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Introduction

Intratumor heterogeneity is one of the recognized characteristics of human tumors, and occurs on multiple levels, such as the genetic, protein and macroscopic level in a wide range of tumors, including breast, colorectal (CRC), non-small cell lung (NSCLC), prostate, ovarian, pancreatic, gastric, and brain cancer and renal clear cell carcinoma (1). In recent years, many studies have focused on the heterogeneity found in primary tumors and related metastases with the consideration that evaluation of metastatic rather than primary sites could be of clinical relevance. Numerous reports have evaluated the genetic heterogeneity in primary tumors and corresponding metastases in a range of solid tumors such as breast cancer (2–9), CRC (10–13) and NSCLC (14,15). Heterogeneity in primary tumors and related metastases may result in the failure of antitumor therapies, particularly in targeted therapies for the treatment of cancer (16).

However, without a suitable tumor model we cannot elucidate whether such heterogeneity results in different responses to anticancer therapy. In a previous study, we successfully established the patient-derived tumor tissue (PDTT) xenograft models of colon carcinoma with lymphatic and hepatic metastases (17). The ideal biological characteristics of such PDTT xenograft models, as previously described (17), confirmed our hypothesis that such PDTT models would help us investigate the underlying mechanism of the differences in heterogeneity-related anticancer therapy response in primary colon carcinoma and its corresponding lymphatic and hepatic metastases.

In this study, PDTT xenograft models of colon carcinoma with lymphatic and hepatic metastases were used to evaluate the response to EGFR- and VEGF-targeted therapies. We also investigated heterogeneity in primary colon carcinoma tissue and its corresponding lymphatic and hepatic metastatic tissues from the same metastatic colon carcinoma patient focusing on the cell signaling pathway proteins.

Materials and methods

Reagents and drugs

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).

Patient and tissue samples

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.

Establishment of xenografts and treatment protocol

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 extraction and mutation analyses

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).

Immunohistochemistry

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.

Western blotting

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.

Statistical analysis

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.

Results

PDTT xenograft models of primary colon carcinoma and related metastases have different response rates to dual-inhibition of EGFR and VEGF

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).

Figure 1 KRAS mutation analysis by pyrosequencing. (A) Sequence data of KRAS in exon 1 at codons 12 and 13 in standard wild-type sample, and (B) in tumor tissues of primary colon cancer, (C) lymphatic metastasis, and (D) hepatic metastasis.

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.

Figure 2 Response curve of cetuximab, bevacizumab (Avastin), and cetuximab + bevacizumab in PDTT xenograft models of (A) primary colon carcinoma, (B) lymphatic metastasis, and (C) hepatic metastasis, and (D) the response rate to cetuximab, bevacizumab, and cetuximab + bevacizumab in PDTT xenograft models of three tumor sites. Ten mice per group were treated with the corresponding agent. Data shown are the means ± SEM. The differences between control tumor volumes, cetuximab-treated, bevacizumab-treated and cetuximab + bevacizumab-treated tumor volumes and the response rates to the same treatment in xenografts of three tumor sites were analyzed by one-way ANOVA. Experiments were repeated at least twice with similar results.

Heterogeneity in primary colon carcinoma and its corresponding lymphatic and hepatic metastases

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.

Figure 3 Immunohistochemical staining of EGFR. (A) Primary colon carcinoma, (B) lymphatic metastasis (C) hepatic metastasis. Original magnification, ×100.

Figure 4 Immunohistochemical staining of VEGF. (A) Primary colon carcinoma, (B) lymphatic metastasis, (C) hepatic metastasis. Original magnification, ×100.

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.

Figure 5 Immunoblotting data of the proteins Akt, pAkt (Ser308 and Ser473), ERK, pERK (Thr202/Tyr204), MAPK, pMAPK (Thr180/Tyr182), mTOR, pmTOR (Ser2448), EGFR, VEGF, cleaved caspase-3, and GAPDH (as loading control) of (A) primary colon carcinoma and its corresponding (B) lymphatic and (C) hepatic metastases.

Discussion

The main purpose of investigating the heterogeneity in primary tumors and their corresponding metastases is to evaluate the effects of such heterogeneity on the efficacy of anticancer therapy and cancer patients' prognosis. As we have previously reported (1,17,19), the PDTT xenograft model which has a sound establishing method and a retained similarity to the corresponding original donor tumors in histological presentation and biological behavior, such as protein expression, tumor biomarker status, and genomic and genetic status, has the potential to be a good strategy to achieve our purpose.

