New Model of In-situ Xenograft Lymphangiogenesis by a Human Colonic Adenocarcinoma Cell Line in Nude Mice

Colorectal adenocarcinoma is the most common malignant tumor via the lymphatic metastasis in human. Lymphatic metastasis of the tumor is an important reason for uneradicative cure and mortality. Recent studies have found that lymphangiogenesis is a major factor and mechanism for colorectal cancer lymphatic metastasis and recurrence (Achen and Stacker, 2006; Sundar and Ganesan, 2007; Nagahashi et al., 2010). With the


Introduction
Colorectal adenocarcinoma is the most common malignant tumor via the lymphatic metastasis in human.Lymphatic metastasis of the tumor is an important reason for uneradicative cure and mortality.Recent studies have found that lymphangiogenesis is a major factor and mechanism for colorectal cancer lymphatic metastasis and recurrence (Achen and Stacker, 2006;Sundar and Ganesan, 2007;Nagahashi et al., 2010).With the lymphatic vessels, technological progress to define mechanism of lymphangiogenesis, it has been taken attach of the relationship between molecular mechanism of lymphangiogenesis and lymphatic metastasis, as well as the therapy based on antilymphangiogenesis (McColl et al., 2005;Wissmann and Detmar, 2006).In order to study lymphatic metastasis in colorectal cancer and antilymphangiogenic treatment, it is imperative to

New Model of In-situ Xenograft Lymphangiogenesis by a Human Colonic Adenocarcinoma Cell Line in Nude Mice
Jian-Jun Sun & , Wei Jing & , Yan-Yan Ni, Xiao-Jian Yuan, Hai-Hua Zhou, Yue-Zu Fan* colorectal carcinoma in nude mice as a common animal model.However, such a model of subcutaneous tumors and hematogenous metastasis, so it is not exactly the performance of biological characteristics in colorectallymphatic or hematogenous metastasis.In recent years, scholars have reported using an orthotopic model of human colorectal cancer transplantation, in order to improve the human colorectal cancer regional lymph node metastasis rate, but no tumor lymphangiogenesis in animal models have been reported.Our experiment built an orthotopic model of human colorectal cancer transplantation ,on the basis of colorectal cancer subcutaneous xenograft model in nude mice, to explore the possibility of the establishment of human colon adenocarcinoma lymphangiogenesis model in nude mouse, in order to provide experimental models for study of human colorectal cancer lymphatic metastasis mechanisms and treatment.
Orthotopic implantation of tumor cells to nude mouse subcutaneously: 0.2 ml of human colon adenocarcinoma HT-29 single-cell suspension (1 × 10 7 /ml) were injected into the side of nude mice subcutaneously (partial dorsal) to format solid tumors using a sterile syringe.
weeks when the tumor diameter of 1.5 cm, tumor tissue stripped from subcutaneously tissue were placed in saline (10 5 U/L penicillin, 10 5 U/L streptavidinfactors)which containing penicillin and streptomycin, and then removed about 1 mm in diameter, and then transplanted to the next generation of nude mice (2/generation), to be passaged to 4, the subcutaneously transplanted tumor (n = 8) was used as the source of orthotopic transplantation tumor.
Orthotopic transplantation of colon tumor to nude mouse model: 36 nude mice, fasting for 12 h before surgery were divided into two groups.One group underwent colon orthotopic implantation (n = 24); the other group injection of saline in the colon in situ as a negative control (n = 12).Colon orthotopic transplantation method: Anaesthesied with 0.5% pentobarbital sodium (45 mg/kg) by intraperitoneal injection, take a ventral midline incision , cut the abdominal wall, free cecum at the end of the colon , make inward push at the prick of the intestinal wall, to form a local intestinal concave niche; subcutaneous tumor mass of about 1.0 mm in diameter cover the tumor surface the surface of and paved to the cecal wall; put back cecum into the abdominal cavity and then closed the abdomen with disinfection incision.Keep warm of the nude mice, subcutaneous injection of saline to supply water postoperatively fast water for 6 h after surgery, and provided with the high-energy diet to supplement the energy.The nude mice were observed in sub-cage to conscious-sterile and the feeding at laminar The gross observation and HE staining: Nude body condition and activities were observed daily after inoculation, also including stool and abdominal signs; palpation the abdominal cavity in situ in order to observation of the colon tumor growth.The negative control group, orthotopic colon tumor nude mice were for HE staining and immunohistochemical staining for optical microscope examination; another part of the fresh samples were cryopreserved, and used for Western blot and RT-PCR.SABC immunohistochemical staining: Slices of dropping primary antibody anti-LYVE-1 (1:60), anti-D2-40 (1:50), biotinylated secondary antibody and SABC step by step, use DAB color box for coloring, and then conventional staining, dehydration, transparent, the sequestration to be tested.Lymph node a positive control and PBS was used as a negative control instead of primary antibody,.The judgment of results: LYVE-1 and D2-40 positive staining showed lymphatic tubular structures of the lymphatic endothelial cell membrane and cytoplasm with brown-yellow-brown particles.Periphery colon adenocarcinoma, the center and the corresponding normal tissue of the cutting edge was observed under lymphatic vessel density areas, and then under high positive number of lymphatic vessels (each brown-stained endothelial cells or endothelial cell cluster, as long as the separated and neighboring vasculature, tumor cells or other connective tissue, is considered one of lymphatic vessels), that was lymphatic vessel density (LMVD).It center, the surrounding and normal colorectal rectal tissue LMVD.
Western blot blotting: Fresh tumor tissue (n = 8) about of 2 mm in diameter and tumor-free colon tissue (negative control, n = 4) in nude mice, extraction of Protein Assay Kit (Shen neng, Shanghai), determination of the A562, and calculate the protein concentration; Preparation of separating gel, the plot layer of glue in the slot, and then adding the electrophoresis buffer, sample, (Bio-Rad protein3, Bio-Rad, USA).Until the proteins were transferred to PVDF membrane (Millpro), closed membrane by adding primary antibody (Anti-LYVE-1, secondary antibody (1:1000); Adding 1:1 AB developer combined with the two anti-HRP (Tiangen, Bio-Rad, USA) imaging analysis of gray values to calculate the ratio of gray values.
Fluorescent quantitative RT-PCR: Take fresh tumor tissue (n = 8) and nude mice colon tissue (negative control, n = 4) block, extraction of total RNA by RT-PCR for mRNA detection.The primers were designed and synthesized by Invitrogen Corporation.LYVE-1: upstream GAPDH was used as an internal control, the annealing temperature of 56 °C for 40 cycles (94 °C for 5 min, 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, 72 °C for 10 min).We analyzed with the ABI Prism 7300 SDS.The relative mRNA expression levels was calculated by the formula (relative mRNA expression = 2 -Ct ).
Fluorescent quantitative RT-PCR detection: Take 8 tissue block in 2 mm diameter in each group, extracted We analyzed with the ABI Prism 7300 SDS.The relative mRNA expression levels was calculated by the formula (relative mRNA expression = 2 -Ct ).
All data analysis was by the SPSS 13.0.The experimental data was described with ± s and comparing difference between two groups using single-factor analysis

