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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 21, 2002 as doi:10.1096/fj.01-0911fje. |
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vß3 expression confers on tumor cells a greater propensity to metastasize to bone1
INSERM Research Unit 403, Faculty of Medicine Laënnec, Lyon, France; UMR 5533 CNRS, Pessac, France; Laboratoire Franco-Luxembourgeois de Recherche Biomédicale, CNRS/CRP-Santé, Luxembourg, Grand Duchy of Luxembourg; and INSERM Research Unit 349, Lariboisière Hospital, Paris, France
2Correspondence: INSERM Research Unit 403, Faculty of Medicine Laënnec, Rue Guillaume Paradin, 69372 Lyon cedex 08, France. E-mail: clezardin{at}lyon151.inserm.fr
SPECIFIC AIM
To address the role of integrin 
vß3 in cancer-induced bone metastasis, CHO tumor cells stably transfected to express integrin vß3 and B02 breast cancer cells constitutively overexpressing integrin vß3 have been inoculated into nude mice and vß3-dependent cellular mechanisms involved in bone metastasis formation have been investigated.
PRINCIPAL FINDINGS
1. Overexpression of integrin vß3 in CHO tumor cells increases the incidence, number, and area of osteolytic lesions in animals
Mock-transfected CHO dhfr+ cells and CHO cells expressing a functionally active (CHO ß3wt, CHO ß3 15–3, and CHO ß3 2–19) or inactive (CHO ß3
744) vß3 integrin were inoculated i.v. into nude mice. All animals inoculated with CHO cell lines expressing a functionally active vß3 integrin had radiographically detectable bone metastases as opposed to mice inoculated with CHO dhfr+ or CHO ß3744 cells (bone metastasis incidence: 60–66%). The number of osteolytic lesions on radiographs in metastatic CHO ß3wt-, CHO ß3 15–3-, and CHO ß3 2–19-bearing mice was increased 
twofold (10–18 lesions per metastatic animal) compared to that observed with CHO dhfr+- and CHO ß3744-bearing mice (5–9 lesions per metastatic animal). Representative radiographs of hind limbs from metastatic mice are shown in Fig. 1
A. The area of osteolytic lesions on radiographs was increased fivefold in CHO ß3wt-bearing animals compared with that of CHO dhfr+- and CHO ß3744-bearing mice (Fig. 1B
, left panel). Histomorphometric analysis of metastatic hind limbs showed a dramatic decrease in bone volume and a concomitant increase of the tumor burden in CHO ß3wt-bearing mice compared with that of CHO dhfr+- and CHO ß3744-bearing mice (Fig. 1B
, middle and right panels). Histological examination of metaphysis from CHO ß3wt-bearing mice revealed that most of the trabecular bone was destroyed and replaced by tumor cells, whereas metaphysis from CHO dhfr+- and CHO ß3744-bearing mice had small tumor foci and preserved bone trabeculae (Fig. 1C
).
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2. Constitutive overexpression of integrin vß3 in human breast carcinoma cells increases the number and area of osteolytic lesions in animals
Breast cancer cell line B02 has been established from bone metastases caused by MDA-MB-231 cells and constitutively overexpressed vß3 integrin whereas the cell surface expression level of other integrins remained unchanged. All animals inoculated with B02 or MDA-MB-231 cells had radiographically detectable osteolytic bone metastases. The number of osteolytic lesions on radiographs in metastatic B02-bearing mice was increased twofold (9–25 lesions per metastatic animal) compared with MDA-MB-231-bearing mice (3–15 lesions per metastatic animal). Representative radiographs of hind limbs from metastatic mice are shown in Fig. 2
A. The area of osteolytic lesions on radiographs was increased five- to sixfold in B02-bearing animals compared with that of MDA-MB-231-bearing mice (Fig. 2B
, left panel). Histomorphometric analysis of metastatic hind limbs showed a dramatic decrease in bone volume and a concomitant increase of tumor burden in B02-bearing mice compared to MDA-MB-231-bearing mice (Fig. 2B
, middle and right panels). Histological examination revealed that the extent of bone destruction in metaphysis from B02-bearing mice was consistently increased vs. that observed in metaphysis from MDA-MB-231-bearing mice (Fig. 2C
).
