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Mohamedi et al. J Cancer Metastasis Treat 2019;5:37 I http://dx.doi.org/10.20517/2394-4722.2018.81 Page 9 of 15

the case of fibronectin, the control cells could cover 65% of the dish area, and the double transfectants could
cover 18% of the dish area [Figure 3]. The data for the control and double transfectants cells of the MDA-
MB-231 cell line were somewhat similar; the control cells covered 90% of the areas of the type-1 collagen-
and fibronectin-coated plates, while the double transfectants covered 35% and 25% of the type-1 collagen-
and fibronectin-coated plates, respectively [Supplementary Figure 1]. The expression of ADAMTS-1 alone
also showed significant effects on migration only over the fibronectin substrate compared with the control
cells and double transfectants of the MCF-7 and MDA-MB-231 cell lines [Figure 3 and Supplementary
Figure 1, respectively]. The overexpression of fibulin-1 alone showed significant effects compared with the
double-transfected and ADAMTS-1-overexpressing MDA-MB-231 cells over fibronectin and type-1 collagen
[Supplementary Figure 1].

Effects of the fibulin-1/ADAMTS-1 interaction on cellular proliferation
Ki-67 nuclear staining is a good marker to measure the proliferative state of any given cell line. In this study,
we used Ki-67 nuclear staining to evaluate the proliferative properties of all the transfectants generated. In
both cell lines, MDA-MB-231 and MCF-7, ADAMTS-1 overexpression increased cell proliferation, as shown
in Figure 4. The number of Ki-67-positive nuclei in the ADAMTS-1-expressing and control cells of the
MDA-MB-231 cell line was 63 and 48, respectively, whereas that in the ADAMTS-1-expressing and control
cells of the MCF-7 cell line was 82 and 64, respectively. On the other hand, fibulin-1 overexpression was able
to significantly reduce proliferation in only the MCF-7 cells; 49 and 64 Ki-67-positive nuclei were detected in
the fibulin-overexpressing and control cells, respectively. When ADAMTS-1 and fibulin-1 were transfected
together, a reduction in the cell proliferation of both cell lines was observed. Specifically, in MDA-MB-231
cells, 35 and 48 Ki-67-positive nuclei were detected in the double transfectants and control cells, respectively,
whereas in MCF-7 cells, 45 and 64 Ki-67-positive nuclei were detected in the double transfectants and
control cells, respectively [Figure 4].

Effects of the fibulin-1/ADAMTS-1 interaction on mammosphere formation
One way of mimicking a tissue environment using 3D cell culture in vitro is through the formation of
mammospheres, allowing the quantification of the size of these structures as well as the self-renewal
capacity of the cell. In this context, we were able to examine the influence of fibulin-1 and ADAMTS-1 on
the mammosphere formation of breast cancer cells. The fibulin-1/ADAMTS-1 interaction resulted in fewer
and smaller mammospheres than did the other three combinations [Figure 5]. Furthermore, significant
differences in mammosphere forming units (MFUs) were observed among all possible combinations.
It is remarkable that the ability to develop MFUs was almost completely abolished when fibulin-1 and
ADAMTS-1 were coexpressed (no MFUs were detected). Compared with the control cells, cells expressing
fibulin-1 also exhibited a diminished ability to develop MFUs (11 MFUs vs. 4 MFUs). In contrast,
ADAMTS-1 expression slightly increased this capacity. Taken together, our data indicate that fibulin-1 and
ADAMTS-1 affect the ability of this cell line to form mammospheres.

Analysis of ADAMTS-1 expression in breast cancer tissue samples
We performed an immunohistochemical analysis of the expression of ADAMTS-1 and fibulin-1 in a tissue
array containing 69 breast cancer tissue samples, including 18 ductal carcinomas, 3 poorly differentiated
ductal carcinomas, 12 moderately differentiated ductal carcinomas, 1 well-differentiated ductal carcinoma,
20 lobular carcinomas, 6 mucoid carcinomas, 3 colloid carcinomas, 3 apocrine carcinomas, and 3
medullary atypical carcinomas [Supplementary Table 1]. Overall, using serial slides, the results indicated
no colocalization of the proteins regardless of the tumor type or stage [Figure 6]. Consistently, ADAMTS-1
immunoreactivity was almost absent or expressed at residual levels in all types of carcinoma samples [Figure 6].
On the other hand, positive immunostaining for fibulin-1 was distributed in both the tumor cells and the
stromal spaces with the exception of the colloid carcinoma, in which fibulin-1 expression was restricted to
the tumor cells [Figure 6, Top].