Results
After the colon orthotopic implantation of subcutaneously transplanted tumor about two weeks , hard nodules could be palpated in the right lower quadrant, mm at 6 weeks.Some animals with weight loss, lack of exercise and cachexia.Tumor could be found at intestinal wall, pale, round or oval, with the surface of nodular (Figure 1).Average tumor weight (0.76 ± 0.24) g.The rate of Orthotopic implantation was 100%.
Control group injected with saline in the nude mouse colon, there was no tumor growth, and it is hereinafter referred to as tumor-free group.
Tumor free group of normal intestinal mucosa, glands, of colon orthotopic implantation tumor was destroyed, round or oval, actively growing cancer cells showed invasive growth of cancerous tissue, arranged in a group cords, abundant cytoplasm, large nuclei deeply stained mitotic increase in cancer cells between the connective tissue, some areas of vacuolar degeneration and necrosis of cancer cells, the structure was unclear, and could be tissue.
Immunohistochemical staining and LMVD: There was no or little brown tan in tumor free group of lymphatic endothelial cell markers LYVE-1 (Figure 3A), andD2-40 positive expression (Figure 3C), LMVD was very low; Colon orthotopic implantation tumor ,of which LYVE-1 (Figure 3B1, 2), and D2-40 (Figure 3D1, 2) expression was strongly positive.Those of LYVE-1, D2-40 positive pipeline are single endothelial cells, thin wall, large lumen, irregular, often collapse like, in line with the morphological features of lymphatic capillaries, LMVD Figure 3E), and which were distributed at the junction of the tumor tissue and normal tissue, almost no lymphatic material, lymphocytes, tumor cells and their debris were still visible in the lymphatic lumen.
Fluorescent quantitative RT-PCR: Orthotopic implantation nude mouse colon tumor, VEGF-C mRNA, VEGF-D mRNA and VEGFR-3 mRNA in expression which is in line with western blot results (Figure 7).

