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Page 4 of 15 Mohamedi et al. J Cancer Metastasis Treat 2019;5:37 I http://dx.doi.org/10.20517/2394-4722.2018.81

(Sigma-Aldrich) for 16 h at 4 ºC following the manufacturer’s instructions. After three washes in lysis buffer,
the immunoprecipitates were resolved by western blotting.

Invasion assays
In vitro invasion potential was evaluated using 24-well Matrigel-coated invasion chambers with an 8-µm
5
pore size (BD Biosciences). For MCF-7 cells, 5 × 10 cells were allowed to migrate for 96 h using 10% fetal
bovine serum as a chemoattractant. Cells that reached the lower surface were stained with crystal violet. At
least three independent experiments were performed in triplicate for each condition. Cells were counted in eight
randomly selected microscopic fields. In the case of MDA-MB-231 cells, invasion was evaluated after 24 h.

Migration assays
The migratory capacity of cells on the ECM components fibronectin, laminin I, and type-I collagen was
TM
examined using the Radius 24-Well Cell Migration Assay kit (Cell Biolabs) following the manufacturer’s
5
instructions. Briefly, 5 × 10 cells were seeded per well, and migration was monitored by time-lapse
microscopy using a Zeiss Axio Observer Microscopy. Experiments were performed in triplicate, and the
covered area was quantified at different times using ImageJ. For MCF-7 cells, the results were obtained after
24 h of migration. In the case of MDA-MB-231 cells, we used a barrier-migration assay over a period of 24 h
[34]
and the same ECM components .

Cell proliferation determination by Ki-67 staining
Cell proliferation was estimated by the quantification of Ki-67-positive nuclei in MCF-7 and MDA-MB-231
cell cultures, including controls and the different transfectants. Cells were fixed with 4% paraformaldehyde,
blocked with 10% fetal bovine serum and incubated overnight with an anti-Ki67 antibody (Santa Cruz
Biotechnologies). After three washes in phosphate buffered saline (PBS), the cells were incubated with a
secondary Alexa 546-conjugated antibody (Life Technologies) for 1 h. In all samples, DAPI (100 ng/mL)
was added to visualize the DNA in the cell nucleus. Images were obtained using a fluorescence microscope
(Axiovert). After quantification, the data were plotted as an average of Ki-67-positive nuclei in relation to the
total number of nuclei per microscopic field.

Mammosphere cultures
4
A total of 4 × 10 MCF-7 cells were plated in 6-well ultralow attachment plates (Costar) and grown in
MammoCult Basal Medium (Stem Cell Research) supplemented with 10% MammoCult Proliferation
Supplement, 4 µg/mL heparin and 0.5 µg/mL hydrocortisone. After 7 days, mammospheres were collected
[35]
and enzymatically dissociated as previously described . The individual dissociated cells were cultured in
96-well ultralow attachment plates at a density of 20 cells/well. Mammosphere formation was microscopically
monitored daily to ensure that the mammospheres were derived from single cells and not from aggregates.
The number of mammospheres was determined after 7 days of culture.

Human breast cancer tissue array
A breast cancer tissue array containing samples from different tumor stages was employed to evaluate
ADAMTS1 and FBLN1 expression in human breast cancer samples. The tissue array was obtained from the
Institute of Oncology of Asturias Tumour Bank. Written informed consent was obtained from all patients
prior to sample collection. The study was approved by the appropriate institutional review board according
to national and EU guidelines.

Slides were processed for indirect peroxidase immunohistochemistry as follows: sections were deparaffinized,
rehydrated and then rinsed in PBS containing 1% Tween-20. For the detection of fibulin-1 and ADAMTS-1,
the sections were heated in an Envision FLEX target retrieval solution at high pH and 80 °C for 20 min
and then incubated for 20 min at room temperature in the same solution. Endogenous peroxidase activity
(3% H O ) and nonspecific binding (33% fetal calf serum) were blocked, and the sections were incubated
2
2
overnight at 4 °C with primary antibodies diluted 1:100. We used labeled polymer-HRP (ready for use)