br DISCUSSION br In this study


DISCUSSION
In this study, we evaluated the effect of combination therapy with ce-tuximab and the oncolytic herpes virus C-REV on human CRC cell lines and tumor xenografts. Cell viability assays revealed that the cyto-toxicity of C-REV was time and dose dependent (Figure 2A). Viral LY3009120 assays indicated that cetuximab did not interfere with viral replication at any dose, implying that viral replication was not affected by inhibition of the EGFR pathway in vitro (Figure 2C). Combination therapy with cetuximab and C-REV suppressed tumor growth significantly relative to other treatments (Figure 3B). HSV-1 staining revealed that the virus was distributed more efficiently in the combination therapy group than in the C-REV group (Figure 4A). Injection of C-REV prior to cetuximab had no additive effect on tu-mor growth relative to C-REV treatment alone (Figure 3E).
Preclinical studies and clinical trials over the past decades have shown that oncolytic viruses have potential efficacy against various tumors,
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Figure 4. Immunohistochemical Staining of Tumor Samples
(A) Immunohistochemical staining of HSV-1 (arrows) in tumors from the C-REV group and combination G1 group, 3 days post-treatment (200 magnification; scale bars, 100 mm). (B) Quantitative analysis of the results in (A). HSV-1 density in the tumor was assessed at 200 magnification. (C) Immunohistochemical staining for CD31 (arrows) in tumors from the control group, cetuximab group, C-REV group, combination G1 group, and combination G2 group, 14 days post-treatment (100 magnification; scale bars, 100 mm). (D) Quantitative analysis of the results of (C). CD31 density in tumors was assessed at 100 magnification. Data are presented as means ± SD, and statistical differences between groups were evaluated by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Only significant differences are indicated.
Table 1. Fractional Tumor Volume (FTV) following Treatment with Cetuximab and C-REV, Alone or in Combination, in HT-29 Tumor Xenografts
Combination G1
E-FTV/O-FTVa
Day
C-REV
Cetuximab
E-FTV
O-FTV
FTV, fractional tumor volume (mean tumor volume experimental/mean tumor volume control); E-FTV, expected FTV (mean FTV of C-REV) (mean FTV of cetuximab); O-FTV, observed FTV. aSynergic effect: E-FTV/O-FTV > 1.
but the inefficient distribution capability of virus in tumors always limited clinical efficacy.25–27 Oncolytic virus distribution is highly affected by the tumor microenvironment.28 High levels of secretion of extracellular matrix proteins by tumor cells contribute to high interstitial fluid pressure (IFP), which is one of the major factors pre-venting viruses from spreading in tumors.28 Because IFP increases as the tumor grows, the tumor angiogenesis is closely linked to high IFP.29–31 Therefore, antiangiogenic therapy has been proposed as a means to promote more efficient viral distribution. In our previous studies, we reported that bevacizumab and erlotinib, via their antian-giogenic effects, enhanced the distribution of the oncolytic herpes vi-rus C-REV in human breast cancer xenografts and pancreatic cancer xenografts, respectively.20,32
Inhibition of EGFR by tyrosine kinase inhibitors or monoclonal anti-bodies decreases the angiogenic profile of tumor cells,33 whereas over-expression of EGFR on tumor cells is related to the production of angiogenic molecules. Bruns et al.34 reported that cetuximab decreased the production of VEGF and interleukin (IL)-8 in pancre-atic cancer in vitro and in vivo. Cetuximab also reduced VEGF, basic fibroblast growth factor (bFGF), and transforming growth factor a (TGF-⍺) expressions and decreased microvessel count in CRC cells and xenografts.35 Together, these findings indicated that cetuximab has an antiangiogenic effect. In this study, immunohistochemical staining for CD31 revealed that the combination of cetuximab and C-REV decreased tumor angiogenesis relative to C-REV treatment