Tissue microarrays (TMAs)
TMAs of four major types of tumors (breast, colorectum, lung, and prostate) and their corresponding normal tissues were purchased from multiple TMA suppliers including Accurate Chemical and Scientific Corp. (West Bury, NY, USA); Biochain Institute, Inc. (Hayward, CA, USA); US Biomax, Inc. (Rockville, MD, USA); BioVintage, Inc. (San Diego, CA, USA); and Imgenex Corp. (San Diego, CA, USA). We also obtained normal human organ TMA (Catalog # FDA993; US Biomax) with 33 types of organs taken from 3 normal human individuals including 30 types recommended by the US Food and Drug Administration.
The 408 breast tumor samples were obtained from 4 men and 404 women, ranging in age from 23 to 87 years (median at 48 years) at surgery. Histologically, there were 367 cases of invasive ductal carcinoma (90%), 29 cases of invasive lobular carcinoma (7%), and 11 cases of other tumor types (3%). There were a total of 79 normal breast samples from 1 man and 78 women, ranging in age from 18 to 71 years (median at 43 years). Thirty-three (42%) were cancer adjacent normal breast tissue and 46 (58%) were from noncancer individuals.
The 511 colorectal tumor samples were collected from 328 men and 183 women with median age at surgery of 58 years (ranging from 20 to 87 years). Overall, 462 patients (90%) had colon cancer and 49 (10%) had rectal cancer. The vast majority (499 cases) were histologically adenocarcinoma (98%), the remaining 12 cases (2%) being mucinous carcinomas. Additionally, 194 normal colorectal tissues were obtained from 132 men and 62 women with median age of 56 (ranging from 13 to 87 years); of which, 141 (73%) samples were cancer-adjacent normal colorectal tissues and 53 (27%) were colorectal tissues from noncancer individuals.
The 429 lung tumor samples were biopsied from 311 men and 118 women ranging from 28 to 89 years (median at 59 years) at the time of surgery. The most common histological type was nonsmall cell lung cancer (NSCLC) (92%, 395 samples), including 195 adenocarcinoma (49%) and 200 squamous cell carcinoma (51%). The remaining 34 cases were small cell lung carcinoma (SCLC, 8%). There were 68 normal lung tissues collected from 53 men and 15 women ranging from 21 to 81 years (median at 58 years); of which 58 (85%) samples were cancer adjacent normal lung tissues and 10 (15%) were noncancer individual lung tissues.
The age of 227 prostate cancer patients ranged from 20 to 89 years (median age of 66 years). Almost all tumors (225) were adenocarcinma (99%), with only 2 tumors being transitional cell carcinoma (1%). The 128 normal prostate tissues were made up of 66 (52%) cancer adjacent normal prostate tissues and 62 (48%) prostate tissues from noncancer men. Their ages spanned from 19 to 86 yeas with median age of 64 years.
Generation of Flp-In™ T-REx™ 293 cell line stably expressing human Notch-1
The full-length Notch-1 coding region tagged with C-terminal end FLAG peptide was chemically synthesized at DNA 2.0 (Menlo Park, CA, USA). Flanking sequences contained 5′ HindIII and 3′ NotI which were used for directional restriction cloning into pcDNA™ 5/FRT/TO (Invitrogen Corporation, Carlsbad, CA, USA) expression vector. Flp-In™ T-REx™ 293 cells (Invitrogen) contain a single genomic FRT (Flp [Flippase] recombinase target) site and express the Tet repressor which allows for the construction of isogenic recombinants under tetracycline control. Tetracycline-inducible stable expression cell lines were generated by cotransfecting pcDNA5/FRT/TO-HN1 +FLAG and pOG44 (Invitrogen), a plasmid encoding FLP recombinase, in a 1:9 ratio into Flp-In 293 T-REx using FuGENE™ 6 (Roche Diagnostics Corporation, Indianapolis, IN, USA) transfection reagent as per manufacturer’s recommendations. Cell lines with stably integrated cDNAs were then selected with hygromycin B (100 μg/mL) (Mediatech, Inc., Manassas, VA, USA) and were expanded. Expression levels of the Notch proteins were assessed by immunohistochemical (IHC) staining, Western blotting, and flow cytometry.
