Cell lines and culture.
All cell lines used in the present study are isogenic. The normal human prostatic epithelial cell line, RWPE-1, a gift from M. Webber (Michigan State University), is heterogeneous, containing stem/progenitor, intermediate, and differentiated cell types (Tokar et al. 2005
). The WPE-stem cell line (NSCs) was isolated from the RWPE-1 line by single-cell dilution cloning and shows characteristics of urogenital system stem/progenitor cells, with anchorage-independent growth, nonadherent spheroid formation, high expression of p63
, and BMI
, and low expression of K8
, and PSA
(Tokar et al. 2005
). These NSCs also show self-renewal as evidenced by serial sphere passage capacity (data not shown). Arsenic malignantly transformed prostate epithelia (originally termed CAsE-PE cells; in this study termed MECs) were developed from RWPE-1 cells chronically exposed to sodium arsenite (5 μM) and show multiple malignant phenotype characteristics, including hypersecretion of MMP-9, colony formation in agar, and aggressive xenograft malignancies (Achanzar et al. 2002
; Tokar et al. 2010b
To investigate potential effects of neighboring cells on NSCs, NSCs were exposed to MECs or control RWPE-1 cells via a co-culture system (Corning Life Sciences, Suwanee, GA) that did not allow direct contact between the two cell types. NSCs were seeded into the lower compartment of 6-well transwell plates. After 1 day to allow NSCs to attach, malignant or control epithelial cells were seeded on the transwell inserts (collagen-coated 0.4-µm pore PTFE (polytetrafluoroethylene) membrane). The cell layers were approximately 1 mm apart in this system. The MECs were derived as reported by Achanzar et al. (2002)
and used for exposure to NSCs in the co-culture system. The MECs (about 3 million cells) were first washed twice with 5 mL Dulbecco’s phosphate-buffered saline (DPBS) and lifted using 2 mL trypsin and 4 mL DPBS before adding in the insert (75,000 cells/insert). Direct evaluation of conditioned medium from MECs during co-culture (at 3 weeks) revealed no detectable arsenic above background by atomic absorption spectrophotometry.
Cell culture surfaces for SC were coated with type IV collagen (Trevigen, Gaithersburg, MD) and fibronectin (BD Biosciences, Bedford, MD) (2.5 μg of each/mL). Cells were maintained in low-calcium, serum-free medium [keratinocyte serum-free medium (KSFM)] containing 50 μg/mL bovine pituitary extract, 5 ng/mL epidermal growth factor, and 1% antibiotic-antimycotic mixture (all from Gibco, Rockville, MD), to maintain the stem/progenitor cell phenotypic status (Litvinov et al. 2006
). Cells were incubated at 37°C in 5% CO2
with a medium change every 48 hr and passaged weekly. Various assessments were carried out weekly during 3 weeks of co-culture. Sphere assessment continued for an additional week, as it took 1 week for the spheres to form.
Secreted MMP activity is highly correlated with malignant transformation in the cells used in this study (Achanzar et al. 2002
; Tokar et al. 2010a
). After co-culture with MECs, SC were re-plated and grown alone to collect conditioned medium, and secreted MMP-9 or MMP-2 activity was examined by zymography (Tokar et al. 2005
Free-floating sphere formation.
The formation of free-floating spheres of viable cells is characteristic of NSCs and CSCs (Tokar et al. 2005
). Weekly during MEC co-culture, SC were collected by trypsin, filtered through a 40-µm cell strainer (BD Falcon, Franklin Lakes, NJ) to a single cell suspension, and plated in uncoated 6-well plates. Cells were fed every 48 hr. After 1 week, floating spheres and adherent cells were collected separately, and viable cells were quantitated by automated cell counter.
Colony formation in soft agar from cells derived from free-floating spheres is related to the NSC or CSC phenotype (Stingl et al. 2006
) and is indicative of anchorage-independent growth. After noncontact co-culture, floating spheroid cells were separated from adherent cells, and colony formation in soft agar was assessed (Tokar et al. 2005
Branched ductal-like structures and serial passage. After 3 weeks of co-culture, single SC were seeded into uncoated 6-well plates until free-floating spheres formed. Free-floating spheres were collected, dissociated by pipette trituration and filtered through a 40-µm cell strainer. Cells were suspended in 1:1 Matrigel/KSFM growth medium in a total volume of 200 µL, plated in a 24-well plate, and placed in the incubator overnight. The next day, 1 mL of KSFM growth medium was added on top of the solidified Matrigel mixture. The medium was changed every 3 days. Photomicrographs were taken with an inverted microscope after 2 weeks. For serial passage (a measurement of self-renewal capacity typical of NSCs or CSCs), colonies in Matrigel were collected, disassociated into single cells, and re-plated into 24-well plates in the Matrigel mixture as described above. The formation of new colonies in Matrigel was accomplished at least 3 times.
Invasive ability as a malignant phenotype was assessed using a modified Boyden chamber assay (Bello et al. 1997
Assessment of gene expression.
Transcript levels of p63, WNT3
were examined by real-time reverse transcription–polymerase chain reaction (RT-PCR) as described previously (Tokar et al. 2010a
). Cycle time (Ct) values were normalized based on control = 100% using the average values of β-actin (ACTB
) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH
) from the same sample. Primer sequences are provided in Supplemental Material, Table 1
For Western blots, protein was collected with M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL), containing 0.1 mM phenylmethyl sulfonyl fluoride (Sigma-Aldrich, St. Louis, MO) and 1% Protease Inhibitor Cocktail (Thermo Scientific, Rockford, IL). Western blots were performed as described previously (Tokar et al. 2010b
). Protein bands were assessed by ECL Reagent (GE Healthcare, Buckinghamshire, UK).
Immunofluorescence. NSCs co-cultured with MECs were washed with DPBS and fixed with acetone and methanol (1:1, vol:vol). For staining, cells were blocked with normal horse serum (room temperature, 1 hr), incubated with primary antibodies [rabbit anti-K5 (ABcam, Cambridge, MA), mouse anti-p63 (Santa Cruz Biotechnology, Santa Cruz, CA) or mouse anti-VIMENTIN (Sigma-Aldrich)], at 4°C, overnight, rinsed, incubated at room temperature for 1 hr with secondary antibodies labeled with Alexa Fluor 488 or 568 (Molecular Probes, Eugene, OR), and rinsed again. Cells were also stained with DAPI (4´,6-diamidino-2-phenylindole dihydrochloride) (Invitrogen, Eugene, OR) at room temperature for 10 min. After washing with DPBS, photomicrographs were captured with an automated Olympus inverted fluorescence microscope, equipped with a 40× objective (Olympus Corporation, Center Valley, PA). Photomicrographs were processed by CellSens software (Olympus Corporation) with consistent settings (e.g., exposure time) for both control and MEC groups.
IL-6 assessment and treatment. Using normal culture conditions, IL-6 secreted into culture medium by MECs over 24 hr was determined using the human IL-6 ELISA kit (R&D Systems, Minneapolis, MN). NSCs treated with 20 ng/mL IL-6 for 1 week were analyzed for characteristics indicative of cancer phenotype such as MMP-9 activity and expression of K5, p63, PTEN, E-CADHERIN (E-CAD) and VIMENTIN.
Statistical analysis. Data represent means and standard errors (SE). Student’s t-test was used in all comparisons, with a p < 0.05 considered significant.