Cell culture media and supplements were purchased from Invitrogen Corporation (Carlsbad, CA) unless otherwise stated. Chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.
Antibodies used were mouse anti-human claudin 4 (clone 3E2C1; Invitrogen), mouse anti-human β-actin (clone AC-74; Sigma-Aldrich), and normal mouse IgG (clone 3-5D1-C9; AbCam). Secondary antibodies used were FITC-conjugated goat anti-mouse IgG + IgM (Roche Diagnostics, Indianapolis, IN), stabilized horseradish peroxidase-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Rockford, IL), and biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA).
3.2. Cell Lines
Ovarian cancer cell lines SKOV3, ES-2, NIH:OVCAR3, HEY, C-13, OV2008, OVCA429, OVCA433, A2780-S, and A2780-CP (provided by Dr. Barbara Vanderhyden, University of Ottawa, Canada), NIH:OVCAR5 (provided by Dr. Judah Folkman, Harvard Medical School, Boston, MA), CAOV3 (provided by Dr. Robert Bast Jr., University of Texas, Houston, TX), and MA148 (provided by Dr. Sundaram Ramakrishnan, University of Minnesota, Minneapolis, MN) were maintained as previously described [6
]. SKOV3, ES-2, and OVCA429 cell lines were derived from clear cell carcinomas; OV2008 and C-13 cell lines were derived from endometrioid tumors; NIH:OVCAR3, NIH:OVCAR5, OVCA433, CAOV3, HEY, MA148, A2780-S, and A2780-CP cell lines were derived from serous adenocarcinomas [59
Immortalized normal ovarian surface epithelial (NOSE) cell lines 1816–575, 1816–686, IMCC3, IMCC5, and HIO117 (provided by Dr. Patricia Kruk, University of South Florida, Tampa, FL), and IOSE-29 and IOSE-80 (provided by Dr. Nelly Auersperg, University of British Columbia, Vancouver, BC, Canada) were also maintained as described [62
]. Cells were maintained in a humidified chamber at 37 °C with 5% CO2
and were routinely subcultured with trypsin/EDTA.
3.3. shRNA Knockdown of Claudin 4
NIH:OVCAR5 cells were stably transfected with shRNA clone TRCN0000116631 (Open Biosystems, Huntsville, AL) plasmid DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
3.4. Transfection of MA148 Cells with Claudin 4
The claudin 4 coding sequence was amplified from CAOV3 total RNA using the Access one step RT-PCR kit (Promega, Madison, WI) with primers; Forward, AGATCTATGGCCTCCATGGGG; Reverse, TCTAGATTACACGTAGTTGCTGGCAGC, and cloned into the TA cloning vector pCR2.1 (Invitrogen) according to the manufacturer’s instructions. The claudin 4 coding fragment was excised from pCR2.1 by digestion with BglII and XbaI and ligated into the pcDNA3.1 expression vector (Invitrogen), and the sequence and orientation were verified by sequencing with vector primers. The pcDNA3.1-claudin 4 plasmid was transfected into MA148 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Stable clones were selected with neomycin.
3.5. Tissue Samples
Snap-frozen tissue samples and formalin-fixed, paraffin-embedded (FFPE) tissue blocks were obtained from the University of Minnesota Tissue Procurement Facility (TPF) after IRB approval. Snap-frozen tissues were used for isolation of RNA and protein; FFPE tissues blocks were used to optimize immunohistochemical staining. The seven snap-frozen ovarian cancer tissues used for RNA and protein analysis were derived from the primary ovarian tumors of women with stage III/IV ovarian cancer of the serous subtype. The five snap-frozen normal ovarian tissues were obtained from patients with benign leiomyomas, endometriosis, benign peritubal cysts, or other non-ovarian diseases. For immunohistochemistry, 33 serous tumors, 4 clear cell tumors, and 21 normal ovaries were examined. All tissue samples underwent strict quality control measures prior to use in these studies. Namely, tumors were diagnosed by a pathologist at the time of surgery using OCT embedded tissue. The following day, the FFPE H&E slides were reviewed by a pathologist to confirm the accuracy of the diagnosis. A third pathologist reviewed the quality control H&E slides of all TPF cases to confirm the diagnosis of the samples prior to distribution to researchers. Additionally, a pathologist (S.E.P.) reviewed the slides while scoring the IHC staining.
