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. 2013 Jan;20(1):17-24.
doi: 10.1038/cgt.2012.75. Epub 2012 Nov 9.

Combination of vinblastine and oncolytic herpes simplex virus vector expressing IL-12 therapy increases antitumor and antiangiogenic effects in prostate cancer models

B J Passer  1 , T Cheema, S Wu, C-L Wu, S D Rabkin, R L Martuza
Affiliations

Combination of vinblastine and oncolytic herpes simplex virus vector expressing IL-12 therapy increases antitumor and antiangiogenic effects in prostate cancer models

B J Passer et al. Cancer Gene Ther. 2013 Jan.

Abstract

Oncolytic herpes simplex virus (oHSV)-1-based vectors selectively replicate in tumor cells causing direct killing, that is, oncolysis, while sparing normal cells. The oHSVs are promising anticancer agents, but their efficacy, when used as single agents, leaves room for improvement. We hypothesized that combining the direct oncolytic and antiangiogenic activities of the interleukin (IL)-12-secreting NV1042 oHSV with microtubule disrupting agents (MDAs) would be an effective means to enhance antitumor efficacy. Vinblastine (VB) was identified among several MDAs screened, which displayed consistent and potent cytotoxic killing of both prostate cancer and endothelial cell lines. In matrigel tube-forming assays, VB was found to be highly effective at inhibiting tube formation of human umbilical vein endothelial cells. The combination of VB with NV1023 (the parental virus lacking IL-12) or NV1042 showed additive or synergistic activity against prostate cancer cell lines, and was not due to increased oHSV replication by VB. In athymic mice bearing CWR22 prostate tumors, VB in combination with NV1042 was superior to the combination of VB plus NV1023 in reducing tumor burden, appeared to be nontoxic and resulted in a statistically significant diminution in the number of CD31(+) cells as compared with other treatment groups. In human organotypic cultures using surgical samples from radical prostatectomies, both NV1023 and NV1042 were localized specifically to the epithelial cells of prostatic glands but not to the surrounding stroma. These data highlight the therapeutic advantage of combining the dual-acting antitumor and antiangiogenic activities of oHSVs and MDAs.