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3. Integrin vß3 increases tumor cell invasion, migration, and adhesion to mineralized bone and bone matrix proteins, but not tumor cell proliferation
Both CHO ß3wt and B02 cell invasion and adhesion to cortical bone were significantly increased compared to that observed with CHO dhfr+ and MDA-MB-231 cells, respectively. CHO ß3wt and B02 cell adhesion to collagen and bone sialoprotein (BSP) was significantly increased compared to that observed with CHO dhfr+ and MDA-MB-231 cells. In contrast, CHO ß3744 cell adhesion and invasion were substantially reduced vs. that observed with CHO ß3wt cells. Increased CHO ß3wt and B02 cell invasion and adhesion to cortical bone were specifically inhibited in the presence of anti-vß3 antibody LM609, indicating that this gain in invasion and adhesion was vß3 dependent. CHO ß3wt cell migration on BSP was also substantially increased. In contrast, CHO ß3wt, CHO dhfr+ and CHO ß3744 cells proliferated at a similar rate on plastic, collagen, and BSP independent of the expression of a functionally active vß3 complex. B02 proliferative responses to plastic, collagen, and BSP were similar but consistently higher that those observed with MDA-MB-231 cells, suggesting that the increased vß3 expression level in B02 cells was not responsible for this higher proliferative rate.
CONCLUSIONS AND SIGNIFICANCE
The reasons why tumor cells metastasize to bone remain obscure. Here we report that overexpression of integrin vß3 in CHO cells (CHO ß3wt, CHO ß3 15–3, and CHO ß3 2–19) increased the frequency, number, and area of osteolytic bone metastases in nude mice whereas CHO ß3744 cells, expressing a functionally inactive vß3 receptor, had a significantly reduced ability to develop osteolytic lesions. B02 breast cancer cells isolated from bone metastases constitutively and specifically overexpressed integrin vß3 and induced substantially more and larger osteolytic lesions in animals than those bearing MDA-MB-231 tumors. As shown in other studies, early mechanisms of bone metastasis formation are likely to involve invasion of tumor cells into the bone marrow cavity and adhesion of tumor cells to bone. Here we provide evidence that integrin vß3 can promote bone metastasis formation by increasing tumor cell invasion and adhesion to mineralized bone and bone matrix proteins (bone sialoprotein and type I collagen). These results agree with other studies showing that bone sialoprotein and type I collagen are relevant vß3 ligands and that integrin vß3 mediates breast cancer cell adhesion to bone mineral and bone sialoprotein in vitro. Overall, our study indicates that vß3 integrin expression in tumor cells accelerates the development of osteolytic lesions presumably through increased invasion and adhesion to bone. However, MDA-MB-231 (which express a low level of vß3) and ß3-deficient CHO cells both possessed the capacity to form bone metastases, indicating that additional molecular mechanisms may be involved in the homing of tumor cells in bone. Among these potential mechanisms are the chemokines and their receptors, which have a critical role in determining the metastatic destination of tumor cells. For example, the chemokine receptor CXCR4 is up-regulated in human breast cancer tissues and its ligand, the chemokine CXCL12, is produced in high quantity in specific organs (bone marrow, liver, and lung) in which breast cancer metastases are often found. It is therefore most likely that different molecular mechanisms are acting in concert to mediate tumor cell trafficking to bone (Fig. 3
). The role of integrin vß3 in mediating in vitro cancer cell adhesion and migration to bone proteins had been reported earlier. However, this is to the best of our knowledge the first in vivo study that reveals that integrin vß3 may be important for the development of bone metastases.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0911fje; to cite this article, use FASEB J. (June 21, 2002) 10.1096/fj.01-0911fje 
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