br alone Figures C and D
alone (Figures 4C and 4D). HSV can induce angiogenesis in various tumors,17,18,36 and viral infection induces the production of
VEGF.37–42 Hence, the inhibition of angiogenesis by cetuximab improved the antitumor effect of C-REV in the HT-29 xenograft model by promoting efficient virus distribution within the tumor.
In addition, the decrease in EGFR Spectinomycin after treatment with C-REV is related to the therapeutic effects of cetuximab. Liang et al.43 Molecular Therapy: Oncolytics
demonstrated that HSV-1 ICP0 can interact with CIN85 and Cbl, forming a complex that downregulates cell surface levels of EGFR in the absence of EGF. Their result is in accordance with our finding that the expression of EGFR on three CRC cell lines decreased following C-REV infection (Figure 1C; Figure S1). This result may explain that C-REV prior to cetuximab combination therapy had no additive inhibitory effect relative to the C-REV group. Moreover, C-REV-induced angiogenesis was not inhibited by cetuximab in vivo (Figure 4C, combination G2). However, other researchers reported that the interaction between EGFR and PI3K was upregulated and EGFR was transiently activated during HSV-1 infection.44 We also determined that the expression of EGFR in vitro was temporarily increased after C-REV administration but subsequently decreased (Figure S2). Further studies are needed to explore the internal mech-anism underlying this finding.
Our results show that the combination of the anti-EGFR monoclonal antibody cetuximab and the oncolytic virus C-REV induced a syner-gistic antitumor effect in a human CRC xenograft model. Cetuximab enhanced the antitumor activity of C-REV by promoting viral distri-bution and inhibiting angiogenesis. Therefore, cetuximab represents an ideal virus-associated agent for antitumor therapy. Our findings suggest that applying cetuximab prior to C-REV can gain more benefit in tumor growth inhibition. With further investigation, com-bination therapy could be developed into an effective antitumor strat-egy against human CRC.
MATERIALS AND METHODS
Cell Lines and Viruses
The human CRC cell line WiDr and African green monkey kidney cell line Vero were obtained from American Type Culture Collection (Manassas, VA, USA). The human CRC cell line CW2 was obtained from the RIKEN Cell Bank (Tsukuba, Japan). The human CRC cell line HT-29 was kindly donated by Dr. Suguru Yamada (Nagoya Uni-versity, Japan). HT-29, WiDr, CW2, and Vero cells were grown in DMEM (Sigma, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (Gibco). C-REV is a highly attenuated mutant clone derived from HSV-1 strain HF.45 The virus was propagated in Vero cells and stored in aliquots at 80 C. C-REV was diluted in PBS for in vivo and in vitro experi-ments. Viral titers were assayed in Vero cells and expressed as pla-que-forming units per milliliter (PFU/mL).
Proteins were separated by electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes using an iBlot apparatus (Invitrogen, MA). After blocking in skim milk for 1 h, the membrane was washed with TBS with Tween 20 (TBS-T) buffer at room temper-ature (RT). Primary antibodies against EGFR (Abcam, Cambridge, UK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam) were added, and the membranes were shaken gently for 1 h at RT. After three washes with TBS-T, the membranes were incu-bated with horseradish peroxidase (HRP)-labeled secondary antibody (anti-rabbit immunoglobulin G [IgG]) for 1 h at RT, and then they
were washed three times in TBS-T. Immunoreactive bands of proteins were visualized using the ECL Plus Solution (Amersham, Arlington Heights, IL, USA), and signals were recorded by autoradiography.