Anti-Akt, anti-ERK, anti-MAPK and anti-mTOR antibodies, and phosphorylation-specific antibodies against Akt (Ser³⁰⁸ and Ser⁴⁷³), ERK (Thr²⁰²/Tyr²⁰⁴), MAPK (Thr¹⁸⁰/Tyr¹⁸²) and mTOR (Ser²⁴⁴⁸) as well as the antibody against cleaved caspase-3 were purchased from Cell Signaling Technology Inc. (Cell Signaling, Beverly, MA). The antibodies against VEGF and EGFR were purchased from Epitomics Inc. (Burlingame, CA). The antibody against GAPDH was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Chemiluminescent detection system was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). Bevacizumab (Avastin^®) was purchased from Roche, Inc. (Roche, USA). Cetuximab was purchased from Merck, Inc. (Merck, Darmstadt, Germany).

Tumor specimens were obtained at initial surgery from a 40-year-old female colon carcinoma patient with lymphatic and hepatic metastases. Prior written informed consent was obtained from the patient and the study received approval from the Ethics Board of the First Affiliated Hospital, College of Medicine, Zhejiang University. The patient had not received chemotherapy or radiation therapy before surgery. The histological type was determined according to WHO criteria. The tumor was diagnosed as mucinous adenocarcinoma (T3N2M1). The tumor samples of colon carcinoma with lymphatic and hepatic metastases were put into medium immediately after surgical resection under sterile conditions and transported without delay to the animal facility.

Four-to six-week-old female BALB/c nude mice purchased from Slaccas (Slaccas Laboratory Animal, Shanghai, China) were housed in a barrier facility and acclimated to 12-h light/12-h dark cycles for at least three days before use. The use of experimental animals adhered to the ‘Principles of Laboratory Animal Care’ (NIH publication No. 85–23, revised in 1985). All experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang University [approval ID: SYXK(ZHE)2005–0072]. The method to establish the PDTT xenograft models of human colon carcinoma with lymphatic and hepatic metastases were described previously (1,17–20).

Xenografts from this second mouse-to-mouse passage were allowed to grow to a size of 200 mm³, at which time mice were randomized into the following three cohorts: cohort of primary colon carcinoma xenografts, cohort of lymphatic metastasis xenografts, and cohort of hepatic metastasis xenografts. In each cohort, xenografts were randomized into four groups with 10 mice in each group: (a) control (saline 100 μl i.v. + 200 μl i.p., twice per week); (b) bevacizumab (Avastin), 10 mg/kg in 100 μl, i.v., twice per week; (c) cetuximab, 10 mg/kg in 200 μl, i.p., twice per week; (d) cetuximab, 10 mg/kg in 200 μl, i.p., twice per week + bevacizumab, 10 mg/kg in 100 μl, i.v., twice per week. Mice were treated for 21 days, monitored twice per week for signs of toxicity, and were weighed once a week. Tumor size was evaluated twice a week by caliper measurements using the following formula: tumor volume = (length × width²)/2. Relative tumor growth inhibition (TGI) was calculated by relative tumor growth of treated mice divided by relative tumor growth of control mice (T/C). Experiments were terminated on day 30. This experiment was repeated twice with similar results.

DNA was extracted from paraffin-embedded samples of colon carcinoma with lymphatic and hepatic metastases. For every tumor tissue, 10-μm sections were prepared, and an additional representative 2-μm section was deparaffinized, stained with haematoxylin and eosin, and analyzed for detailed morphology. Regions of tumor tissue were marked, and this tissue was extracted with 0.2 M sodium hydroxide in 1 mM edetic acid and neutralized with 100 mM Tris-TE (pH 6.5). After extraction, DNA was purified with Qiagen PCR purification kit (Qiagen, Hilden, Germany). KRAS gene exon 1 was analysed at codons 12 and 13 with pyrosequencing using a previously described assay which has been shown to be of greater sensitivity (21).

Selected tumor specimens were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections (5 μm) were cut, dewaxed, rehydrated, and subjected to antigen retrieval. After blocking endogenous peroxidase activity, the sections were incubated with the primary antibodies against EGFR (1:100) and VEGF (1:100) overnight at 4°C. Immunohistochemistry was performed using the streptavidin-biotin peroxidase complex method (Lab Vision, Fremont, CA). The slides were examined and images were captured using an Olympus BX60 (Olympus, Japan). Sections known to stain positively were incubated in each batch and negative controls were also prepared by replacing the primary antibody with preimmune sera.

