Combination of Paclitaxel and MG1 Oncolytic Virus as a Successful Strategy for Breast Cancer Treatment

Marie-Claude Bourgeois-Daigneault; Lauren Elizabeth St-Germain; Dominic Guy Roy; Adrian Pelin; Amelia Sadie Aitken; Rozanne Arulanandam; Theresa Falls; Vanessa Garcia; Jean-Simon Diallo; John Cameron Bell


Breast Cancer Res. 2016;18(83) 

In This Article


Cell Lines and Culture

Vero kidney epithelial, 4 T1, EMT6 and EO771 murine mammary carcinoma and Hs578T, BT-549 and MDA-MB-231 human mammary carcinoma cell lines (American Type Culture Collection (Manassas, VA, USA)) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Corning cellgro, Manassas, VA, USA) supplemented with 10 % fetal bovine serum (FBS) (Sigma life science, St-Louis, MO, USA) and maintained at 37 °C with 5 % CO2.

Virus Amplification and Purification

MG1-green fluorescent protein (GFP) was purified as previously described.[8] Briefly, Vero cells were infected for 24 h at a multiplicity of infection (MOI) of 0.01. Supernatants were then filtered using a 0.2-um bottle top filter (Millipore, MA, USA) prior to 1.5-h centrifugation at 30100 g. The pellet was resuspended in Dulbecco's phosphate-buffered saline (DPBS) (Corning cellgro, Manassas, VA, USA) and aliquots were stored at −80 °C.

PAC Treatment

PAC was purchased from Accord healthcare Inc. (Durham, NC). The cells were pre-treated at a concentration of 2 uM in culture media for 4 h prior to infection, unless specified otherwise. For in vivo experiments, animals were treated intraperitoneally (IP) with 2 mg/kg or 10 mg/kg of PAC as specified (see figure legends).

Virus Titration

Titers were obtained by plaque assay. Briefly, serial dilutions of the samples were transferred to monolayers of Vero cells. Following an incubation of 1 h, cells were overlaid with 0.5 % agarose/DMEM supplemented with 10 % FBS. Plaques were counted 24 h later. For in vivo experiments, tumors and organs were collected 48 h post treatment, homogenized in PBS using a tissue homogenizer, then serially diluted and virus quantified as described above.

In Vitro IFNβ Treatment and Quantification

Monolayers of tumor cells were treated with 250 U/mL of murine IFNβ (PBL interferon source, Piscataway, NJ, USA) 4 h prior to virus infection. The production of IFNβ by tumor cells was quantified using the ELISA mouse IFNβ kit (R&D systems, Minneapolis, MN, USA) following the manufacturer's protocol. The samples were generated by pre-treating the cells with PAC as described above and infecting them for 24 h at an MOI of 0.1.

Coomassie Blue Staining/Viability Assay

At 72 h post infection, cells were fixed for 30 minutes using fixative solution (3:1 methanol-acetic acid). The fixative was then replaced by the Coomassie Blue staining solution (3:1 methanol-acetic acid, 0.1 % Commassie Blue dye) for 30 minutes. The plates were washed and dried overnight prior to scanning. For quantification, the Coomassie Blue staining was solubilized using 10 % SDS, and serial dilutions were performed and transferred to a 96-well plate for reading using a Fluoroscan plate reader at 450 nm.


For nuclear staining, cells were cultured and treated on coverslips for 72 h. Cells were then washed with cold PBS and fixed using ice-cold methanol-acetone (1:1). Nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI) included in the Prolong gold anti-fade (Molecular Probes) used to mount the coverslips onto slides. Live images of MG1-GFP infected cells were acquired using an EVOS Fl cell imaging system (ThermoFisher Scientific) microscope 24 h post infection.

Flow Cytometry

For quantification of virus infection, cells were processed as previously described.[15] Briefly, cells were harvested and fixed using IC fixation buffer (eBioscience) 24 h after PAC treatment and infection with MG1-GFP at an MOI of 0.01. Cells were then washed twice and resuspended in FACS buffer (3 % FBS, PBS) for analysis using a Cyan ADP 9 flow cytometer (Beckman Coulter, Mississauga, ON, Canada).

In Vivo Experiments and Tumor Models

Balb/c mice were used (Charles River Laboratories) for the 4 T1 and EMT6 murine tumor models. For orthotopic implantation of the tumors, 2 × 105 cells were injected into the second left mammary fat pad. For the EO771 tumor model, 1 × 106 cells were implanted into the second left mammary fat pad of C57/Bl6 mice. For treatments, the virus and drug preparations were diluted to the appropriate concentration in a total volume of 100 uL of PBS and injected IP or intratumorally (IT) using insulin syringes (The Stevens Co, Montreal, QC, Canada). All experiments were performed in accordance with the University of Ottawa animal care and veterinary services guidelines.

Histological Analysis

Tumors were collected 48 h after treatment and fixed in 10 % buffered formalin phosphate (Fisher Scientific, Waltham, MA, USA) for 48 h. Paraffin-embedded sections were stained using hematoxylin and eosin or the specified antibodies. For antibody staining, the sections were rehydrated through graded alcohol and heat-mediated antigen retrieval was performed in citrate buffer (sodium citrate 10 mM, pH 6). Tissue sections were stained as described previously[16] using a rabbit anti-VSV (made in house) and rabbit anti-caspase-3 (Cell signalling technology) antibodies.

TNBC Ex-vivo Samples

Patient-derived TNBC xenografts were grown into NOD/SCID mice as described previously.[17,18] When the tumors reached 1500 mm3 in size they were collected and cores were generated as described previously.[19] The cores were treated ex-vivo with MG1 (103 plaque-forming units (pfu)) and PAC and culture supernatant was collected 48 h later to titer the virus output.

Tumor Measurements and Survival Experiments

The length and width of the tumors were measured using digital calipers (Fowler). The formula (length × width2)/2 was used to calculate tumor volumes. The mice were sacrificed when they displayed respiratory distress, significant weight loss, ulceration, or discomfort, or when the tumor volume reached 1500 mm3 in size.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 6.0 software (see figure legends). Error bars represent standard error of the mean.