br Epidermal growth factor receptor EGFR signaling is involved in
Epidermal growth factor receptor (EGFR) signaling is involved in apoptosis, angiogenesis, cell proliferation, migration, and invasion.19 We previously applied a combination therapy of C-REV and erloti-nib, an EGFR tyrosine kinase inhibitor, against human pancreatic cancer; the results revealed that erlotinib enhanced C-REV distribu-tion within the tumor by inhibiting virus-induced angiogenesis.20 Sal-omon et al.21 reported EGFR overexpression (72%–82% above normal tissue) in CRC. Cetuximab, a monoclonal antibody that binds to the extracellular domain of EGFR, has been applied widely to sup-press tumor growth. Inhibition of EGFR activation leads to downre-gulation of the RAS/RAF/MEK/ERK and PI3K/AKT pathways, which decrease vascular endothelial growth factor (VEGF) promoter activity. Inhibition of EGFR activity by cetuximab induces an
Correspondence: Hideki Kasuya, MD, PhD, FACS, Department of Surgery II,
Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku,
E-mail: [email protected]
Molecular Therapy: Oncolytics Vol. 13 June 2019 ª 2019 The Authors. 107 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Figure 1. Evaluation of EGFR Expression
(A and B) EGFR expression in three human colorectal cancer cell lines (HT-29, WiDr, and CW2) was measured by western blotting (A) and flow cytometry (B). (C) EGFR expression in HT-29 was detected by flow cytometry after C-REV infection. GAPDH was used as the endogenous control.
antiangiogenic effect by decreasing VEGF production.22 Moreover, cetuximab can induce antibody-dependent cellular cytotoxicity (ADCC) through Fcg receptors on immune effector cells, such as nat-
ural killer (NK) Prostaglandin J2 and macrophages. In CRC, cetuximab-mediated ADCC activity is correlated with the expression of EGFR.23,24
In this study, we combined C-REV and cetuximab to treat human CRC, and we evaluated the antitumor efficacy of this regimen. Cetux-imab treatment prior to C-REV treatment strongly inhibited tumor growth by enhancing virus spread and preventing angiogenesis.
EGFR Expression Level in CRC Cell Lines
We compared the expression level of EGFR among three CRC cell lines, HT-29, WiDr, and CW2, by western blotting and flow Molecular Therapy: Oncolytics
cytometry (Figures 1A and 1B). HT-29 expressed the highest level of EGFR and CW2 the lowest. To determine whether C-REV treat-ment affects EGFR expression, we performed flow cytometry to detect EGFR after C-REV infection. In our flow cytometry analysis, dead cells after C-REV treatment were eliminated by gating of for-ward scatter and side scatter. Only living cells were detected and recorded. EGFR expression of all three cell lines was reduced r> 3 days after C-REV (MOI 1) infection (Figure 1C; Figure S1). This result suggested that C-REV infection directly modulates EGFR expression in CRC cell lines.
Cytotoxicity of Cetuximab and C-REV in CRC Cell Lines and Cetuximab Has No Effect on Viral Replication
Because the expression of EGFR differed among the CRC cell lines, we used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-mide (MTT) assay to evaluate the sensitivity of HT-29, WiDr, and CW2 to C-REV, cetuximab, and their combination. C-REV exerted a strong cytotoxic effect on all three cell lines, and its effect was time- and dose- dependent. Cetuximab alone had a slightly cytotoxic effect in vitro (Figure 2A), and combination therapy with cetuximab and C-REV had no additive effect (Figure 2B).
To determine whether cetuximab affects viral replication in CRC cell lines, we titered virus from infected cells in order to assess viral repli-cation. We infected three cell lines with C-REV (MOI 1), and we co-incubated them with various concentrations of cetuximab (5, 10, and 20 mg/mL) for 3 days. Cetuximab had no effect on viral replication in any of the three cell lines (Figure 2C).
Combination Therapy with Cetuximab and C-REV Exerts a Strong Antitumor Effect in HT-29 Tumor Xenografts
Next, we evaluated the in vivo antitumor efficacy of combination ther-apy with cetuximab and C-REV. To determine combination therapy with cetuximab and C-REV, we chose HT-29 tumor xenografts, as HT-29 expressed the highest level of EGFR among the cell lines we examined. We applied two kinds of treatment regimens to our tumor model (Figures 3A and 3D), and we compared their efficacy. C-REV was injected intratumorally at the same time in both regimens (days 1, 4, and 7), and cetuximab was injected intraperitoneally prior to (com-bination G1) or after C-REV (combination G2).