Cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA) unless otherwise stated. Chemicals were purchased from Sigma Chemical (St Louis, MO) unless otherwise stated.
Monoclonal antibodies used were mouse antihuman nectin 4 (clone 337516; R&D Systems, Minneapolis, MN), mouse antihuman nectin 4 (clone N4.61; provided by M.L.), mouse antihuman nectin 1 (clone CK8; Invitrogen), mouse antihuman ADAM17 (clone 111623; R&D Systems), and normal mouse IgG (clone 3-5D1-C9, AbCam, Cambridge, MA).
Polyclonal antibodies used included biotin-conjugated goat antihuman nectin 4 (BAF2659, R&D Systems), horseradish peroxidase–conjugated rabbit antimouse IgG (ab5762, AbCam), and biotin-conjugated goat antimouse IgG F(ab')2 fragment (Jackson ImmunoResearch, West Grove, PA).
Ovarian cancer cell lines SKOV3, ES2, OVCAR3, HEY, C13, OV2008, OVCA429, OVCA433, A2780s, and A2780cp (provided by Barbara Vanderhyden, PhD, University of Ottawa, Ottawa, Canada); NIH:OVCAR5 (provided by Judah Folkman, MD, Harvard Medical School, Boston, MA); CAOV3 (provided by Robert Bast Jr, MD, University of Texas, Houston); and MA148dsRed2 (provided by Sundaram Ramakrishnan, PhD, University of Minnesota, Minneapolis) were maintained as previously described.19–22
SKOV3, ES2, and OVCA429 cell lines were derived from clear cell carcinomas; OV2008 and C13 cells were derived from endometrioid tumors; OVCAR3, OVCAR5, OVCA433, CAOV3, HEY, MA148, and A2780s/cp cell lines were derived from serous adenocarcinomas.
Immortalized normal ovarian surface epithelial (NOSE) cell lines 1816-575, 1816-686, HIO117, HIO135, IMCC3, IMCC5, and 3173-11 (provided by Patricia Kruk, PhD, University of South Florida, Tampa) and IOSE-80 (provided by Nelly Auersperg, MD, PhD, University of British Columbia, Vancouver, Canada) were also maintained as described.23,24
Cells were maintained in a humidified chamber at 37°C with 5% carbon dioxide and were routinely subcultured with trypsin/EDTA.
Snap-frozen and formalin-fixed, paraffin-embedded (FFPE) tissue blocks were obtained from the University of Minnesota Tissue Procurement Facility after institutional review board approval. RNA was isolated from snap-frozen tissues, while FFPE tissues were used to optimize immunohistochemical staining. The five ovarian cancer tissues were of the serous subtype. The 7 normal tissues were benign leiomyoma (n = 3), endometriosis (n = 1), benign peritubal cyst (n = 1), and normal ovary (n = 2). All tissue samples underwent strict quality control measures before use in these studies. Namely, tumors were diagnosed by a pathologist at the time of surgery using Tissue Tek OCT (Sakura Finetek, Torrance, CA)–embedded tissue. The following day, the FFPE H&E-stained slides were reviewed by a pathologist (S.E.P.) to confirm the accuracy of the diagnosis. A third pathologist reviewed the quality control H&E-stained slides of all University of Minnesota Tissue Procurement Facility cases to confirm the diagnosis of the samples before distribution to researchers. In addition, a pathologist (S.E.P) reviewed the slides while scoring the immunohistochemical staining.
Blood and Ascites Samples
Staff at the University of Minnesota Tissue Procurement Facility ensured that all patients signed an institutional review board–approved consent form before surgery. Blood was collected immediately before surgery from women with abdominal masses suspected to be ovarian cancer. Blood samples were collected from 51 women with serous ovarian cancer (5 stage 1, 7 stage 2, 28 stage 3, 10 stage 4, and 1 unstaged; 4 Silverberg grade 1, 9 Silverberg grade 2, 37 Silverberg grade 3, and 1 ungraded), 14 women with clear cell ovarian cancer (8 stage 1, 1 stage 2, 4 stage 3, and 1 not staged; 14 Silverberg grade 3), 18 women with low malignant potential (LMP) ovarian tumors (10 LMP serous, 3 LMP mucinous, and 5 LMP with no subtype noted), and 51 women with benign gynecologic disease (12 benign endometriosis, 10 benign mucinous cystadenoma, 6 fibroma, 6 benign serous cystadenoma, 17 benign disease not specified). Ascites fluid was collected from 10 women with serous ovarian cancer.
