ADAMTS-18 in the host tissues exerts little effect on breast tumor progress in a murine 4T1 breast cancer model

Background In this study, we aimed to identify a novel extracellular proteinase ADAMTS-18 that could be a potential tumor suppressor gene. Results We successfully constructed Adamts-18 knockout mice with BALB / c background. RT-PCR analysis showed syngeneic mammary tumor cell line 4 T1 per se has weakly endogenous ADAMTS-18 expression. Orthotopic inoculation of 4 T1 cells within the mammary fat pad of host mice, we found no significant difference in tumor growth and metastasis between Adamts-18 knockout mice and widetype control. Conclusions We did not confirm that ADAMTS-18 in the host tissues is relevant for breast tumor progress in a murine 4 T1 breast cancer model.


Background
Matrix metalloproteinases are traditionally linked to tumor dissemination through their ability to degrade most extracellular matrix (ECM) components, thus facilitating tumor cell invasion and metastasis [1]. However, recent studies suggest that some metalloproteinases contribute to inhibit tumorigenesis [2]. ADAMTS-18 (a disintegrin and metalloproteinase with thrombospondin motif 18), as yet no known function or substrate, is a member of ADAMTS family of secreted proteinases. It has been shown that ADAMTS-18 gene was inactivation in many carcinomas especially breast tumors and, therefore, is regarded as a novel functional tumor suppressor [3]. We previously reported that the C-terminal ADAMTS-18 fragment induces oxidative platelet fragmentation, dissolves platelet aggregates, and protects against carotid artery occlusion and cerebral stroke [4]. To further study the role of ADAMTS-18 in vivo, we generated Adamts-18 knockout mice (unpublished data). The aim of this study was to investigate the effect of ADAMTS-18 on tumorigenesis in a murine 4 T1 breast cancer model.

Animal studies
In our laboratory Adamts-18 heterozygote mice (Adamts-18 +/-) with C57BL6/129SV were developed (unpublished data). They backcrossed to wild-type BALB/c mice for 7 generations to obtain BALB/c background Adamts-18 +/mice. Adamts-18 +/mice with BALB/c background are intercrossed to generate Adamts-18 knockout mice (Adamts-18 -/-) and wildtype control (Adamts-18 +/+ ) for further experiment. Syngeneic mammary tumor cell line 4 T1 cells were orthotopically implanted within the mammary fat pad of females aged 8-10 weeks. Cells implantation in the mammary fat pad was achieved using a (1/2)-inch 26 gauge needle and gentle pressure during delivery of 100 μl cells. Mice were watched daily to monitor the growth of the tumor and once a substantial size of tumor mass was found, the mice were sacrificed and the tumor mass were collected and weighted. The surface metastatic nodules on the lungs and other organs were counted, and then histologic analyses were performed. All procedures in animal experiments were approved by the Institutional Animal Care and Use Committee of East China Normal University (ECNU). All the mice used in this study are female and maintained in a specific pathogen-free facility at ECNU.

Western blotting
Proteins from the brain, kidney, liver or xenograft tumors of both mice were separated on a 12 % SDS-PAGE under reducing conditions and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked in blocking buffer (PBS, 0.5 % Tween-20, and 5 % non-fat dry milk powder) and then incubated with rabbit anti-ADAMTS18 IgG (sc-68416, Santa Cruz Biotechnology, Inc.) for 1 h at RT. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT. The immunoreactive bands were visualized with enhanced chemiluminescence (ECL) western blot kit.

Histological analysis
Tumor tissues were fixed in 10 % formaldehyde in phosphate-buffered saline (PBS), pH 7.4, dehydrated and embedded in paraffin and sectioned with a microtome. Tumor malignant degree and mitotic counts in sections were evaluated by hematoxylin and eosin (H.E) staining. Intratumoral vascular density was assessed by

Statistical analysis
P values were determined through the two-tailed Student's t test. Data are presented as mean ± SD. Differences were considered statistically significant when P < 0.05.

Results and discussion
The potential role of ADAMTS-18 in tumor genesis is first suggested by the genetic analysis [5]. It has been shown that the loss of 16q23 region is strongly associated with a variety of cancers. Since ADAMTS-18 is one of these genes located around 16q23 region, it has been studied as a candidate oncogene [3,[5][6][7][8][9][10]. Because ADAMTS-18 is often inactivated in breast tumors, we therefore develop Adamts-18 knockout mouse with BALB/c background, and inoculate syngeneic mammary tumor cell line 4 T1 cells to evaluate the role of ADAMTS-18 in breast cancer. First, Adamts-18 heterozygote mice (Adamts-18 +/-) with C57BL6/129SV background were backcrossed to wild-type BALB/C mice for 7 generations to obtain Adamts-18 +/mice with BALB/ c background. They are intercrossed to get Adamts-18 knockout mice (Adamts-18 -/-) and wildtype control (Adamts-18 +/+ ) for further experiment (Fig. 1a-b). Immunoblot analysis for ADAMTS-18 expression in different tissues from both mice further confirmed the knockout effectiveness (Fig. 1c). The results of orthotopic inoculation of syngeneic mammary tumor cell line 4 T1 cells (10 6 /mouse) within the mammary fat pad showed that the percentage of tumor incidence reached 100 % after six and seven generations (Fig. 1d). In the seventh generation of mice, 10 of 10 mice bear tumor when the inoculation doses of 4 T1 cells were 10 6 per mice (Fig. 1e). Representative photograph of breast tumor (Fig. 1f) and hematoxylin-eosin (HE) staining are shown (Fig. 1g).
Many ADAMTSs have proved to be anti-angiogenic through their metalloproteinase-dependent and -independent activities, which contribute to inhibit cancer development and progression. ADAMTS-1 displays both types of mechanism [11]. It induces the generation of anti-angiogenic fragments by cleaving matrix-bound thrombospondins-1 and -2 [12], and also sequesters angiogenic factors such as the basic fibroblast growth factor (bFGF) and vascular endothelial growth 1(VEGF165) [13,14]. ADAMTS-9 also demonstrates metalloproteinasedependent and -independent activities, but the mechanism remains unclear [15]. Other ADAMTSs, including ADAMTS-2, -4, -5, and -12, displays metalloproteinaseindependent inhibition of neovascularization, which is closely related to their thrombospondin type 1 sequence repeat (TSR) domain [16][17][18][19]. The peptides (termed Adamtsostatins) corresponding to the TSRs of some family members have anti-angiogenic properties, indicating that this may be a general activity of ADAMTSs. In this study, we didn't find difference in microvessel densities of tumors between Adamts-18 +/+ and Adamts-18 -/mice. Nevertheless, more sophisticated assays for angiogenesis such as wound healing, tube formation, and ischemia assays are needed in future studies.

Conclusions
The current data suggest that the murine 4 T1 breast tumor cell per se has weakly endogenous ADAMTS-18 expression, and ADAMTS-18 in the host mouse exerts little effect on breast tumor progression in murine 4 T1 breast cancer model.