or AC29 (n = 10) (B) tumor-bearing mice treated with GLV-0b347 i.p. compared to control animals (mock). (C) Presence of ascites in AC29 mice (n = 10) treated with GLV-0b347 compared to control animals (mock). Each symbol indicates one mouse. The strength of agreement is given by the number of crosses: (^x) indicates poor (≤0.2) agreement, (^xx) fair (>0.2–0.4) agreement.

Figure 10

AB12 tumor cells in BALB/c mice after mock treatment show tumor deposits (indicated by white arrows) at the parietal and visceral peritoneum (A) compared to GLV-0b347-treated animals with hardly any tumor left over (B) at the time of necropsy. (C,D) Depict AC29 cells in CBA/j mock-treated mice with excessive tumor load ((C), white arrows) compared to virus-treated mice without any signs of residual tumors (D).