In our previous study, we established the PDTT xenograft models of colon carcinoma with lymphatic and hepatic metastases (17). The biological characteristics of such PDTT xenograft models, as previously described (17), confirmed our belief that such PDTT models would aid in our investigation of the underlying mechanism of heterogeneity-related anticancer therapy response differences in primary colon carcinoma and its corresponding lymphatic and hepatic metastases. Based on this hypothesis and considering that the KRAS gene status in the three tumor sites are all wild-type, the drug sensitivity of bevacizumab (Avastin) in combination with cetuximab in the primary colon carcinoma and its corresponding lymphatic and hepatic metastases was evaluated in this study using the PDTT xenograft models.

In the present study we also investigated the heterogeneity in primary colon carcinoma and its corresponding lymphatic and hepatic metastases focusing on the cell signaling pathway proteins using immunohistochemical staining and western blotting. We found that the expression levels of EGFR, VEGF, Akt/pAkt, ERK/pERK, MAPK/pMAPK, and mTOR/pmTOR were different in primary colon carcinoma and matched lymphatic and hepatic metastases, although the KRAS gene status in all was wild-type.

With regard to CRC, the therapeutic benefit of EGFR-targeted monoclonal antibodies such as cetuximab and panitumumab has been established in various studies (25–27). Notably, no correlation was observed between the expression levels of EGFR and therapeutic success (25–27), and even patients with tumors apparently lacking EGFR expression responded to antibody therapy in up to 25% of the cases (28–30). In our study, xenografts of primary colon carcinoma and its corresponding lymphatic and hepatic metastases all responded to cetuximab (Fig. 2A–C). However, no significant difference could be observed in these groups (Fig. 2D) although they have different expression levels of EGFR (Fig. 3 and 5).

Across a wide range of human tumors and/or cell lines, expression of VEGF has been shown to lead to the development and maintenance of a vascular network that promotes tumor growth and metastasis. Moreover, a large and growing body of evidence indicates that both VEGF gene expression and production are associated closely with poor prognosis (31–35). However, no correlation was observed between the expression levels of VEGF and clinical outcomes of VEGF targeted therapy (36). Our findings show that primary colon carcinoma and its corresponding lymphatic and hepatic metastases have different expression levels of VEGF (Fig. 4 and 5), but they all responded to bevacizumab (Fig. 2A–C), and no significant difference was observed in these groups (Fig. 2D).

Moreover, xenografts of primary colon carcinoma and its corresponding lymphatic and hepatic metastases have different response rates to treatment of bevacizumab in combination with cetuximab (Fig. 2D) although all xenografts responded to the dual-inhibition of EGFR and VEGF (Fig. 2A–C). Our results demonstrated that dual-inhibition of EGFR and VEGF could result in significantly different response rates in primary colon carcinoma and corresponding metastases if the EGFR and VEGF expression levels are different in these tumors. Our results indicate that heterogeneity in primary colon carcinoma and its corresponding lymphatic and hepatic metastases may result in differences in response to dual-inhibition of EGFR and VEGF.

In this study, we investigated heterogeneity in primary colon carcinoma and its corresponding lymphatic and hepatic metastases focusing on the cell signaling pathway proteins, and we found that the levels of EGFR, VEGF, Akt/pAkt, ERK/pERK, MAPK/pMAPK, and mTOR/pmTOR were different in primary colon carcinoma and matched lymphatic and hepatic metastases. Furthermore, with the help of PDTT xenograft models, we demonstrated that such heterogeneity would result in different responses to anti-EGFR and anti-VEGF targeted therapies. The PDTT xenograft model could be a good in vivo tool to examine whether the primary tumors and corresponding metastases have different responses to the same anticancer drugs.

Acknowledgements

This work was supported by the State Key Basic Research and Development Program of China (973 Program, Grant No. 2009CB521704), National High-tech Research & Development Program of China (863 Program, Grant No. 2006AA02A245), National Natural Science Foundation of China (Grant No. 81000894), Zhejiang Provincial Science and Technology Projects (Grants No. 2009C13021, 2011C23087), Zhejiang Provincial Medical and Healthy Science and Technology Projects (Grants No. 2011KYB137), Science Research Fund of Taizhou (Grant No. A102KY09), Science Research Fund of Shaoxing (Grants No. 2011D10013) and Science Research Fund of Zhuji (Grants No. 2011CC7874).

References