Blood and ascites samples were processed by standard protocols, divided into aliquots, and stored at −80°C. Before use in this study, the pathology reports for the blood and ascites samples were reviewed by a gynecologist/oncologist (M.A.G or P.A.A.) to ensure that the samples were placed into the correct category.
Reverse Transcriptase–Polymerase Chain Reaction
Total RNA was extracted from cell lines and ovarian tissue samples using the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. A 189-base-pair sequence corresponding to nectin 4 was amplified with the following primers: forward, 5'-CAAAATCTGTGGCACATTGG-3'; and reverse, 5'-GCTGACATGGCAGACGTAGA-3'. One-step RT-PCR was performed with the RT-PCR Access kit (Promega, Madison, WI), with conditions as follows: 45 minutes at 45°C; 1 cycle of 94°C, 2 minutes; 55°C, 1 minute; 68°C, 1 minute; 25 cycles of 94°C for 30 seconds, 55°C for 1 minute, and 68°C for 1 minute; and a final extension at 68°C for 7 minutes. Expression of nectin 4 in MCF7 breast cancer cell lines has been reported and was used as a positive control.18
Expression of β-actin in the samples confirmed that messenger RNA (mRNA) was not degraded and that similar amounts of mRNA were loaded.
Real-time quantification of nectin 4 was performed using the SYBRGreen assay (Bio-Rad Laboratories, Hercules, CA) and the iQ5 Real-Time PCR thermocycler (Bio-Rad). We amplified 2 µL of complementary DNA in a 25-µL reaction containing 13 µL of iQ SYBRGreen Supermix (Bio-Rad), 1 µL each of nectin 4 forward and reverse primers (forward, TGCTCAAGTGCCTGAGTGAA; reverse, AGACGTAGATGCCGCTGTG), and 8 µL of nuclease-free water. Following an initial denaturation step of 95°C for 3 minutes, 40 cycles of PCR were performed under the following conditions: 95°C, 10 seconds (denaturation), and 57°C, 30 seconds (annealing/extension). Product size was verified by agarose gel electrophoresis. Threshold cycle (Ct) values were calculated according to the iQ5 real-time detection software. Standard curves for nectin 4 and β-actin were generated by plotting Ct vs the log of the initial starting amount of RNA in nanograms.25
Each sample was normalized to the amount of β-actin mRNA present in the sample, and the relative amount of each sample was determined as a fold-change increase over the lowest expressing cell line (1816-575).
Cells (1 × 106) were incubated with 1 to 2.5 µg of mouse anti–nectin 4 (clone 337516), mouse anti–nectin 1, mouse antihuman ADAM17, or a control mouse IgG for 30 minutes at 4°C. Cells were washed and then incubated with biotin-conjugated goat antimouse IgG F(ab')2 fragment for 30 minutes at 4°C. After washing, cells were incubated with allophycocyanin-conjugated streptavidin for 20 minutes at 4°C. After washing, cells were resuspended and run on a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ) and analyzed using CellQuest Pro software (Becton Dickinson), gating on live cells by forward and side scatter.
Total protein extracts were derived from confluent monolayers of cells in 50 mmol/L tris(hydroxymethyl)aminomethane (Tris), 150 mmol/L sodium chloride, 1 mmol/L EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland), and 1 mmol/L phenylmethylsulfonyl fluoride and then stored at –80°C. Fifty micrograms of total cell lysate were separated on a 10% SDS Tris-hydrochloride polyacrylamide gel and then blotted onto a polyvinylidene difluoride membrane (GE Healthcare Limited, Piscataway, NJ). Membranes were blocked with 5% powdered milk (Roundy’s, Milwaukee, WI) in PBS, as previously described,26,27
and then incubated in 1 µg/mL mouse antihuman nectin 4 (clone N4.61) overnight, followed by a 2-hour incubation in horseradish peroxidase–conjugated rabbit antimouse antibody diluted 1/5,000. Protein was visualized using the Super Signal West Femto kit (Thermo-Fisher Scientific, Rockford, IL) according to the manufacturer’s instructions. Membranes were exposed to autoradiography film (Midwest Scientific, Valley Park, MO) and developed.