3.6. Isolation of Spheroids from the Ascites of Ovarian Cancer Patients
Ascites fluid was obtained from the University of Minnesota Tissue Procurement Facility after IRB approval. Spheroids were isolated from ovarian cancer patient ascites as previously described [5
]. Briefly, ascites was centrifuged at 500–700 × g for 10 min and erythrocytes were lysed by resuspending cells in lysis buffer (10 mM potassium bicarbonate, 155 mM ammonium chloride, 0.1 mM EDTA, pH 7.5) for 5 min. Remaining cells were collected by centrifugation, washed with PBS and viably frozen (10% dimethyl sulfoxide in fetal bovine serum) and stored in liquid nitrogen until use.
3.7. RNA Extraction and 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 372 bp sequence corresponding to claudin 4 was amplified from 200 ng of total RNA using the following primers: Forward, 5′ TGATATCACCTCTGGGACTGT’; Reverse, 5′ CAGAAACCACAAAGAAGGAAG. One-step RT-PCR was performed with the RT-PCR Access kit (Promega, Madison, WI), with conditions as follows: 45 min at 45 °C; 1 cycle of 94 °C, 2 min; 56 °C, 1 min; 72 °C, 1 min; 35 cycles of: 94 °C, 30 sec; 56 °C, 1 min; 72 °C, 1 min; and a final extension at 72 °C for 7 min. Expression of β-actin in the samples confirmed that RNA was not degraded and that similar amounts of RNA were loaded [β-actin primers (Forward, 5′GGCCACGGCTGCTTC; Reverse, 5′GTTGGCGTACAGGTCTTTGC)]. These experiments were performed at least two times.
3.8. Quantitative Reverse Transcriptase Polymerase Chain Reaction
Real time quantification of claudin 4 was performed using the SYBR-green assay (Bio-Rad Laboratories, Hercules, CA) and the iQ5 Real-Time PCR thermocycler (Bio-Rad). Two micrograms of total RNA was used for cDNA synthesis using an oligo dT primer and Superscript III first-strand synthesis kit (Invitrogen) according to the manufacturer’s specifications. Two microliters of cDNA was amplified in a 25 μL reaction containing 1 μL each of claudin 4 forward and reverse primers (Forward, CTTCATCGGCAGCAACATT; Reverse, AGCAGCGAGTCGTACACCTT), and 13 μL of iQ SYBR green supermix (Bio-Rad). Following an initial denaturation step of 95 °C for 3 min, 40 cycles of PCR were performed under the following conditions: 95 °C, 10 sec (denaturation) and 52 °C, 30 sec (annealing/extension). All real-time PCR reactions were run in duplicate and melt curve analysis was performed to determine amplification of a single product. Data was normalized to the amount of β-actin present in the sample, determined in a separate reaction [primers β-actin forward: AGAGCTACGAGCTGCCTGAC; β-actin reverse: GGATGTCCACGTCACACTTC; and annealing temperature 54 °C]. Transcript levels were quantitated using cRNA standard curves for claudin-4 and β-actin [64
] and the relative amount of each sample was determined as a fold-change increase over the lowest expressing cell line (1816–575). Expression values reported in are the average of two experiments, except samples IMCC3, IMCC5, HIO117 and IOSE-29 which were run in duplicate in a single experiment.