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Figures

Figure 1
Figure 1
Cytotoxic effects of MDA’s. (A) HUVEC (human umbilical vein endothelial cells) primary cultures and endothelial cell lines MS1 (mouse pancreatic), C166 (mouse yolk sac), HBME-1 (human bone marrow) and (B) Human CWR22 and PC3 prostate cancer cell lines were treated with increasing concentrations of the indicated MDA’s. MTS assays were performed on day 3 to determine the extent of cell killing.
Figure 2
Figure 2
Analysis of NV1023 and NV1042 replication and effects on prostate cancer cell killing (A) Single burst assays were performed using either NV1023 or NV1042 (MOI =1.5) on CWR22 or PC3 cells over a 72 hr period. Virus (cell pellet plus supernatants) on days 1, 2 and 3 post-infection and viral titers (pfu/ml) determined by plaque assay on Vero cells as described previously. Input virus: ~4×105 plaque forming units (pfu; indicated by arrow). (B) Cell susceptibility assays were performed using NV1023 or NV1042 on either CWR22 (left) or PC3 (right) prostate cancer cells. Cell lines were inoculated with the indicated MOI’s of virus and cell killing was evaluated by MTS assay on day 3 (error bars represent S.E.M.). (C) PC3 (open bar) or CWR22 (solid bar) prostate cancer cells were treated with increasing concentration of VB (0.01-100 nM) and MTS assays were performed to generate dose-response curves. (D) Supernatants collected from single burst assays were assayed for IL-12 derived from NV1042 by ELISA at the indicated time points. NV1023 was used as a control vector to normalize for background andnormal goat IgG control was also included to demonstrate specificity for murine IL-12. (E) Prostate cancer (PC3 and CWR22) and normal prostate epithelial (PrEC) cells were incubated in the presence or absence of non-toxic concentrations of VB (0.1 nM) for 12h and thereafter, cells were infected with NV1023 (MOI of 1.5) for 72 hrs. Virus titers were determined by plaque assay on Vero cells. Note that virus replication is not altered in the presence of VB and is negligible in PrEC.
Figure 3
Figure 3
Combination-Index analysis. Fraction affected (Fa) versus combination index plots were generated using the method of Chou and Talalay to determine the extent of synergy if any for either vinblastine (VB), docetaxel (Doc) or epothilone-B (EpoB) in combination with NV1023 or NV1042 in CWR22 (upper panels) and PC3 (lower panels) cell lines. (+) indicates simultaneous oHSV and drug addition; (→) indicates oHSV inoculation 24 hrs before drug treatment; (←) indicates drug treatment 24 hrs prior to oHSV infection. Synergistic effects are defined as combination index (CI) < 1 additive effects are CI=1, and antagonistic effects are CI>1. Note that the dotted line in each plot indicates a reference point of a CI value of 1.
Figure 4
Figure 4
Increased antiangiogenic effects ofNV1042/vinblastine combination in an in vitro tube formation assay. HUVEC cells were plated on matrigel-coated plates and either remained untreated (Neg.) or treated with NV1023 or NV1042 (MOI 1), vinblastine (1nM) or their combination. Twentyhrs later, tube formation was scored. Representative fields are shown for each condition (A) and the number of tubes/field was quantified (B). Each treatment was performed in triplicate. Student’s t test was used to determine statistical significance between the indicated control and treatment groups. Bars represent average ± S.E.M..*, P< 0.01 for NV1023+VB and NV1042+VB verses NV1023 alone, NV1042 alone, VB alone or untreated.
Figure 5
Figure 5
In vivo efficacy studies. (A) Schematic illustrating the dosing and scheduling of single and combination treatment regimes in mice bearing s.c. CWR22 tumors. (B) Assessment of CWR22 tumor growth. Virus [NV1023 or NV1042; 5×105 plaque-forming units (pfu)] or virus suspension buffer (PBS with 10% glycerol) was injected intratumorally on days 13 and 15 after tumor implantation. Vinblastine was administered intraperitoneally at 0.35 mg/kg on day 15 for nine consecutive days. *, P< 0.05 for mock (n = 8) versus NV1023 only (n = 8) and VB only (n = 7); **, P< 0.01 for mock verses NV1042 only (n = 6) and NV1023 + VB (n = 6); ***, P< 0.01 for NV1042+VB (n = 7) verses NV1042 only and NV1023 + VB at day 32. (C) Evaluation of CD31+ staining (brown) as a function of treatment condition. Mice (n=3/group) were treated as described above and at day 30 post-implantation tumors were harvested, frozen and cut into tissues sections. Tissue sections were stained with a rat anti-mouse CD31 antibody and were counterstained with hematoxylin. (D) Quantification of CD31-positive staining. Tumor microvessel density was quantified for all treatment groups. Tumor microvessel density was compared between all treatment groups and untreated control group. (Mean ± S.E.M); * P< 0.04 for NV1023 verses NV1042; **, P<0.03 for NV1042 verses NV1042/VB.
Figure 6
Figure 6
Analysis of NV1023 and NV1042 infection of prostate cancer surgical samples. Tissues were incubated with NV1023 (1 × 106pfu) or NV1042 (1 × 106pfu) for 1 h in direct contact and thereafter, placed on a semi-submersed collagen sponge for 3 days. (A) Tissue specimens were stained with anti-HSV-1 gC (upper panels) or anti-cytokeratin-8/18 (lower panels) antibodies. Sections were counterstained with hematoxylin. Note the partial overlap between anti-HSV gC and cytokeratin-8/18+ staining. (B) Evaluation of NV1023 or NV1042 replication in prostate organ cultures. Each color dot indicates a different prostate surgical specimen. Horizonal line denotes the average viral titer for the two groups, which are statistically not significant. Note that infection with NV1023 or NV1042 resulted in similar viral titers. The five specimens examined represent Gleason Scores of either 3+3=6 or 3+4=7.
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