2-1 Isolation of fibroblasts from normal and cancer breast tissue
Tumor and normal tissues specimens were obtained from stage II TNBC patients between the ages of 30 and 50 years undergoing lumpectomy and mastectomy at Ordibehesht surgery clinic, Isfahan, Iran. Written informed consent was obtained from each participants before surgery. The proposal was approved by the ethics committee of Isfahan University of Medical Sciences. The tumor samples were isolated from tumor zone (within tumor boundary) and normal samples isolated from normal zone (at least 10 mm distal from tumor boundary) and were taken to lab in Dulbecco's Modified Eagle's medium (DMEM)/F12 + 10% FBS medium, sliced and digested with enzyme mixture contained 160µg/ml of Collagenase IV and 25µg/ml Hyaluronidase (19). Digested samples were incubated at 37º with 5% CO2 overnight and then cultured in DMEM/F12 + 10% FBS for about a week.
2-2 Cell lines
BT-20. In 1958, BT-20 cell line derived from a 74-year-old Caucasian female with TNBC breast cancer, caused by invasive ductal carcinoma in mammary glands.
MDA-MB-231. This is an epithelial, human breast cancer cell line that is highly aggressive and invasive. MDA_MB-231 cell line was established from a pleural effusion of a 51-year-old Caucasian female with TNBC breast cancer.
HUVEC. The human umbilical vein endothelial cells, HUVEC, are endothelial like cells that are usually used for angiogenesis assays.
All the cell lines were purchased from Pasteur Institute of Iran, cultured in DMEM + 10% FBS medium and incubated at 37º with 5% CO2 for further assays.
2-3 Immunocytochemistry (ICC)
Alpha actin smooth muscle (α-SMA) is known as a specific marker widely expressed in fibroblast (20). To identify CAFs and NFs, the α-SMA (cat no. ab9654. Abcam) biomarker antibody were used and analyzed by ICC test based on the company instructions.
2-4 Transfection of miR-200c-3p mimics and scramble
MiR-200c-3p mimics (small, chemically modified double-stranded RNAs) (Cat No. MCH01501) and scramble (Cat No. MIR 7902) were purchased from abm and Mirus companies, respectively. Scramble is conjugated by FITC to measure the transfection rate. CAF and NF were transfected with mimic-miR-200c and scramble using the lipofectamine 2000 reagent (Invitrogen, Cat No. 13778030), according to the manufacturer’s instructions.
2-5 Real-time RT-PCR analysis
Trizol reagent (Invitrogen, USA) use to RNA extraction, cDNA was synthesized and expression levels of mature has-mR-200c-3p was evaluated in NF, CAF, miR-200c transfected NF and miR-200c transfected CAF cells with ExiLENT SYBR® Green master mix kit (Exiqon, Denmark) using StepOne Plus™ real-time PCR (Applied Biosystems, USA). Fold change was calculated using Livak method and ready to use primers were purchased from Exiqon and the SNORDs was used as a house keeping gene to normalize the data.
2-6 Co-culture of isolated fibroblastand TNBC cell lines (BT-20 and MDA-MB-231)
Study plan. There were five sets of triplicates in this study. The first set was TNBC breast cancer cells (BT-20 and MDA-MC-231) that were not co-cultured with fibroblast which served as the negative control. The second set was co-cultured with un-transfected NF. The third set was co-cultured with scramble transfected NF. The fourth set was co-cultured with miR-200c transfected NF. The fifth set was co-cultured with un-transfected CAF. The other set was co-cultured with scramble transfected CAF and the last set was co-cultured with transfected CAF. For co-culture, 3 ×103 CAF and NF cells were plated in the upper well of transwell and 2×103 BT-20, MDA-MB-231 or HUVEC cells were seeded in lower well.
MTT assay. To investigate the proliferation of BT-20 and MDA-MB-231 cells after co-culturing with fibroblast, based on sets described above, MTT test was carried out. Two days after co-culturing, the upper wells were removed, then 50 µl MTT solution (5 mg/ml) (Sigma-Aldrich Co., USA) was added to each culture well. After 4-hour incubation, 200µl DMSO was added and incubated for more 30 min, and the absorbance was read at 570 nm by ELISA reader.
Wound healing assay. The directional cell migration of BT-20 and MDA-MB-231 cells was evaluated using wound healing assay after co-culturing with fibroblast, according to the groupings described above. BT-20 and MDA-MB-231 cells were plated. After 24 h, the monolayer was scratched by a sterile pipette tip and the wells were obliterated of debris. Later, the co-culture was performed based on study plan section and allowed the cells to close the wound for 24, 48 and 72 h. Wound closure was measured with microscopic photography in 10 randomized field. The results were reported and analyzed by the imageJ software (version 1.52i).
HUVEC capillary tube formation assay. To run the assay, 100µl of Geltrex (Cat No. A1413202 Gibco) was added to the lower plate of transwell to form a 3D vessel- like tubes, then 10 5 HUVEC cells were seeded top of the Geltrex. Co-culture was performed based on study plan section and allowed the HUVEC cells to form tubes after about 24 and 48 h. Image analysis was carried out using the Angioquant software (version v1.33). The size, length and number of branches were measured and reported.
2-7 Statistical analysis
The data were analyzed using one-way ANOVA and Mann–Whitney U test. All results were shown as mean ± SD and P < 0.05 was considered as statistically significant.