3.9. Western Immunoblotting
Protein was extracted from snap-frozen tissues in T-PER (Tissue Protein Extraction Reagent; Thermo Fisher Scientific) containing a protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland). Total protein extracts were also derived from confluent monolayers of cells in 50 mM Tris, 150 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail (Roche Applied Science), and 1 mM PMSF then stored at −80 °C. Protein concentration was determined using the BCA Protein Assay (Thermo Fisher Scientific). Fifty micrograms of protein were separated on a 10% SDS Tris-HCl polyacrylamide gel or a 4–20% Tris-HCl Criterion precast gel (BioRad), then blotted onto a polyvinylidene difluoride membrane (GE Healthcare Limited; Piscataway, NJ). Membranes were blocked with 5% powdered milk (Roundy’s Inc.; Milwaukee, WI) in PBS as previously described [65
], and then incubated in 0.167 μg/mL mouse anti-human claudin 4 (clone 3E2C1) overnight, followed by a 2-hr incubation in horseradish peroxidase conjugated goat anti-mouse antibody diluted 1/5000. Protein was visualized using the Super Signal West Femto kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Membranes were exposed to autoradiography film (Midwest Scientific; Valley Park, MO) and developed. Blots were reprobed with an antibody against β-actin as a loading control.
3.10. Immunohistochemical Staining of Tissues
FFPE tissue sections were deparaffinized and rehydrated through a series of xylene and ethanol washes as previously described [66
]. Antigen retrieval was performed in a citrate buffer (Biocare, Concord, CA) and endogenous peroxidase activity was blocked with hydrogen peroxide. Slides were incubated with mouse anti-human claudin 4 monoclonal antibody (clone 3E2C1; Invitrogen) or normal mouse IgG1 (clone 3-5D1-C9; AbCam) at 0.25 μg/mL overnight. Slides were washed and incubated with biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA), followed with an avidin:biotin complex (Vector Laboratories). Staining was visualized with 3,3′-diaminobenzidine (Biocare). Slides were examined by a pathologist (S.E.P.) in a blinded manner and assigned a score of 0 (no staining); 1 (<10% of neoplastic cells staining); 2 (10–50% of neoplastic cells staining); or 3 (>50% of neoplastic cells staining). FFPE blocks of human intestine were used as a positive control for claudin 4 antibody staining.
3.11. Tissue Microarrays
TMA slides containing 0.6 mm duplicate core samples for 500 ovarian cancer patients were provided by the Cheryl Brown Ovarian Cancer Outcomes Unit (University of British Columbia; Vancouver, BC, Canada). Patients included in the TMA were chosen based on having been optimally cytoreduced at initial surgery with no macroscopic residual disease remaining. Due to these criteria, a significant proportion of early stage cases were present on the TMA relative to the general population of patients with ovarian cancer. 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 prior to this analysis. Hematoxylin and eosin stained slides for all cases were reviewed by a gynecologic pathologist (C.B.G.) to confirm diagnosis, stage, tumor cell type, and grade prior to TMA inclusion to ensure that the current diagnostic criteria for subclassification of ovarian cancer based on cell type were uniformly applied [67
]. 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.
]. Patients were followed for a median of 4.6 (0.1–18) years after the initial surgery. Three-tiered grading of ovarian cancer tissues was done using the Silverberg grading system at the time of review of the complete slide sets for all cases on the ovarian TMAs [69
Tissue microarray slides were treated and stained identically to the individual tissue sections, and scored in a blinded manner as described above. In cases where the duplicate core samples received different scores, results were averaged for analysis. For some analyses, scores of 1, 2, and 3 were grouped and considered positive (“binarized data”).
3.12. TMA Statistical Analysis
Differential expression for claudin 4 across the histopathological subtypes was assessed with the Pearson Chi-Square 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 p < 0.05. All statistical calculations were computed with JMP v. 6.0.3 (SAS Institute Inc., Carey, NC).
