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. 2006;8(2):R20.
doi: 10.1186/bcr1398. Epub 2006 Apr 11.

Tumor-specific expression of alphavbeta3 integrin promotes spontaneous metastasis of breast cancer to bone

Erica K Sloan  1 , Normand Pouliot, Kym L Stanley, Jenny Chia, Jane M Moseley, Daphne K Hards, Robin L Anderson
Affiliations

Tumor-specific expression of alphavbeta3 integrin promotes spontaneous metastasis of breast cancer to bone

Erica K Sloan et al. Breast Cancer Res. 2006.

"V体育官网入口" Abstract

Introduction: Studies in xenograft models and experimental models of metastasis have implicated several beta3 integrin-expressing cell populations, including endothelium, platelets and osteoclasts, in breast tumor progression. Since orthotopic human xenograft models of breast cancer are poorly metastatic to bone and experimental models bypass the formation of a primary tumor, however, the precise contribution of tumor-specific alphavbeta3 to the spontaneous metastasis of breast tumors from the mammary gland to bone remains unclear VSports手机版. .

Methods: We used a syngeneic orthotopic model of spontaneous breast cancer metastasis to test whether exogenous expression of alphavbeta3 in a mammary carcinoma line (66cl4) that metastasizes to the lung, but not to bone, was sufficient to promote its spontaneous metastasis to bone from the mammary gland V体育安卓版. The tumor burden in the spine and the lung following inoculation of alphavbeta3-expressing 66cl4 (66cl4beta3) tumor cells or control 66cl4pBabe into the mammary gland was analyzed by real-time quantitative PCR. The ability of these cells to grow and form osteolytic lesions in bone was determined by histology and tartrate-resistant acid phosphatase staining of bone sections following intratibial injection of tumor cells. The adhesive, migratory and invasive properties of 66cl4pBabe and 66cl4beta3 cells were evaluated in standard in vitro assays. .

Results: The 66cl4beta3 tumors showed a 20-fold increase in metastatic burden in the spine compared with 66cl4pBabe V体育ios版. A similar trend in lung metastasis was observed. alphavbeta3 did not increase the proliferation of 66cl4 cells in vitro or in the mammary gland in vivo. Similarly, alphavbeta3 is not required for the proliferation of 66cl4 cells in bone as both 66cl4pBabe and 66cl4beta3 proliferated to the same extent when injected directly into the tibia. 66cl4beta3 tumor growth in the tibia, however, increased osteoclast recruitment and bone resorption compared with 66cl4 tumors. Moreover, alphavbeta3 increased 66cl4 tumor cell adhesion and alphavbeta3-dependent haptotactic migration towards bone matrix proteins, as well as their chemotactic response to bone-derived soluble factors in vitro. .

Conclusion: These results demonstrate for the first time that tumor-specific alphavbeta3 contributes to spontaneous metastasis of breast tumors to bone and suggest a critical role for this receptor in mediating chemotactic and haptotactic migration towards bone factors VSports最新版本. .

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Figures

Figure 1
Figure 1
αvβ3 integrin expression in tumor lines of the metastasis model. Cell surface expression of integrin subunits was determined by measuring the specific fluorescence by flow cytometry as described in Materials and methods. Shaded area, isotype; dotted line, αv; solid line, β3.
Figure 2
Figure 2
Tumor expression of αvβ3 integrin promotes spontaneous metastasis to the spine but not primary tumor growth. Cells (1 × 104 in 20 μl of 50% Matrigel) were inoculated into the mammary gland of Balb/c mice (15 mice per group). (a) Primary tumor volumes were measured three times per week. (b) Primary tumor weights were recorded on the day of harvest (day 39). (c) In vitro proliferation of 66cl4pBabe and 66cl4beta3 cells. The tumor burden in the (d) spine and (f) lungs was measured by real-time quantitative PCR as described in Materials and methods. Each point represents one mouse, but not all points are distinguishable. The average metastatic burden for each group is shown with a line. *P = 0.01. (e) H&E of a 66cl4beta3 spontaneous femoral metastasis. A metastatic nodule (M) in proximity of a blood vessel (BV) is seen invading through the cortical bone (C). Scale bar = 100 μm.
Figure 3
Figure 3
Tumor αvβ3 integrin enhances osteoclast recruitment and osteolysis following intratibial injection of tumor cells. (a) The relative tumor burden was measured by real-time quantitative PCR 14 days following direct intratibial injection of 66cl4pBabe and 66cl4beta3 cells. Representative H&E-stained sections through tibias injected with (b) 66cl4beta3 or (c) 66cl4pBabe cells. Osteoclasts are marked with arrows. B, bone; T, tumor. Scale bar = 50 μm. (d) Serial sections adjacent to those stained by H&E were stained for tartrate-resistant acid phosphatase (TRAP) activity, and the active osteoclasts (TRAP-positive) present per millimeter of bone in tibias injected with either 66cl4beta3 or 66cl4pBabe cells were counted. Results of two independent experiments are shown. n = 4 mice/group per experiment *P < 0.001.
Figure 4
Figure 4
αvβ3 integrin mediates mammary tumor cell adhesion to vitronectin but not to endothelial cells. (a) Short-term adhesion to different matrices. (b) Effect of neutralizing antibodies against β3 integrin on short-term adhesion to vitronectin. (c) Adhesion of 66cl4pBabe and 66cl4beta3 cells to a monolayer of bEnd.3 endothelial cells in the absence of serum. The experiments were repeated at least twice and the results represent the mean ± standard deviation of a representative experiment performed in triplicate wells. *P < 0.001.
Figure 5
Figure 5
Expression of αvβ3 integrin promotes tumor cell migration and invasion. Migration and Matrigel invasion assays were performed in Transwell migration chambers (8 μm pore size). (a) Haptotactic migration in response to collagen IV, osteopontin and vitronectin and the effect of neutralizing β3 antibodies. (b) Effect of the matrix metalloproteinase inhibitor AG3340 (10 μM) on the haptotactic migration of 4T1.2 and 66cl4beta3 cells. (c) Chemotactic migration of 66cl4pBabe and 66cl4beta3 cells towards a monolayer of bone stromal cells seeded in the lower chamber. (d) Invasion of cells through Matrigel in response to a serum chemotactic gradient. All assays were performed in duplicate wells and the cells counted from three fields of view/membrane at 40× magnification (a, b and d) or 20× magnification (c). The experiments were repeated at least twice and the data represent the mean number of migrated cells ± standard deviation of six fields of view/condition from a representative experiment performed in duplicate wells. *P < 0.01, **P < 0.001.
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