Matrigel basement membrane

The invasiveness of HeyA8, HeyA8-MDR, and SKOV3IP1 cells was determined as previously described [83 (link)]. Briefly, cells were treated with 50 nM of the indicated oligonucleotide, and 48 hours later, live cells were collected and washed with serum-free media. Fixed numbers of the viable cells (4 × 10⁵ cells), were resuspended in 1 mL of serum-free RPMI-1640 medium and added onto 6-well plate Transwell inserts (8-μm-pore size; Fisher Scientific) coated with a Matrigel basement membrane (0.7 mg/mL; BD Biosciences). Lower chambers were filled with 2 mL of medium supplemented with 15% FBS. After 24 hours, non-invading cells on the upper surface of the filter were removed with cotton swabs. Cells that invaded through the Matrigel onto the lower side of the filter were fixed, stained with the Hema-3 Stain System (Fisher Scientific), and photographed. The number of cells that invaded the lower side of the filter was counted in 5 fields and expressed as the mean number of cells from triplicate measurements.

A precooled 96-well plate was first filled with 50 μL of precooled PBS, followed by the addition of 50 μL Matrigel Basement Membrane (BD Biosciences). HLECs were diluted with conditioned medium, and 60,000 cells were introduced into each well. Lymphangion formation was observed to begin at 6 h and reach completion around 10 h. HO-8910 and A2780 cells were then seeded onto the HLECs in a separate 96-well plate for 30 min, washed twice with PBS to remove free cells, and lysed by the addition of red blood cell lysate for 10 min. The absorbance at 690 nm was measured, and the NC group was normalized to 1.

Dissociated tumor cells mixed 1:1 with Matrigel Basement Membrane (BD Biosciences) were transplanted orthotopically in the inguinal mammary gland of 10–/12‐week‐old NSG mice and when tumors reached 5 mm of diameter mice were randomized for mock, h‐RANKL (0.75 mg/kg, 4–6 doses, twice per week; Amgen Inc.), h‐RANK‐Fc treatment (10 mg/kg, three times per week; Amgen Inc.) or denosumab (10 mg/kg, three times per week; XGEVA^®). Docetaxel (Hospira/Actavis, 20 mg/kg) was administered once per week together with dexamethasone (0.132 mg/kg, Merck), to reduce the chemotherapy‐induced inflammation. All the drugs were administered intraperitoneally. Tumor growth was monitored and measured with a caliper once per week. Tumor volume was then calculated as follows: π × length × width²/6 in cm³. Treatment was interrupted when tumors regress below 3 mm of diameter. Mice were sacrificed and tumors were surgically removed 24 h after treatment completion. In combination treatments with Docetaxel, mice were sacrificed once relapsing tumors reached 10 mm of diameter.

The invasion bility of SGC-7901 and HGC-27 cells was measured using 24-well transwell units with polycarbonate filters (pore size, 8.0 μm; Corning, NY, USA). Matrigel^® basement membrane (BD Biosciences, San Diego, CA, USA) was mixed with the serum-free RPMI at a ratio of 1:8, and 100 μL of the mixture was used to coat the upper transwell inserts. Then, the cells were seeded at 1 × 10⁵ in the upper insert with 100 μL serum-free RPMI medium, and placed in the lower chambers with 600 μL of complete media. The cells were allowed to culture for 24 h at 37°C, and non-invasive cells were removed using a cotton swab. The filters were fixed using 4% (v/v) paraformaldehyde, the cells were stained with 0.05% (v/v) crystal violet solution, and counted under a microscope.