Discussion
markers have been successively found and the molecular mechanism of lymphangiogenesis have been revealed step by step, thus the role of lymphangiogenesis in tumor metastasis, recurrence, treatment is gradually being recognized, the related research is also increasingly in-depth (Kowanetz and Ferrara, 2006;Mylona et al., 2007;Zwaans and Bielenberg, 2007;Liersch et al., 2010).Because the tumor metastasis animal models is important in cancer metastasis research, as well as lymphatic (node) metastasis as the main path of the cancer spreading take more and more attach of colorectal cancer metastasis, it is inevitably put forward higher requirements of animal models in the related mechanisms research of colorectal cancer metastasis, especially lymphatic metastasis (Tsutsumi et al., 2001;Flatmark et al., 2004;Sasaki et al., 2008).But in the past, with using of fresh cancer tissues or cell lines established in nude mice subcutaneous tumor model can not be the occurrence of deep invasion and lymph node metastasis (Fidler, 1990), while the use of nude mouse colon subserosal injection of human colorectal cancer cell lines produce orthotopic implantation of tumor, can be simulated colorectal cancer distant organ metastasis, lymphatic way to transfer is not obvious, colorectal situ lymph node micrometastasis and lymphatic generated, the development process falls far short.The reason is that tumor metastasis requires cellcell and cell-matrix interactions between the activated cell secretory protein degradation enzymes, degradation of the matrix membrane and interstitial matrix, activation of the mobility of tumor cells invasion and metastasis-related mechanism involved in (Flatmark et al., 2004).Therefore, we needed a tumor lymphangiogenesis in animal models, to knot the study of the mechanism of rectal cancer lymphangiogenesis and lymphatic metastasis, drug intervention and anti-lymphatic therapy.
Recent 10 years, the development of tumor lymphatic of lymphatic endothelial cells, molecular signals involved in lymphatic vessels generate, and tumor control or intervention based on these mechanisms.LYVE-1 (Jackson et al., 2001;Prevo et al., 2001;Achen et al., 2006), D2-40 (Breiteneder et al., 1999;Renyi et al., 2000;Achen et al., 2006;Kowanetz and Ferrara, 2006;Wissmann and Detmar, 2006) are the most important lymphatic endothelial cell-specific markers.While LYVE-1, D2-40 and VEGFR-3 expression is positive lymphangiogenesis rather than angiogenesis.VEGF-C and VEGF-D and VEGFR-3 are recognized as lymphatic endothelial growth factor and receptors in the lymphatic-VEGFR-3 signaling pathway (Kubo et al., 2000;Achen and Stacker, 2006;Achen et al., 2006;Kowanetz and Ferrara, 2006;Wissmann and Detmar, 2006;Sundar and Ganesan, 2007;Nagahashi et al., 2010).Our results suggest that the models of orthotopic transplantation of colorectal cancer in nude mice showed LYVE-1, D2-40 immunohistochemical staining strongly positive, LYVE-1, D2-40, VEGF-C, VEGF-D and VEGFR-3 protein capillaries and the VEGF-C, -D/VEGFR-3 signaling axis control mechanism, which proved that the model of orthotopic transplantation of colorectal cancer in nude mice was a colorectal adenocarcinoma lymphangiogenesis animal model.This experiment was successfully established an orthotopic transplantation and lymphatic metastasis model of human colorectal cancer in nude mice, it showed tumorigenesis in a short time, the high rate of tumor formation and more stable tumor pathology and biological characteristics.At the same time, observing the process of lymphangiogenesis and early lymphatic metastasis, the model simulated the natural growth of process, not only in the signaling pathway but also in the morphology of lymphangiogenesis, objectively metastasis in vivo.It provides an ideal animal model for in-depth study of lymphangiogenesis in colorectal cancer, lymphatic metastasis and antimetastatic therapy.

Figure 1 .
Figure 1.The Nude Mice with Xenografts and the Xenografts of Human Colonic Adenocarcinoma Cell Line HT-29 in Nude Mice.A: subcutaneous xenograft, which was used to establish in-situ xenograft.B: in-situ colonic xenograft.C: control mouse without in-situ colonic xenograft

Figure 3 .
Figure 3. Immunohistochemical Staining of LYVE-1 and D2-40 (SABC method), and Number of Lymphangiogenetic Microvessels (LMVD) in the Insitu Colonic Xenografts and the Control Colon Without Xenograft in Nude Mice.A: LYVE-1 expression was strong positive expression of LYVE-1 in the in-situ colonic xenografts was observed, and these positive tubular structures accorded with thin wall, large lumen, irregular morphological features of lymphatic capillaries, and distribution in tumor tissue lymphocyte, tumor cells and their fragments in the other lumen

Figure 4 .Figure 5 .Figure 7 .
Figure 4.Western Blot Analysis of LYVE-1 and D2-40 in the In-situ Colonic Xenografts (1) and the control colon without xenograft (2) in nude mice.The expression of LYVE-1 than that of the control group (both *P<0.01)