RPMI-1640 and L-15 medium were obtained from Mediatech, Inc. (Herndon, VA). Fetal bovine serum (FBS), low glucose DMEM, insulin-transferrin-selenium-X (ITS-X), and ALBUMax1 were purchased from Invitrogen (Carlsbad, CA). MCBD 201 medium, ascorbic acid 2-phosphate, and dexamethasone were from Sigma-Aldrich (St. Louis, MO). Epidermal growth factor (EGF) and platelet derived growth factor-BB (PDGF-BB) were from R&D Systems (Minneapolis, MN).
Human bronchioloalveolar carcinoma cells (H358) and human lung alveolar carcinoma cells (SW1573) were obtained from American Type Culture Collection (Manassas, VA). H358 was cultured in RPMI 1640 and SW1573 was cultured in L-15, both supplemented with 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. Human UCMSCs were prepared from human umbilical cord Wharton’s jelly obtained from a local hospital with an appropriate Kansas State University Institutional Review Board guidance. hUCMSCs were prepared and cultured as described in our previous study [14
]. The culture medium for hUCMSCs was low glucose DMEM containing 37% MCDB 201, 2% FBS, 1% ITS-X, 1.5 g/mL ALBUMax1, 10 nM dexamethasone, 50 μM ascorbic acid 2-phosphate, 1 ng/mL EGF, 10 ng/mL PDGF-BB, 100 units/mL penicillin and 100 μg/mL streptomycin. The cells were incubated in 5% CO2
humidified air at 37°C. SW1573 cells were maintained with L-15 media in humidified air at 37°C without CO2
Gene transduction with adenoviral vectors
The fiber-modified adenoviral vectors encoding genes for human IFN-β were prepared in the laboratory of Dr. F. Marini. The gene transduction to hUCMSCs was done by following the procedure in our previous study [14
]. Twenty four hours after gene transduction, the IFN-β-hUCMSCs were used for the experiments described below.
Effect of hUCMSC and IFN-β-hUCMSC co-culture on lung cancer cells
H358 or SW1573 were seeded in normal growth media at 3 × 105 cells per well in 6-well plates. After allowing the cancer cells to attach to culture vessels, 3 × 105 hUCMSCs or IFN-β-hUCMSCs were seeded into Transwell cell culture inserts (3.0 μm pore size, BD Biosciences, San Jose, CA). The cells were cultured with the growth medium for hUCMSCs for 72 hrs, cancer cells were lifted, and the number of the cancer cells was counted using a hemocytometer.
Effect of hUCMSCs and IFN-β-hUCMSCs conditioned media on lung cancer cells
The normal growth media were conditioned by culturing IFN-β-hUCMSCs or hUCMSCs for 24 hrs. The conditioned media were applied to the lung cancer cell lines which were seeded 24 hrs before. Neutralizing antibody against IFN-β (mouse anti-human IFN-β monoclonal antibody, Chemicon, Temecula, CA) was added to the conditioned media at 5 μg/ml. The viable cancer cell number was counted after 72 hrs of culture. The proportion of apoptotic cells was analyzed at different time points using an Annexin V-FITC apoptosis detection kit (Biovision, Mountain View, CA) with fluorescence activated cell sorting (FACS). Briefly, cells were washed with cold PBS and re-suspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl and 2.5 mM CaCl2) at a concentration of 5–10 × 106 cells/ml. Cells were incubated at room temperature with 5 μl each of Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for 5 minutes, and analyzed on a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA).