3.13. Spheroid Formation in Vitro
Spheroids were cultured using the liquid overlay method, as previously described [52
]. Briefly, 96-well tissue culture plates were coated with 100 μL of 0.5% w/v SeaKem LE agarose (Lonza, Walkersville, MD) in serum-free culture media, to prohibit cell adhesion to the substratum. Plates were allowed to cool for at least 30 min at room temperature. Cells grown in monolayer cultures were released with 0.5% trypsin, 2 mM ethylenediaminetetraacetic acid (Invitrogen) and resuspended in complete cell culture media. The cell suspension was run through a 70 μm cell strainer (BD Biosciences, Bedford, MA) to remove residual clumps. Cells were counted with a hemocytometer, then diluted to 2000 to 170,000 cells/mL. Cell suspensions were layered on top of the agarose-coated plates at a volume of 100 μL/well and then incubated at 37 °C.
3.14. Immunocytochemical Staining of Spheroids
Spheroids either isolated from the ascites of ovarian cancer patients or formed in vitro
from ovarian cancer cell lines were embedded in thrombin clots and 20 μm OCT-frozen sections were stained as previously described [52
]. Alternatively, spheroids were fixed and stained in a 96-well plate prior to mounting onto slides. Briefly, spheroids were washed three times with Dulbecco’s phosphate buffered saline (DPBS) containing calcium and magnesium, and centrifuged at ~200 × g for 5 min. Cells were fixed in 200 μL of ice cold 100% methanol overnight at −20 °C, then rehydrated with 3 washes of DPBS containing calcium and magnesium. Cells were blocked with 1% normal goat serum, 0.3% Tween-20 in DPBS containing calcium and magnesium for 1 hr, then incubated in 100 μL of primary antibody at 2.5 μg/mL in blocking buffer overnight at 4 °C with gentle agitation. Cells were washed three times in blocking buffer and incubated in a 1:100 dilution of secondary antibody (FITC-conjugated goat anti-mouse IgG + IgM; Roche Diagnostics) overnight at 4 °C in the dark. Cells were washed three times in blocking buffer and incubated in 100 μL of a 2.86 × 10−7
M DAPI solution for 5 min, then washed three times in DPBS containing calcium, magnesium, and 0.3% Tween-20 with a final wash in SlowFade equilibration buffer (Invitrogen, Eugene, OR). Cells were mounted in 1× SlowFade reagent in PBS containing 50% glycerol.
3.15. FITC-Dextran Paracellular Permeability Imaging
Spheroids were formed in 6-well plates by coating plates with 2 mL agarose as described above. Cells were plated in 2 mL complete media at concentrations from 10,000 to 500,000 cells per well and spheroids were formed for 48 to 96 hr at 37 °C. Thirty minutes prior to visualization, a 4 kDa conjugate FITC-dextran (Sigma-Aldrich) was added to a final concentration of 0.05–0.1% [33
]. Spheroids were imaged in situ
on an Olympus FluoView FV1000 upright confocal microscope with a 20X water immersion objective and 488 nm laser. Fluorescence intensity profiles were generated with Image J 1.37v software.
In this study, we validated our previous gene microarray data, showing that claudin 4 RNA and protein is overexpressed in ovarian cancer tissues and cell lines compared to tissues and cell lines from normal ovaries. We also demonstrated that claudin 4 is differentially expressed across histological subtypes of ovarian cancer; however, no difference in survival was observed between claudin 4 positive vs.
negative tumors. Claudin 4 was also expressed in ovarian cancer spheroids isolated from the ascites of patients. The parental NIH:OVCAR5 ovarian cancer cell line expressed high levels of claudin 4 and was able to form compact spheroids in vitro
more rapidly than when the cell line was treated with shRNA targeting claudin 4, causing low levels of claudin 4 to be expressed. These results demonstrate a role for claudin 4 in spheroid formation and integrity, and lead us to speculate that claudin 4 may play a role in mediating chemoresistance in spheroids by increasing tight junction barrier function or activation of prosurvival signaling. Furthermore, as the majority of cases of ovarian cancer examined exhibited elevated levels of claudin 4 protein expression, this supports the use of claudin 4 as a therapeutic target, and we postulate that blocking claudin 4 function may increase the efficacy of chemotherapy delivered intraperitoneally. Indeed, several groups have reported using claudin 4 as a target for delivery of toxins and fluorescent molecules to ovarian and breast cancer cells [70