Paraffin embedment of Notch-1-transfected cells
Actively growing Notch-1 stably transfected Flp-In™ T-REx™-293 cells were induced by doxycycline (2 μg/mL) for 48 hours and harvested through trypsinization. The cell pellets were then fixed in 10% neutral buffered formalin (ThermoFisher, Pittsburgh, PA, USA) for 16–24 hours. The cells were processed with 70% ethanol, 95% ethanol, 100% ethanol, xylenes, and paraffin in a tissue processor (Sakura, Torrance, CA, USA). The cells were then embedded in paraffin, cut into 5 μm sections, and mounted onto Superfrost® Plus glass slides (ThermoFisher).
Cell lysates from full-length Notch-1-transfected Flp-In™ T-REx™-293 cells, with or without induction of doxycycline (2 μg/mL) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 3%–8% Tris-Acetate gel (Invitrogen) and transferred to nitrocellulose membrane using the iBlot system (Invitrogen). The blots were blocked overnight (16–20 hours) at 4°C in a 1% bovine serum albumin-containing 1X Tris-Tween buffered saline (TTBS) solution. After two rinses with 1X TTBS, the blots were incubated with rabbit anti-Notch-1 polyclonal antibody (Cat. # sc-6014-R; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at a concentration of 1 μg/mL for 2 hours. After six 5-minute washes in 1X TTBS, the blots were incubated with goat-anti-rabbit-HRP (R&D Systems, Inc., Minneapolis, MN, USA) at a 1:2000 dilution for 1 hour, followed by six more 5-minute washes with 1X TTBS. Antibody binding was detected by an enhanced chemiluminescence kit (Pierce Biotechnology, Inc., Rockford, IL, USA).
Paraffin embedded cell or tissue sections were deparaffinized by incubation at 60°C overnight followed by immersion in two changes of xylene (ThermoFisher) and two changes of absolute ethanol (Pharmco Products Inc., Brookfield, CT, USA), and rehydrated in distilled water. Antigen retrieval was performed in a calibrated steam pressure cooker (Decloaking Chamber, BioCare Medical, Walnut Creek, CA, USA) for 30 seconds at 125°C in 1X Target Retrieval Solution (Dako-Cytomation, Carpinteria, CA, USA). All additional steps (unless specified) were done at room temperature in a hydrated chamber. Endogenous peroxidase was blocked with peroxidase block from an EnVision™ + Rabbit Kit (DakoCytomation) for 10 minutes. The slides were also immersed in 5% nonfat dry milk for 30 minutes to minimize nonspecific binding due to hydrophobic interaction. The slides were then incubated with the rabbit anti-Notch-1 polyclonal antibody overnight at 4°C followed by labeled polymer from an EnVision + Rabbit Kit for 30 minutes. The slides were developed with diaminobenzidine and hydrogen peroxidase for 3 minutes and counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA).
Scoring system and statistical analysis
The semi-quantitative method of staining intensity of Notch-1 protein on normal and tumor cells was manually scored as negative (–, no staining), weak (+), moderate (++), and strong (+++) by two of the investigators (YL and JB) according to a prespecified intensity scale, with discrepancies agreed upon by consensus. Representative examples for the different scores are provided in .
Figure 1 Specificity of the anti-Notch-1 antibody. A) Western blots. The cell lysates from full-length Notch-1-transfected Flp-In™ · T-REx™ 293 cells, both doxycycline (DOX)-uninduced (−) and induced (+) were used to test the specificity (more ...)
The chi-square test was used to determine the differentiation of Notch-1 expression between cancer patients and normal population. It was also used in analyzing the clinicopathological correlations for parameters with two categories. When evaluating their associations to tumor stage and grade, a chi-square test for trend is utilized. A P-value of less than 0.05 was considered to indicate statistical significance. All tests were performed using GraphPad Prism (version 5.01 for Windows; GraphPad Software, San Diego, CA, USA).