Western blot analysis
Total cellular protein was prepared using lysis buffer (1% Triton X-100, 0.1% SDS, 0.25M sucrose, 1mM EDTA, 30mM Tris-HCl, pH 8.0) supplemented with protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN). Protein samples were separated by 10% SDS-PAGE gels, electroblotted onto nitrocellulose membrane (GE Healthcare Bioscience Corp., Piscataway, NJ), and blocked with 5% nonfat dry milk in 0.1% Tween20 in PBS (PBST) overnight at 4°C. The membranes were incubated with anti-caspase-8 or -9 or anti-cleaved caspase-3 antibodies (Cell Signaling Technology, Inc., Danvers, MA) at 1:1000 dilution with 5% nonfat dry milk in PBST overnight at 4°C and then with a horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody (GE Healthcare). The protein expression signal was detected with Pierce ECL Western Blotting substrate (Pierce, Rockford, IL). Actin was used as the loading control for samples by re-probing with an anti-actin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Orthotopic lung cancer model
Having determined the effect of hUCMSCs or IFN-β-hUCMSCs conditioned medium in vitro, the orthotopic H358 tumor model was used to evaluate the in vivo effect of hUCMSCs or IFN-β-hUCMSCs. H358 tumors were developed in the lungs of 6-week-old female CB17 SCID mice (Charles River Laboratories, Inc. Wilmington, MA). In brief, 1 × 106 H358 cells were injected twice, with an interval of eight weeks, through the lateral tail vein. One week after the second injection of H358 cells, all mice were randomly distributed in the following groups: PBS, hUCMSCs, or IFN-β-hUCMSCs. Either 200μl PBS or 3 × 105 cells/200 μl PBS (either hUCMSCs or IFN β-hUCMSCs) were injected through the tail vein every 5 days; these injections were repeated a total of 4 times. Both hUCMSCs and IFN β-hUCMSCs were labeled with red fluorescent SP-DiI (10 μg/ml for overnight) prior to injection. All animals were sacrificed two weeks after the final treatment and lungs were collected. The tumor burdens were evaluated by having two independent individuals blindly count the number of tumors on the surface of the lung under a dissection microscope. All animal experiments were conducted with appropriate approval of the Institutional Animal Care and Use Committee and Institutional Biosafety Committee.
Histopathological analysis of tumors in the lung
All of the lung tissues were fixed with 10% formalin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E) for histological examination. Some selected sections were deparaffinized and stained with 4′-6-diamino-2-phenylindole for 30 min as a nuclear stain. Red fluorescent SP-DiI labeled hUCMSCs were distinguished under epifluorescence microscopy (Nikon Eclipse, Boyce Scientific, Inc). Images were captured using a Roper Cool Snap ES camera and Metamorph 7 software. For quantitative evaluation of tumor nodules in the lungs, three microscopic view areas were randomly selected in the H&E tissue sections; numbers of tumor nodules in these areas were blindly counted under a microscope by two independent individuals. Data were presented as the average number of tumor nodules per view. To assess the effect of the treatments on tumor size, 10 tumors were randomly selected in the H&E tissue sections and the area of the tumors was calculated using the NIH Image J analysis software.
Immunohistochemical analysis of cell division and apoptosis of tumors
Cell division in tumor tissues was evaluated by determining Ki-67 positive cells in tumors. After deparaffinization of sections, heat-induced epitope unmasking was performed in citrate buffer followed by incubation with 0.3 % H2O2/methanol for 20 minutes to block endogenous peroxidase activity. Sections were incubated with polyclonal anti-Ki-67antibody (Abcam, Cambridge, MA) at 1:100 dilution for 1 hr at 37°C. Then, sections were reacted with a biotin-conjugated anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA) at 1:100 dilution for 1 hr at 37°C, followed by reaction with the avidin-biotin peroxidase complex reagent (Vector Laboratories). Reactions were developed with 3, 3′-diaminobenzodine tetrahydrochloride (Sigma) and counterstained lightly with hematoxylin. To determine the Ki-67 labeling index, 10 nodules were selected randomly by light microscopy and the area of Ki-67 positive cells in each nodule was calculated using the NIH Image J analysis software. The index was assessed as the percentage of Ki-67-positive area in the tumor.
To determine apoptosis in the tumors, the DeadEndTM colorimetric TUNEL system (Promega, Madison, WI) was used according to the manufacturer’s protocol with a slight modification. The sections were counterstained with methyl green. Ten tumor nodules were selected randomly, and the number of TUNEL positive cells in each nodule was counted. The apoptotic index was assessed as the percentage of TUNEL positive cells/tumor cells.
Data are expressed as mean ± SE (standard error). For all in vitro and in vivo experiments, statistical significance was assessed by Tukey-Kramer Pairwise Comparisons test. If not otherwise stated, all experiments reported represent two independent replications performed in triplicate. Statistical significance was set at * p < 0.05; ** p < 0.05; *** p < 0.001.