Cell lines and culture
Except where otherwise specified, all reagents were obtained from Sigma (Sigma Chemical Co., St. Louis, USA).
All manipulations described below were repeated 82 times: 82 cell culture dishes with monolayer were formed, and 82 compacted fragments were formed (47 from ZR-75-1 cells and 35 from MDA-MB-231 cells).
Cells of ZR-75-1 and MDA-MB-231 were purchased from American Type Culture Collection (ATCC; Manassas, USA) and grown in AIM V Medium (Thermo Fisher Scientific, Waltham, USA) supplemented with 10% fetal bovine serum (FBS) and Amphotericin B at 37 °C in a humidified chamber with 5% CO2. Culture media were renewed every 48 h.
Formation of the model of solid tumors from monolayer was previously described . Briefly, the cells after three times of cell passages were in vitro cultured for ten days without cell passages. Culture medium was renewed every 24 h after a cell layer was formed. A cell scraper (Greiner Bio-one, Frickenhausen, Germany) was used for harvesting the cell layer and forming the model tissue for the following cryopreservation. This method was applied as well to the cryopreserved cells back to in vitro culture, albeit without cell passage, before xenotransplantation of the chorioallantoic membrane.
Cryopreservation (freezing and thawing) of the model tissues
Cryopreservation of compacted fragments of cancer cells was performed based on the protocols for cryopreservation of fragments of human ovarian tissue  with adjustment and peculiarities as described below. The model tissues were cryopreserved similarly to the process for ovarian fragments.
The harvested tissues were placed for 5 min (ZR-75-1 cells) and 10 min (MDA-MB-231 cells) into the standard 5 ml cryo-vials (Thermo Fisher Scientific, Rochester, USA) previously filled by 4.5 ml freezing solutions (medium L-15 supplemented with 6% dimethyl sulfoxide, 6% ethylene glycol and 0.15 M sucrose) precooled to 4 °C. Then compacted cells tissues were frozen in IceCube 14S freezer (SyLab, Neupurkersdorf, Austria). The slow cooling profile started at -6 °C, the samples were then cooled from − 6 to -34 °C at a rate of -0.3 °C/min. The used freezing protocol included auto-seeding step at -6 °C. At -34 °C cryo-vials were finally plunged into liquid nitrogen and stored until thawing.
For thawing of samples, cryo-vials were removed from liquid nitrogen and held for 30 s at room temperature, they were then immersed in a 100 °C (boiling) water bath for 60 s. The exposure time in the boiling water was visually controlled by the presence of ice in the medium; as soon as the ice was in form of 2 to 1 mm apex, the cryo-vial was removed from the boiling water, at which point the final temperature of the medium was between 4 and 10 °C. Within 10 s after thawing, about 90% of freezing medium was removed from cryo-vials and then these cryo-vials were filled by thawing solution pre-warmed to 37 °C (basal medium containing 0.5 M sucrose). Then cryo-vials were put into thermostat at 37 °C for 7 min (ZR-75-1 cells) and for 15 min (MDA-MB-231 cells) for stepping removal of cryoprotectants. After dehydration, about 90% of thawing medium (0.5 M sucrose) was removed from cryo-vial and then these cryo-vials were filled step wisely by basal (culture) medium. For stepwise rehydration (slow addition of basal medium to the solution of sucrose with cells), it was used the same, previously published ‘dropping’ methodology . The final concentration of sucrose was 0.05 M, resulting in almost isotonic conditions. After rehydration, cells were digested by 6 ml 0.05% Trypsin-EDTA, 5 min at 37 °C in a humidified chamber with 5% CO2. After washing and centrifuged, the cell pellet was resuspended in 10 ml culture medium by full pipette and then transferred into a 10 cm cell culture dish to allow adhesion overnight.
