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  • BODIPY-Cholesterol br able to distinguish endometrial cancer cases

    2022-07-25


    140 able to distinguish endometrial cancer cases from controls ≤ 2 years of diagnosis, and to
    141 determine whether there was a temporal relationship in abundance of these BODIPY-Cholesterol among cases
    142 as time of cancer diagnosis approached.
    ACCEPTED MANUSCRIPT
    161 Materials and Methods
    162 We performed a nested case-control study within the screening arm of the PLCO Cancer
    164 recruited from 10 screening centers across the United States. Participants were randomized to
    165 either a screening or non-screening arm; participants randomized to the screening arm provided
    166 serum samples upon enrollment and at five subsequent medical examinations. Incident cancers
    167 were determined by participant self-report and confirmed by review of the participant’s medical
    168 record and pathology report. All participants provided written informed consent and the PLCO
    169 Cancer Screening Trial was approved by the institutional review board of the National Cancer
    171 We evaluated postmenopausal women randomized to the screening arm of the PLCO
    172 Cancer Screening Trial that met the following inclusion criteria: intact uterus, available serum
    173 sample, no previous cancer diagnosis, and completion of intake questionnaire. Participants also
    174 had to provide written consent allowing biochemical study of their serum. Endometrial cancer
    175 cases were defined as having a primary diagnosis of an invasive epithelial tumor of the uterus
    176 between the initial screening visit and January 1, 2010. Participants with a preexisting
    177 malignancy at enrollment or a mesenchymal uterine tumor were excluded.
    178 Controls were matched 1:1 based on age, race, study site, year of blood draw, and year of
    179 randomization. Controls were restricted to women with no history of a hysterectomy and were
    180 required to be alive at time of diagnosis of their matched case.
    181 Serum samples from 552 participants were provided by the PLCO central bank and were
    182 stored at -80˚C until use. No more than two freeze- thaw cycles were allowed for each sample.
    183 Samples were blinded and randomized for processing and analysis. Serum samples were thawed
    ACCEPTED MANUSCRIPT
    184 on ice and highly abundant proteins were immunodepleted using Multiple Affinity Removal
    185 Human-14 Spin Cartridge (Agilent Technologies) according to manufacturer’s instructions. The
    186 unbound protein fraction was eluted and buffer-exchanged with 100 µL x 6 of 100 mM
    187 triethylammonium bicarbonate (TEAB) in a Microcon-10 (Millipore) spin-column filter. A total
    189 were then transferred to 0.5 mL MicroTubes (Pressure Biosciences) and incubated at 99 ⁰C for
    190 30 min, after which they were cooled to ambient temperature and 1 µL of SMART Digest
    191 Trypsin (Thermo Scientific) was added to each. The MicroTubes were capped with MicroPestles
    192 (Pressure Biosciences) and digestion was performed in a Barocycler 2320EXT (Pressure
    193 BioSciences) by cycling between 45 kpsi for 50 s and atmospheric pressure for 10 s for 60 cycles
    194 at 50 ⁰C. Peptide digests were dried by vacuum centrifugation, resuspended in 100 mM TEAB,
    195 and quantified using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific).
    196 Equivalent amounts of peptide were labeled with tandem-mass tag (TMT) isobaric labels 197 (TMT11plex™ Isobaric Label Reagent Set, ThermoFishe r Scientific) as per manufacturer’s
    198 recommendations. TMT-labeled plexes were fractionated using high pH reversed-phase liquid