br Materials and Methods br
Materials and Methods
RWPE1 Spheroid Culture
RWPE1 ML162 were acquired from ATCC (Manassas, VA) in 2014, used at passage <20, and were maintained in RPMI 1640 medium and 10% fetal bovine serum. Cells were transduced with lentivirus that contained full miR-183 family cluster sequence or a control vector and sorted with fluorescence-activated cell sorting for green fluorescent pro-tein expression.19 These cells were grown in a 50% Matrigel (Corning, Corning, NY) suspension for 8 days, dissociated with Dispase (Stemcell Technologies, Vancouver, Canada), suspended in Histogel (Thermo Fisher, Waltham, MA), formalin fixed, and paraffin embedded before ISH.
TMA and Prostate Tissue Specimens
The Outcome TMA was constructed by the National Cancer
Instituteesponsored Cooperative Prostate Cancer Tissue Resource.25,26 This TMA was designed as a case-control
study for biochemical recurrence after prostatectomy. The specimens were collected between 1988 and 2002. All pa-tients with biochemical nonrecurrence were followed up for
a minimum of 5 years and five serum prostate-specific an-tigen (PSA) measurements. Recurrence was defined as a postsurgical PSA value 0.4 ng/mL or two consecutive values 0.2 ng/mL. The original TMA contained 404 pa-tients with four tumor cores per patient; however, many cores have been depleted. Data were collected from 133 patients, 56 of whom had both cancer and benign epithelium present. Cores with a diameter of 0.6 mm were taken from tumor regions of tissue. The number of cores analyzed per
patient ranged 1 to 4 (mean, 2.4 cores). The TMA is pub-licly available and completely deidentified through the Cooperative Prostate Cancer Tissue Resource.
The Murphy TMA was constructed based on patients un-dergoing radical prostatectomy at the Jesse Brown Veterans Affairs Medical Center for clinically localized PCa. Collabo-rating pathologists performed centralized pathologic review and assembled the TMA from the formalin-fixed, paraffin-embedded prostatectomy specimen with pathologic and clin-ical data. Cores were selected from the highest Gleason grade region of the prostatectomy specimen with care to punch cores from areas of >75% tumor epithelium and from the contra-lateral normal benign epithelium. The prostatectomy tissues were collected between 2013 and 2017. Cores with a 1-mm diameter were taken from tumor and benign regions of tissue. The TMA contains cores from 66 patients with three tumor cores and two benign cores per patient. Fifty-five patients were analyzed, and the number of cores analyzed per patient ranged 2 to 4 (mean, 3.7 cores). Patients consented to the use of their tissues for PCa research. Specimens are deidentified. The tis-sue collection was approved by the Jesse Brown Veterans Affairs Institutional Review Board.
Additional deidentified prostatectomy tissues analyzed were part of a cohort of University of Illinois at Chicago (UIC) patients and the Cooperative Human Tissue Network approved by the UIC Office for the Protection of Research Subjects under UIC Institutional Review Board 2013-0341 as previously described.27
Immunofluorescence and Staining
A 5-mm tissue section adjacent to the section used for ISH was probed for rabbit polyclonal cytokeratin 5 (KRT5, clone Poly19055, BioLegend, San Diego, CA) and mouse mono-clonal pan-cytokeratin AE1/AE3 (ab27988, Abcam, Cam-bridge, UK) antibodies diluted to 1:200. Antigens were retrieved using sodium citrate buffer, pH 6, 100 C for 5 mi-nutes at 5 psi. Alexafluor 555e and 488elabeled secondaries (Invitrogen, Carlsbad, CA) were used at 1:200, followed by DAPI nuclear counterstain. Slides were imaged on the Vectra Automated Multispectral Imaging System (PerkinElmer, Waltham, MA) at the Research Histology and Tissue Imaging Core at UIC. The other adjacent section was hematoxylin and eosin (H&E) stained and scanned with Aperio AT2 (Leica, Wetzlar, Germany) at the Research Histology and Tissue Im-aging Core.
The protocol from the miRCURY LNA miRNA ISH optimi-zation kit (Exiqon, Vedbaek, Denmark) was followed with modifications. Formalin-fixed, paraffin-embedded TMA sec-tions (5 mm) were placed onto hydrophilic slides, baked over-night at 60 C, deparaffinized, and incubated for 20 minutes at 37 C with 15 mg/mL of proteinase K for protein digestion. Digoxigenin-labeled miR-182 LNA probe (80 nmol/L),
912 ajp.amjpathol.org - The American Journal of Pathology
High miR-182 in Low-Risk Prostate Cancer
digoxigenin-labeled U6 LNA probe (10 nmol/L) (positive control), or no probe (negative control) was incubated at 48 C for 60 minutes followed by stepwise 5-minute washes in saline-sodium citrate buffer at 42 C ( 1, 0.5, 0.2, and then 0.2) at room temperature. Slides were blocked and incubated for 60 minutes with alkaline phosphataseeconjugated anti-digoxigenin antibody (Sigma-Aldrich, St. Louis, MO) at 1:200. Alkaline phosphatase was visualized with Vector Red alkaline phosphatase substrate (Vector Laboratories, Burlingame, CA) for 90 minutes and stopped with KTBT buffer. Slides were counterstained with DAPI.