Oncolytic reovirus as a combined antiviral and anti-tumour agent for the treatment of liver cancer

Objective Oncolytic viruses (OVs) represent promising, proinflammatory cancer treatments. Here, we explored whether OV-induced innate immune responses could simultaneously inhibit HCV while suppressing hepatocellular carcinoma (HCC). Furthermore, we extended this exemplar to other models of virus-associated cancer. Design and results Clinical grade oncolytic orthoreovirus (Reo) elicited innate immune activation within primary human liver tissue in the absence of cytotoxicity and independently of viral genome replication. As well as achieving therapy in preclinical models of HCC through the activation of innate degranulating immune cells, Reo-induced cytokine responses efficiently suppressed HCV replication both in vitro and in vivo. Furthermore, Reo-induced innate responses were also effective against models of HBV-associated HCC, as well as an alternative endogenous model of Epstein–Barr virus-associated lymphoma. Interestingly, Reo appeared superior to the majority of OVs in its ability to elicit innate inflammatory responses from primary liver tissue. Conclusions We propose that Reo and other select proinflammatory OV may be used in the treatment of multiple cancers associated with oncogenic virus infections, simultaneously reducing both virus-associated oncogenic drive and tumour burden. In the case of HCV-associated HCC (HCV-HCC), Reo should be considered as an alternative agent to supplement and support current HCV-HCC therapies, particularly in those countries where access to new HCV antiviral treatments may be limited.


Isolation of PBMC using step-density gradient separation
Blood from healthy donors was diluted 2:1 with HBSS and layered onto Lymphoprep TM , then centrifuged for 25 min with no brake. The white cell layer was isolated and washed twice with HBSS.
PBMC were used immediately.

Reovirus replication and UV-irradiation
Cell lines and primary hepatocytes were incubated with Reo at 1 PFU/cell for 24 or 72 hours. Cells and supernatants were then collected and lysates generated by 3 cycles of freeze-thaw. Virus concentration was quantified using a standard L929 plaque assay.
Reo stocks were exposed to a range of durations of 254 nm UV light from an 8 watt bulb housed in a Stratalinker ® UV Crosslinker 1800 (Stratagene). Inhibition of the viral lifecycle was determined by standard L929 plaque assay. The minimum duration of UV exposure necessary to abrogate productive infection was 2 minutes, and this was used to make UV-Reo.

MTT assays
Cells were treated for 72 hr for the assessment of cytotoxicity from reovirus infection, or as stated in other experiments. Following these treatment schedules, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (Sigma) was added at 0.5 mg/ml. Plates were incubated for 4 hr, following which the media was removed and the cells solubilised using DMSO (Sigma). Optical density (OD) absorbance readings were determined using a Thermo Multiskan EX plate reader (Thermo scientific) at 550 nm absorbance.

Characterisation of immune cell populations in mixed liver cells and LMC; mixed liver cells or LMC
were harvested, washed in FACS buffer and labelled with combinations of the antibodies described in table 1.  min. Data were acquired immediately.

Preparation of mixed liver cells, primary enriched hepatocytes and LMC
Ex-vivo normal liver and HCC samples were cultured in RPMI supplemented with 10 % FCS (Biosera) To obtain LMC, the single cell suspensions were subjected to two rounds of density step-gradient centrifugation using Lymphoprep TM 800 x g for 25 min with no brake. Cells were collected from the interface layer and washed in HBSS prior to culture. To obtain enriched hepatocytes, the pelleted cells from the first density gradient centrifugation were were re-suspended in ACK buffer for 1 min, washed in cold HBSS and cultured. Plateable cryopreserved human hepatocytes (TRL/Lonza) were utilised in Fig 1c, d, e.