Observation of cell growing and morphology
Cryopreserved ZR-75-1 and MDA-MB-231 cells were seeded in AIM V medium and at a concentration of 1 × 104 cells/ml in 96-well plates compare with the controls, respectively. Cells were allowed to adhere overnight. We measured cell growing by using Cell Counting Kit-8 (CCK-8) and observed consecutively for five days. From day 1 to 5, ten µl CCK-8 solution was added to each well of one plate at a fixed time and incubated for 4 h, then the OD at 450 nm (reference 650 nm) was determined by a multimode reader machine (Tecan Group Ltd., Maennedorf, Zurich, Switzerland). Culture media were renewed every 48 h. Results were plotted with the time axis as the abscissa and the cell count as the vertical axis. Each experiment was repeated three times. For the morphology change, cells were maintained in the 10 cm culture dish to observe under microscopy each day. Images were taken by EVOS FL Auto 2 Cell Imaging System (Thermo Fisher Scientific).
Assessment of cell motility
Cell motility was determined using thewound-healing assay and 3D transmembrane migration and invasion experiment. The wound-healing test was implemented with a well-established artificial gap on the confluent cell monolayer. A density of 1 × 106 cells/ml in 140 µl suspension of both cell lines was seeded in a 35 mm µ-Dish ibidi Culture Insert (ibidi GmbH, Planegg, Bavaria, Germany) with 70 µl in each well, incubated for 24 h and obtained the cell layers. After removal of the insert, the µ-Dish was washed with PBS twice to remove cell debris and non-attached cells and filled with 2 ml of 1% FBS-supplemented cell-free medium. Time-lapse measurement of the wound area between the cell layers was performed every 24 h for ZR-75-1 and every 2 h for MDA-MB-231 cells to calculate cell front velocity. Experiments were carried out in triplicate at least three times.
Cell invasion was determined in vitro to evaluate the ability of tumor cells to transmigrate a layer of the reconstructed extracellular matrix. Corning transwell inserts were used to accomplish the cell invasion assay, according to our previous study . Polycarbonate filters (6.5 mm in diameter, 8 µm pore size) were coated with type I rat tail collagen (100 µg/ml; BD Biosciences, Franklin Lakes, USA) for 1 h at 37 °C by the manufacturer’s protocol. The fresh and cryopreserved cells were resuspended and seeded into the upper compartment of the insert in the serum-free culture medium, respectively. ZR-75-1 cells were seeded at 2 × 105 cells/well and cultured for 72 h; MDA-MB-231 cells were seeded 5 × 104 cells/well and cultured for 8 h. The lower chamber was filled with 600 µl of the appropriate culture medium supplemented with FBS as a chemoattractant. After incubation, the upper insert with cells was washed with PBS, fixed with 4% formaldehyde, and permeabilized with methanol at room temperature. Cells were then stained with 0.1% crystal violet solution and were gently rinsed with PBS and wiped by cotton-tipped swabs then dried in the air. Penetrative cells went through the polymerized collagen layer to the bottom of the polycarbonate membranes and were counted in five different fields of view under a microscope. For the migration assay, cells were treated using the same procedure, except that the transwell membrane was not coated with collagen. Samples in each group ran in triplicate. Each experiment was performed at least three times.
Immunofluorescent (IF) staining
Antibodies were purchased from Biolegend. Twenty-five × 104 cells were first seeded on cover glasses in 6-well plates. After 48 h, the culture medium was aspirated, and cells were fixed with 2% paraformaldehyde for 20 min at room temperature. After washing twice by PBS, the cells were incubated in 0.5% Triton X-100 in PBS for 10 min for permeabilization and blocked by cell staining buffer (Biolegend, San Diego, USA) for 30 min. Then the coverslips were transferred into a humidified chamber and incubated with Alexa Fluor 488-conjugated anti-human Ki-67 antibody, Alexa Fluor 594 anti-human Epithelial cadherin (E-cadherin) antibody, Alexa Fluor 647 anti-GATA3 antibody and Alexa Fluor 488 anti-Vimentin antibody overnight at 4 °C, or with Alexa Fluor 488-conjugated Flash Phalloidin (F-Actin) in room temperature for 1 h. After washing twice, the coverslips were mounted on glass slides with 25 µl of mounting medium with 4’,6-diamidino-2-phenylindole (Abcam, Cambridge, UK). The slides were analyzed by a Leica SP8 confocal microscope. Images were taken using LAS X software (Leica Microsystems, Wetzlar, Germany).