Protein expression profiles were analyzed by western blotting as previously described (22–24). Briefly, lysates for immunoblotting were prepared by adding lysis buffer [50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, 0.02% sodium azide, and 0.1% SDS] containing protease and phosphatase inhibitors (Sigma, St. Louis, MO) to the tumor tissue homogenized in fluid nitrogen. After centrifugation at 15,000 rpm at 4°C for 10 min, the supernatants were collected, and the protein concentration was determined using Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Protein extracts of tumor lysates (30 μg) were added to a loading buffer [10 mmol/l Tris-HCl (pH 6.8), 1% SDS, 25% glycerol, 0.1 mmol/l mercaptoethanol, and 0.03% bromophenol blue], boiled, and separated on 8–12% (w/v) polyacrylamide gels in the presence of SDS. Molecular weights of the immunoreactive proteins were estimated based on the relative migration with colored molecular weight protein markers (Amersham Pharmacia Biotech, Piscataway, NJ). Following electrophoresis, the protein blots were electro-transferred to PVDF membranes (Millipore, Billerica, MA). The membranes were then blocked at room temperature with 5% nonfat milk in TBS [10 mmol/l Tris-HCl (pH 7.5), 0.5 mol/l NaCl, and 0.05% (v/v) Tween-20] buffer for 1 h. The primary antibodies were diluted at 1:1,000 and the membranes were incubated with primary antibodies overnight at 4°C. The antibodies tested were anti-Akt, anti-ERK, anti-MAPK, anti-mTOR antibodies, anti-EGFR, anti-VEGF, anti-cleaved caspase-3, and phosphorylation-specific antibodies against Akt (Ser³⁰⁸ and Ser⁴⁷³), ERK (Thr²⁰²/Tyr²⁰⁴), MAPK (Thr¹⁸⁰/Tyr¹⁸²) and mTOR (Ser²⁴⁴⁸). The following day, the membranes were washed and incubated for 1 h at room temperature with rabbit immunoglobulin G-horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), at a final dilution of 1:5,000. After washing thrice with TBS, antibody binding was visualized using enhanced chemiluminescence detection system (SuperSignal West Pico, Pierce) as described by the manufacturer and autoradiography. To show equal protein loading, the blots were stripped and reprobed for GAPDH. This experiment was repeated three times with similar results.

Drug sensitivity data are presented as the mean ± SEM and analyzed by SPSS 16.0 software. Difference among mean of the groups is determined with one-way ANOVA. Comparison is considered to be statistically significant at p<0.05.

It is necessary to ascertain the molecular basis for the response to cetuximab in metastatic colon carcinoma. For this purpose, we assessed the mutation status of the KRAS gene exon 1 at codons 12 and 13 in primary colon carcinoma and its lymphatic and hepatic metastases. Our results revealed that the KRAS gene status in the three tumor sites are all wild-type (Fig. 1).

We subsequently evaluated the therapy response of cetuximab in combination with bevacizumab in the primary colon carcinoma and its corresponding lymphatic and hepatic metastases using the PDTT xenograft models. Our results showed that all xenografts of primary colon carcinoma and corresponding lymphatic and hepatic metastases in nude mice responded to the dual-inhibition of EGFR and VEGF (Fig. 2A–C). However, dual-inhibition of EGFR and VEGF resulted in significantly different relative TGI in xenografts of primary colon carcinoma (22.2%) and corresponding lymphatic (9.6%) and hepatic metastasis (9.9%) (Fig. 2A–D). Our results demonstrate that primary colon carcinoma and its corresponding lymphatic and hepatic metastases have different response rates to anti-EGFR and anti-VEGF therapies.

Immunohistochemical staining revealed that EGFR and VEGF expression levels in primary colon carcinoma tissue are different from those in its lymphatic and hepatic metastases. Our findings reveal that the expression levels of EGFR (Fig. 3) and VEGF (Fig. 4) in metastatic tissues were higher than those in primary colon carcinoma tissue.

In this study, the expression levels of the EGFR and VEGF downstream signaling pathway proteins were further determined using western blotting. The expression levels of EGFR, VEGF, pAkt, and mTOR in metastatic tissues were found to be higher than those in primary colon carcinoma tissue (Fig. 5), while the expression levels of Akt, ERK, MAPK, and pmTOR in primary colon carcinoma tissue were higher than those in metastatic tissues (Fig. 5). Hepatic metastasis had the highest expression levels of pERK and pMAPK (Fig. 5). Our results indicate that the heterogeneity of EGFR- and VEGF-related signaling pathway proteins exist in primary colon carcinoma and its corresponding lymphatic and hepatic metastases. Furthemore, our findings indicate that heterogeneity in primary colon carcinoma and its corresponding lymphatic and hepatic metastases may partially make contribute to differences in response to anti-EGFR or anti-VEGF targeted therapies.