Injected s.c. in the flank with a 0.2 mL inoculum of

Injected s.c. in the flank with a 0.2 mL inoculum of 5 ?10 6 viable USC-HN1 cells. Three weeks after implantation, tumors were removed and the tissue was either flash frozen in liquid nitrogen or fixed in 10 neutral buffered formalin overnight at room temperature for paraffin-embedded procedures.ImmunohistochemistryTumor biopsies were mechanically dissociated into small tissue fragments using scissors and forceps. Fragments were then further dissociated enzymatically in 20 ml of RPMI-1640 medium containing 10 FCS, and 0.2 micron membrane filtered 0.01 hyaluronidase, 0.1 collagenase, and 0.01 DNase (Sigma Chemical Co., St. Louis, MO) for 40 min in a 37 water bath with intermittent mixing. After digestion, the cells and fragments were washed once in complete medium (RPMI-1640 medium containing 20 FCS, 1 Antibiotic-Antimycotic Solution, 10 ug/ml ciprofloxacin HCl, and 10 g/ml Gentamicin Sulfate), treated for 30 seconds in 30 ml sterile filtered RBC lysis buffer (8.3 g NH4Cl, 1 g KHCO3, and 0.037 g EDTA/L dH2O), washed again in complete medium, and seeded in two T-75 flasks. After 2-3 days of incubation in a humidified 5 CO2 37 incubator, the fragments wereFor immunohistochemistry (IHC) studies, cytospin preparations of USC-HN1 cells and tissue sections of USCHN1 tumors grown in Nude mice were used and compared to stained, fixed slides of the original tumor. USC-HN1 cells grown for 24 hr directly on sterile printed 25 ?75 mm glass slides (Bellco Glass, Inc., Vineland, NJ) were fixed sequentially with 2 parafolmaldehyde (Polysciences, Inc., Warrington, PA) for 10 min at room temperature and acetone for 5 min at -20 . For tissue sections, excised heterotransplanted USC-HN1 tumors from Nude mice were fixed overnight in 10 neutral buffered formalin and embedded in paraffin blocks. For cell culture and tissue morphology studies, Wright-Giemsa and hematoxilin RRx-001 eosin stains were used, respectively on air-dried cytospin preparations. In addition, USC-HN1 cytospin preparations and 5 micron tissue sections were stained with monoclonal antibodies against human CD44 (clone DF1485 DakoLiebertz et al. Head Neck Oncology 2010, 2:5 http://www.headandneckoncology.org/content/2/1/Page 4 ofCorp., Carpinteria, CA), E-cadherin (clone 4A2C7 Invitrogen, Carlsbad, CA), epidermal growth factor receptor (EGFr) (clone E30 Biogenex, San Ramon, CA), keratin (clone AE1/AE-3 Covance, Berkeley, CA), p53 (clone 1801 CalBiochem, San Diego, CA), and Rb (clone RbG3-245 BD Biosciences, San Diego, CA). Observation, evaluation and image acquisition were made using Leica DM2500 microscope (Leica Microsystems, http://www. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/16989806 leica-microsystems.com) connected to an automated, digital SPOT RTke camera and SPOT Advanced Software (SPOT Diagnostic Instrument Inc., http://www. diaginc.com). Images were further resized and brightened for publication using Adobe Photoshop software (Adobe, http://www.adobe.com).Flow cytometryCytogeneticsKaryotype analysis was performed by the Division of Anatomic Pathology, City of Hope (Duarte, CA) using cultured USC-HN1 cells.Polymerase chain reaction (PCR) viral screenSingle cell suspensions (1 ?106 cells in 100 l) in FACS buffer (1 FCS in PBS) were stained with FITC, PE, PerCPCy5.5, and APC conjugated antibodies. For intracellular staining, cell surface staining was performed first, followed by buffer fixation/permeabilization (eBioscience, San Diego, CA) and intracellular staining. Antibodies used were: CD24 (ML5), CD74 (M-B741), E-cadher.