In order to validate the analysis, replicates cell culture were obtained from three different donors according to the methodology described in our previous study. [22], 23 and 24 The squamous cell carcinoma (CAL27) was obtained from American Type

Culture Collection (ATCC VA, USA). This study was conducted following the approval of the Ethical Committee of São Leopoldo Mandic Institute and Dental Research Centre, Campinas, Brazil (Protocol # 09/0014). The obtained cells were cultured in Dulbecco’s modified Eagle medium (DMEM®) (Sigma, St. Louis, MO, USA) and supplemented with 1% antimycotic–antibiotic solution (10,000 units of penicillin, 10 mg of streptomycin and 25 μg of amphotericin B per ml in 0.9% sodium chloride; Sigma®), containing 10% of donor calf serum (DCS; GIBCO®, MLN8237 in vitro Buffalo, NY), plated in 60-mm diameter plastic culture dishes and incubated under standard cell culture conditions (37 °C, 100% humidity, 95% PLX4032 in vitro air, and 5% CO2) following the used protocol for this cell lineage culture25. When both cells had reached confluence, they were detached with 0.05% trypsin and subcultured at a density of 110 cells/mm2

in the polystyrene plate (96-wells), at the same time, for the following experiment. To certify the formation of in situ-like neoplasic areas in which neoplastic cells of squamous cell carcinoma are surrounded by benign myoepithelial cells from pleomorphic adenoma, the cells were examined by phase contrast microscopy, in each studied phase (3, 5, 7, 9, 13 and 16 days after initial culture) and also immunostained with vimentin and AE1/AE3, markers for tumoral benign myoepithelial cells and squamous cell carcinoma lineage, respectively. As control, the malignant (CAL27) and the myoepithelial cells were isolated cultured and the supernatants were collected for the following experiments. To observe the neoplasic areas formation,

cells grown Metalloexopeptidase on coverslips were fixed in methanol for 6 min at 20 °C, rinsed in PBS followed by blocking with 1% bovine albumin in phosphate buffer saline (PBS) for 30 min at room temperature. The primary polyclonal antibodies used were vimentin (1:50, anti-rabbit, Sta. Cruz®) and AE1/AE3 (1:75, anti-mouse, Dako®). Control staining reaction was performed using PBS in substitution to the primary antibody. The secondary antibodies used were Alexa Fluor 488 anti-rabbit IgG and Alexa Fluor 594 anti-mouse IgG (Invitrogen, USA). After washing, preparations were mounted using Vectashield® DAPI-associated (4′-6-diamidino-2-phenylindole) (Vector®) and observed on a Zeiss Axioskop 2 conventional fluorescence microscope (Carl Zeiss MicroImaging GmbH, Germany) equipped with 63× Plan Apochromatic 1.4NA and 100× Plan Apochromatic 1.4NA objectives in standard conditions (Carl Zeiss, Oberköchen, Germany). After 3, 5, 7, 9, 13 and 16 days, supernatants from the cell culture were harvested and centrifuged at 5000 × g for 15 min at 4 °C.