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. 2013 Mar 26:11:79.
doi: 10.1186/1479-5876-11-79.

Growth inhibition of different human colorectal cancer xenografts after a single intravenous injection of oncolytic vaccinia virus GLV-1h68

Klaas Ehrig  1 , Mehmet O Kilinc, Nanhai G Chen, Jochen Stritzker, Lisa Buckel, Qian Zhang, Aladar A Szalay
Affiliations

Growth inhibition of different human colorectal cancer xenografts after a single intravenous injection of oncolytic vaccinia virus GLV-1h68

Klaas Ehrig et al. J Transl Med. .

Abstract

Background: Despite availability of efficient treatment regimens for early stage colorectal cancer, treatment regimens for late stage colorectal cancer are generally not effective and thus need improvement. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) strains is a promising new strategy for therapy of a variety of human cancers.

Methods: Oncolytic efficacy of replication-competent vaccinia virus GLV-1h68 was analyzed in both, cell cultures and subcutaneous xenograft tumor models.

Results: In this study we demonstrated for the first time that the replication-competent recombinant VACV GLV-1h68 efficiently infected, replicated in, and subsequently lysed various human colorectal cancer lines (Colo 205, HCT-15, HCT-116, HT-29, and SW-620) derived from patients at all four stages of disease. Additionally, in tumor xenograft models in athymic nude mice, a single injection of intravenously administered GLV-1h68 significantly inhibited tumor growth of two different human colorectal cell line tumors (Duke's type A-stage HCT-116 and Duke's type C-stage SW-620), significantly improving survival compared to untreated mice. Expression of the viral marker gene ruc-gfp allowed for real-time analysis of the virus infection in cell cultures and in mice. GLV-1h68 treatment was well-tolerated in all animals and viral replication was confined to the tumor. GLV-1h68 treatment elicited a significant up-regulation of murine immune-related antigens like IFN-γ, IP-10, MCP-1, MCP-3, MCP-5, RANTES and TNF-γ and a greater infiltration of macrophages and NK cells in tumors as compared to untreated controls.

Conclusion: The anti-tumor activity observed against colorectal cancer cells in these studies was a result of direct viral oncolysis by GLV-1h68 and inflammation-mediated innate immune responses. The therapeutic effects occurred in tumors regardless of the stage of disease from which the cells were derived. Thus, the recombinant vaccinia virus GLV-1h68 has the potential to treat colorectal cancers independently of the stage of progression.

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Figures

Figure 1
Figure 1
Analysis of viral titers after infection of five different CRC lines with GLV-1h68 in culture. Cells were infected at an MOI of 0.1 and samples were collected at various times after infection. Titers were averaged from triplicates and normalized to pfu/1x106 cells. Averages plus standard deviations are plotted.
Figure 2
Figure 2
Cell viability of human colorectal cancer lines after GLV-1h68 infection. Cells were infected in culture at MOIs of 0.1 (A) and 1.0 (B) and assayed for viable cells using the MTT assay over a time course of 72 hours. Viability was measured in two sets of triplicates and averaged. Averages were normalized against the uninfected controls at each time-point which were considered to be 100% viable.
Figure 3
Figure 3
Analysis of cell susceptibility to GLV-1h68 cytotoxicity in HCT-15 and HCT-116 CRC cells. A) Plaque forming ability in CV-1 cells (standard), HCT-15 (low cytotoxicity), and HCT-116 (high cytotoxicity). All cell lines were tested in triplicates with a dose of 1x108 pfu of GLV-1h68 for 24 hours at 37°C. Cells were then harvested and plaque forming ability was determined by standard plaque assay. Average titers were determined from triplicates and expressed as pfu/mL. Averages plus standard deviations were plotted. B) Cell proliferation of HCT-15 and HCT-116. Cells were seeded at a concentration of 1x103 cells/well and cell proliferation was followed for 24, 48, 72, 96 and 120 hours. Cell lines were tested in quadruplicates and averages plus standard deviations are plotted. C) Replication of GLV-1h68 at low and high MOIs. Cells were infected at an MOI of 0.01 (low) or 10 (high) and samples were collected at 6, 24, and 48 hours after infection. Average titers were determined in from triplicate and normalized to pfu/1x106 cells. Averages plus standard deviations are plotted.
Figure 4
Figure 4
Fluorescence microscopy of virus-mediated Ruc-GFP expression in HCT-116. HCT-116 cells were infected at an MOI of 1.0 and monitored for 72 hours. (BF) shows bright field microscopic images of the morphology of virus-infected cells. (GFP) expression in infected cells was visualized by direct fluorescence microscopy; propidium iodide (PI) was used as a marker for dead cells. Colocalization of (GFP) and (PI) signal is shown in the (merged) images. All pictures were taken at 40x magnification.
Figure 5
Figure 5
Flow cytometry analysis of GLV-1h68-infected HCT-116 cells. Cells were infected at MOIs of 0.1 or 1.0. Data represents the average distribution of uninfected/infected [GFP neg/pos] and viable/dead [PI neg/pos] cells over the course of 72 hours post infection.
Figure 6
Figure 6
Effects of a single administration of GLV-1h68 on tumor growth of HCT-116 tumor-bearing mice. HCT-116 cells were implanted subcutaneously in the right hind leg of athymic nude mice and GLV-1h68 treatment was evaluated versus PBS treatment. Average tumor volume (ATV) for GLV-1h68-treated (n=10) and for PBS treated control mice (n=5) is plotted over time after treatment and one-way analysis of variance (ANOVA) was used to compare the data. P ≤ 0.05 was considered statistically significant; * = P ≤ 0.05, *** P ≤ 0.0005.
Figure 7
Figure 7
Virus-mediated Ruc-GFP expression at the local tumor site of HCT-116-bearing mice. A) Analysis of GFP fluorescence intensity in GLV-1h68-treated mice during the course of the experiment. GFP intensity is determined by using a four level visual scoring system; 0) no GFP signal, 1) one spot, 2) two or three local spots, 3) diffuse signal from half the tumor, 4) strong signal from whole tumor. Average scores with standard errors for groups of five mice at each time point are presented. B) Fluorescence imaging of GFP expression at the local tumor site is shown for one representative mouse.
Figure 8
Figure 8
Histochemical staining of HCT-116 tumor sections. Microscopic images of histological sections of representative HCT-116 tumors from an untreated mous at 21 days post-injection [A] and GLV-1h68-treated mice [B] at 21 and 42 days post-injection [dpi] are shown. Images were obtained under bright field (BF) or fluorescence microscopy (Phalloidin-TRITC, GFP). Digital images were processed with GIMP2 (Freeware) and merged to yield pseudocolored images (merged). Whole tumor cross-sections (thickness = 100 μm) were labeled with Phalloidin-TRITC to detect de novo synthesis of actin as an indicator for live cells. GFP fluorescence indicates GLV-1h68 replication in the tumor.
Figure 9
Figure 9
Flow cytometry analysis of innate immune cells in untreated and GLV-1h68-treated tumors. A) Representative flow cytometry plots of macrophage and NK cell populations in uninfected (n=4) and GLV-1h68-infected (n=5) HCT-116 tumors from flow cytometry analysis experiments at 21 days post treatment are shown. B) Quantification of total F4/80low CXCR4pos macrophages and CD19pos DX5pos NK cells per gram tumor after normalization by counting beads to quantify the total amount of cells per sample in untreated or GLV-1h68-treated HCT-116 tumors at 21 days post treatment. Average values with standard deviations are plotted. * = P≤0.05, ** = P≤0.005.
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