Assessment of a novel VEGF targeted agent using patient-derived tumor tissue xenograft models of colon carcinoma with lymphatic and hepatic metastases

Abstract

The lack of appropriate tumor models of primary tumors and corresponding metastases that can reliably predict for response to anticancer agents remains a major deficiency in the clinical practice of cancer therapy. It was the aim of our study to establish patient-derived tumor tissue (PDTT) xenograft models of colon carcinoma with lymphatic and hepatic metastases useful for testing of novel molecularly targeted agents. PDTT of primary colon carcinoma, lymphatic and hepatic metastases were used to create xenograft models. Hematoxylin and eosin staining, immunohistochemical staining, genome-wide gene expression analysis, pyrosequencing, qRT-PCR, and western blotting were used to determine the biological stability of the xenografts during serial transplantation compared with the original tumor tissues. Early passages of the PDTT xenograft models of primary colon carcinoma, lymphatic and hepatic metastases revealed a high degree of similarity with the original clinical tumor samples with regard to histology, immunohistochemistry, genes expression, and mutation status as well as mRNA expression. After we have ascertained that these xenografts models retained similar histopathological features and molecular signatures as the original tumors, drug sensitivities of the xenografts to a novel VEGF targeted agent, FP3 was evaluated. In this study, PDTT xenograft models of colon carcinoma with lymphatic and hepatic metastasis have been successfully established. They provide appropriate models for testing of novel molecularly targeted agents.

Figure 1. Expression of CDX-2 and CK-20 in the tumor tissues of lymphatic (A and B) and hepatic metastasis (C and D).

Original magnifications, ×200.

Figure 2. Representative H&E stained tissues of primary colon carcinoma (A and B) and its corresponding lymphatic (C and D) and hepatic (E and F) metastases and their early-generation of xenograft tumors.

G0, the primary tumors; G3, the third generation of xenografts. Original magnifications, ×100.

Figure 3. VEGF immunohistochemical staining of primary tumor tissues of primary colon carcinoma (A and B) and its corresponding lymphatic (C and D) and hepatic (E and F) metastases and their early-generation xenograft tumor tissues.

G0, the primary tumors; G3, the third generation of xenografts. Original magnifications, ×100.

Figure 4. EGFR immunohistochemical staining of primary tumor tissues of primary colon carcinoma (A and B) and its corresponding lymphatic (C and D) and hepatic (E and F) metastases and their early-generation xenograft tumor tissues.

G0, the primary tumors; G3, the third generation of xenografts. Original magnifications, ×100.

Figure 5. Exemplified immunoblotting data of the proteins Akt, pAkt (Ser³⁰⁸ and Ser⁴⁷³), ERK, pERK (Thr²⁰²/Tyr²⁰⁴), MAPK, pMAPK (Thr¹⁸⁰/Tyr¹⁸²), mTOR, pmTOR (Ser²⁴⁴⁸), EGFR, VEGF, Casepase-3, PCNA and GAPDH (as loading control) of primary colon carcinoma and its corresponding lymphatic and hepatic metastases and their early-generation xenograft tumor tissues.

G0, the primary tumors; G3, the third generation of xenografts.

Figure 6. Gene expression profiling of patient-derived tumor tissues and corresponding xenografts.

A, hierarchical clustering based on 140 probe sets differentially expressed between primary colon carcinoma and its xenograft. B, hierarchical clustering based on 70 probe sets differentially expressed between colon carcinoma lymphatic metastasis and its xenograft. C, hierarchical clustering based on 145 probe sets differentially expressed between colon carcinoma hepatic metastasis and its xenograft. PCT, primary colon carcinoma. PCT Xe, primary colon carcinoma xenograft. Lym, colon carcinoma lymphatic metastasis. Lym Xe, colon carcinoma lymphatic metastasis xenograft. Hep, colon carcinoma hepatic metastasis. Hep Xe, colon carcinoma hepatic metastasis xenograft.

Figure 7. Response curve of FP3 and Avastin in the PDTT xenograft models of primary colon carcinoma (A), lymphatic metastasis (B), and hepatic metastasis (C).

Ten mice per group were treated with the corresponding agent according to Materials and Methods. Data shown are means ± SEM. The differences between control tumor volumes, FP3-treated, and Avastin-treated tumor volumes were analyzed by using one-way ANOVA. ^*** p<0.001, versus control. Experiments were repeated at least two times with similar results.

Figure 8. FP3 decreased vascular structure in the xenograft model of primary colon carcinoma.

Vasculature was examined by angiography with immunostaining for endothelial cells (using anti-CD31 antibody; bar = 100 µm), and pericytes (using anti-α-SMA antibody; bar = 100 µm). There was a paucity of vessels identified in FP3-treated tumors.

Figure 9. FP3 decreased vascular structure in the xenograft model of colon carcinoma lymphatic metastasis.

Vasculature was examined by angiography with immunostaining for endothelial cells (using anti-CD31 antibody; bar = 100 µm), and pericytes (using anti-α-SMA antibody; bar = 100 µm). There was a paucity of vessels identified in FP3-treated tumors.

Figure 10. FP3 decreased vascular structure in the xenograft model of colon carcinoma hepatic metastasis.

Vasculature was examined by angiography with immunostaining for endothelial cells (using anti-CD31 antibody; bar = 100 µm), and pericytes (using anti-α-SMA antibody; bar = 100 µm). There was a paucity of vessels identified in FP3-treated tumors.

Figure 11. Effects of FP3 and Avastin on the expression of VEGF in the PDTT xenograft models of primary colon carcinoma (A–C), lymphatic metastasis (D–F), and hepatic metastasis (G–I).

Original magnifications, ×100.

Figure 12. Effects of FP3 and Avastin on the expression of PCNA in the PDTT xenograft models of primary colon carcinoma (A–C), lymphatic metastasis (D–F), and hepatic metastasis (G–I).

Original magnifications, ×100.

Figure 13. Effects of FP3 and Avastin on the expression of EGFR in the PDTT xenograft models of primary colon carcinoma (A–C), lymphatic metastasis (D–F), and hepatic metastasis (G–I).

Original magnifications, ×100.

Figure 14. Effects of FP3 and Avastin on the expression of VEGFR-2 in the PDTT xenograft models of primary colon carcinoma (A–C), lymphatic metastasis (D–F), and hepatic metastasis (G–I).

Original magnifications, ×100.

Figure 15. Western blotting analysis showing the effects of FP3 and Avastin on the expression of VEGFR-2 protein in the PDTT xenograft models of primary colon carcinoma (A), lymphatic metastasis (B), and hepatic metastasis (C).