TMA slides containing 0.6-mm duplicate core samples for 500 ovarian cancer cases were provided by the Cheryl Brown Ovarian Cancer Outcomes Unit (University of British Columbia, Vancouver, Canada). Cases included in the TMA were chosen based on having been optimally cytoreduced at initial surgery with no macroscopic residual disease remaining. Owing to these criteria, a significant proportion of early-stage cases were present on the TMA relative to the general population. None of the patients received neoadjuvant therapy, and all received platinum-based chemotherapy following surgery. The 500 cases included on the TMA were collected up to 18 years before this analysis. H&E-stained slides for all cases were reviewed by a gynecologic pathologist (C.B.G.) to confirm diagnosis, stage, tumor cell type, and grade before TMA inclusion to ensure that the current diagnostic criteria for subclassification of ovarian cancer based on cell type were uniformly applied.28,29
Samples displaying multiple cell types (mixed tumors) were excluded from the study. Details regarding the cohort used for these TMAs are provided in and in Gilks et al.30
Patients were followed up for a median of 4.6 years (0.1–18 years) after the initial surgery.
Subtype, Stage, Silverberg Grade, and Nectin 4 Score of Tissue Microarrays*
Immunohistochemical Staining of Tissues
Slides were dried, and tissue sections were deparaffinized and rehydrated as previously described.26
Staining was performed manually without antigen-retrieval procedures. Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide, and then slides were incubated with 10 µg/mL mouse antihuman nectin 4 (clone 337516) or normal mouse IgG diluted in 1:5 Sniper/PBS (Biocare, Concord, CA) overnight at 4°C. Slides were incubated with horseradish peroxidase–conjugated rabbit antimouse polyclonal secondary antibody for 20 minutes. Staining was visualized with Vulcan Fast Red (Biocare), and hematoxylin was used as a counterstain. Slides were dehydrated through a series of ethanol and xylene washes, and then coverslipped with VectaMount (Vector Laboratories, Burlingame, CA).
Tissues were graded on a 4-point scale by a pathologist (S.E.P.) in a blinded manner. Sections with no staining were scored as 0, those with less than 10% of cancer cells staining were scored as +1, tissues with 10% to 50% of the cancer cells staining were scored as +2, and tissues with more than 50% of the cancer cells staining were scored as +3. For most of the analysis, the data were binarized to create 2 groups, in which all positive scores (+1, +2, and +3) were grouped together and compared with the negatively scored tissues.
TMA Statistical Analysis
Differential expression for nectin 4 across the histopathologic subtypes was assessed with the Pearso n χ2 statistic. Univariable relapse-free survival for the entire cohort and each histopathologic subtype was examined with Kaplan-Meier survival curves. Results significant in univariable analysis were subjected to multivariable relapse-free survival using the Cox proportional hazards test. The level of significance for all comparisons was a P value less than .05. All statistical calculations were computed with JMP, version 6.0.3 (SAS Institute, Cary, NC).
Enzyme-Linked Immunosorbent Assay
A sandwich ELISA was used to detect soluble nectin 4 in patients’ serum and ascites samples. Ninety-six well tissue culture plates were coated with 10 µg/mL of mouse antihuman nectin 4 monoclonal antibody (clone N4.61). After blocking the wells with PBS containing 1% bovine serum albumin (R&D Systems), 100 µL of serum or ascites was incubated for 12 hours at 4°C and washed, and then 0.5 µg/mL biotinylated goat antihuman nectin 4 antibody was added. Streptavidin-peroxidase in PBS–bovine serum albumin was incubated for 1 hour at 37°C. Next, 100 µL of peroxidase substrate was added (One Step ABTS, Pierce, Rockford, IL), and optical density was read at 405 nm. Three to five wash steps were performed between incubations with PBS containing 0.5% polysorbate (Tween) 20. Analyses were done in duplicate. The nectin 4 concentration was calculated using serial dilution of recombinant human nectin 4-Fc protein as previously described.14
ELISA Statistical Analysis
Serum nectin 4 levels were compared among serous, clear cell, LMP, and benign gynecologic disease by using the Kruskal-Wallis test. Subsequent pairwise comparisons were conducted using the Wilcoxon rank sum test.
Receiver-operating characteristic (ROC) curves were constructed for nectin 4 alone, CA125 alone, and the combination of CA125 and nectin 4. The Youden index was used to place the optimal threshold point for nectin 4 to distinguish between a serous ovarian cancer mass and benign gynecologic disease on the curve; the corresponding sensitivity and specificity of the test were then determined. Exact P values for the classification table of the test results by the known status were then calculated.