Enhanced efficacy with azacytidine and oncolytic BHV-1 in a tolerized cotton rat model of breast adenocarcinoma

Abstract

Oncolytic viruses selectively replicate in cancer cells by exploiting biochemical differences between normal and tumor cells. Treatment with epigenetic modifiers such as 5-Azacytidine, a DNA methyltransferase inhibitor, increases the replication and cytotoxicity of oncolytic viruses in vivo and in vitro. The cotton rat is an attractive animal to study oncolytic viruses, as syngeneic models of breast adenocarcinoma and osteosarcoma are well established, and many features of primary and secondary tumor growth recapitulate human disease. Treatment of LCRT breast cancer cells with 5-Azacytidine increases bovine herpesvirus type 1 (BHV-1)-mediated cytotoxicity in vitro, with Chou-Talalay analysis indicating a very strong synergy. In vivo, BHV-1 monotherapy delayed tumor growth but did not improve survival of cotton rats with subcutaneous breast adenocarcinomas. However, combination therapy significantly decreased the incidence of secondary lesions, with enhanced tumor cell clearance and evidence of immune cell infiltration compared to BHV-1 monotherapy. Together, these results warrant further investigation of BHV-1 combination therapy with epigenetic modifiers for the treatment of breast cancer, particularly in the context of the prevention and treatment of secondary lesions.

Figure 1

Bovine herpesvirus type 1 (BHV-1) replication and cytotoxicity on LCRT. LCRT cells in 96-well plates were mock or infected with BHV-1 at the indicated MOIs for 1 hour at 37 °C. (a) GFP expression, as a marker of virus replication, was detected using a Typhoon Bioanalyzer (Amersham Biosciences, Piscataway, NJ) at 2 days pi. (b) Cell metabolism, as a measure of cell viability, was assessed using MTT at 2 days pi. Fluorescence was detected using a SpectraMax i3 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA) and the fold change in fluorescence relative to untreated, uninfected controls was calculated. Data were collected in triplicate and are represented as the mean.

Figure 2

Bovine herpesvirus type 1 (BHV-1)-mediated cytotoxicity with and without 5-Aza treatment on normal primary human cell lines Ventressca and human embryonic lung (HEL) cells. (a) Ventressca or (b) HEL cells were seeded into 96-well plates and were mock or infected with BHV-1 at the indicated MOIs for 1 hour at 37 °C. Cell metabolism, as a measure of cell viability, was assessed using MTT at 2 days pi. Fluorescence was detected using a SpectraMax i3 Multi-Mode Microplate Reader (Molecular Devices) and the fold change in fluorescence relative to untreated, uninfected controls was calculated. Data were collected in triplicate and are represented as the mean, n = 2. *P = 0.05

Figure 3

5-Aza enhances bovine herpesvirus type 1 (BHV-1) replication and cytotoxicity on LCRT. (a) LCRT cells were treated with 5-Aza at 1 or 3 μmol/l. After 14 hours, whole cell lysates were harvested for western blot analysis with Dnmt1 primary antibody. Actin served as a loading control. Positive (+ve) control HeLa whole cell lysate. LCRT cells in 96-well plates were treated with 5-Aza at 0.5, 1, or 3 μmol/l for 14 hours, then mock or infected with BHV-1 at MOI 3 or 5 for 1 hour at 37 °C. (b) GFP expression, as a marker of virus replication, was detected using a Typhoon Bioanalyzer (Amersham Biosciences) 2 days pi and (c) cell metabolism, as a measure of cell viability, was assessed using MTT at 2 days pi. Fluorescence was detected using a SpectraMax i3 Multi-Mode Microplate Reader (Molecular Devices) and the fold change in fluorescence relative to untreated, uninfected controls was calculated. Error bars represent mean + SEM, n = 3. *P = 0.05

Figure 4

Bovine herpesvirus type 1 (BHV-1) viral burst increases with 5-Aza treatment in LCRT cells. Cells were infected with BHV-1 at MOI 5 for 1 hour at 37 °C. Cell-associated virus particles were collected 1, 2, and 3 days pi and titrated on naive MDBK monolayers. Error bars represent mean + SEM, n = 3. *P = 0.05.

Figure 5

Kaplan-Meier survival and tumor volumes for cotton rats treated with 5 × 10⁶ pfu BHV-1. When tumors reached treatable size they were treated with 5 × 10⁶ pfu BHV-1 intratumoral (i.t.) daily for 5 days. Tumors were measured every 2 days until animals reached end point. (a) Kaplan-Meier estimates of survival and (b) tumor volumes of cotton rats treated with PBS and (c) 5 × 10⁶ pfu i.t. BHV-1 are shown. BHV-1, bovine herpesvirus type 1.

Figure 6

Kaplan-Meier survival curve and tumor volumes for cotton rats (CRs) treated with 5 × 10⁶ plaque-forming unit (pfu) bovine herpes virus type 1 (BHV-1), 2 mg/kg 5-Aza, or 5 × 10⁶ pfu BHV-1 and 2 mg/kg 5-Aza combination therapy. 5 × 10⁶ LCRT cells were implanted into CRs by subcutaneous injection. When tumors reached treatable size, they were injected with 5 × 10⁶ pfu BHV-1 intratumoral (i.t.) one dose daily for 5 days, one dose 5-Aza (2 mg/kg) intraperitoneal (i.p.), or pretreated with 5-Aza (2 mg/kg) i.p. 1 day prior to i.t. injection of 5 × 10⁶ pfu BHV-1, one dose daily for 5 days. Tumors were measured every 2 days until animals reached end point. (a) Kaplan-Meier estimates of survival and (b) tumor volumes of CRs treated with 5 × 10⁶ pfu BHV-1 i.t. (c) 2 mg/kg 5-Aza i.p. or, (d) 5 × 10⁶ pfu BHV-1 i.t. and 2 mg/kg 5-Aza i.p. are shown. *Indicates animals for which vacuous cavities were found in primary tumors upon necropsy.

Figure 7

Representative histological analysis of primary tumors from bovine herpesvirus type 1 (BHV-1), 5-Aza and combination therapy treated cotton rats. Tumors were excised from phosphate-buffered saline (a,e), BHV-1 (5 × 10⁶ pfu; (c,g)), 5-Aza (2 mg/kg; (b,f)) and combination treated (d,h–j) animals at endpoint, fixed and H&E or periodic-acid Schiff (PAS) stained for histological analysis. Images were captured at 1× magnification (a–d; tumor cell clearance, H&E) using a Leica Aperio AT Turbo slide scanner or at 20× (e–h; immune cell infiltration, H&E) or 40× (i,j; large granulated lymphocytes, PAS) magnification with a Leica DM IRE2 microscope. Scale bars = 0.3 mm (a–d), 1 mm (e–h), 0.5 mm (i,j).