Western blotting (WB)
Cultured cells were incubated in Accutase at 37 °C for 5–10 min followed by resuspension and centrifugation. Cell lysis was conducted using lysis buffer: RIPA buffer (Thermo Fisher Scientific) with protease inhibitor cocktail. Cell lysates were separated by centrifuging at 20000 g, 30 min at 4 °C. Protein concentrations were measured via Bradford test and adjusted to 20 µg/20 µl in one sample by 4X sodium dodecyl sulfate-containing laemmli sample buffer, then heated in boiling water for 5 min. Later, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to separate the total protein and then separated protein was transferred on nitrocellulose membrane. We used the pre-cast 4–12% polyacrylamide gradient gels (Thermo Fisher Scientific) and the Trans-Blot® Turbo™ Turbo membrane (Biorad, Hercules, USA) in the transfer system according to the manufacturer instruction. After blocking, the membrane was incubated in primary antibodies diluted to 1:2000 by 5% Bovine Serum Albumin in PBST (0.1% Tween-20 in PBS), at 4 °C overnight. The P53, E-cadherin, GATA3, and Vimentin antibodies were purchased from Cell Signaling Technologies (Danvers, Massachusetts, USA). The following day, the fluorescent secondary antibodies (LI-COR, Lincoln, NE, USA) were used to incubate at room temperature for 2 h. Bands were visualized using Odyssey Clx (LI-COR). Image J software (http://developer.imagej.net) was used to estimate the band density.
Model of solid tumors tissue CAM-Xenotransplantation: stimulation of angiogenesis
Preparation of the chick embryo chorioallantoic membrane (CAM) for transplantation of cancer cells were performed as described early [15, 16]. Briefly, fertilized eggs of White Leghorn chickens were purchased at a local hatchery and incubated at 37 °C-38 °C with 60% relative humidity for three days. On day 5, each egg was washed with warm 70% ethanol and opened a small window with 1.0 cm diameter on the sharp pole of the shell. We sealed the window by a 2 × 2 cm medical fabric tape only on the edge of the opening, and the egg was allowed to continue the incubation. The following day, a 1-mm-thick sterile silicone ring with an inner diameter of 5 mm was laid on the exposed chorioallantoic membrane. We divided 54 well-incubated 6-day-old chicken embryos randomly into four groups, 12 eggs in each group, and six as blank controls. Both initial and cryopreserved MDA-MB-231 model tissues were adjusted to two concentrations: 4 × 106 and 8 × 106 cells/egg. Then we grafted the four groups of tissues into pre-treated chicken embryos on the relative avascular region of CAM, which was group 1: 4 × 106 fresh cells; group 2: 8 × 106 fresh cells; group 3: 4 × 106 cryopreserved cells; group 4: 8 × 106 cryopreserved cells. We used 40 µl PBS as blank. The five-millimeter inner diameter silicon rings were used to restrict the displacement of the grafts along with the chick embryo movement. The medical tape closed the window and continued to incubate for six days. The survival of the embryos, the tumor formation rate and the induction of angiogenesis were observed. The tumors with a diameter of ≥ 0.3 cm was considered positive, and the tumor formation rate was calculated. At the same time, the CAM xenograft specimens were fixed in situ with 4% paraformaldehyde and removed. The neovascularization in the tumor area was observed under a microscope on the 6th day of in vivo culture. The field of blood vessels distributed radially within a radius of 1 cm from the tumor tissue was quantified by Image J software. The relative density of blood vessels was calculated by the formula: Vascular density = vascular area/CAM area. Tumor volume was measured under an inverted microscope by the formula: Tumor volume = 1/2 × (major axis × minor axis2).
Data analysis was executed with SPSS 23.0 software (IBM Corp., Armonk, USA). Differences among the groups were tested by Student’s t-test or two-way ANOVA. Data are expressed as mean ± standard deviation. The level of statistical significance was set at p < 0.05.