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  • br Corresponding author University of Alabama at Birmingham

    2022-04-25


    ∗ Corresponding author: University of Alabama at Birmingham, Department of Surgery, School of Medicine, 1824 6th Avenue South, Wallace Tumor Institute Suite 310H, Birmingham, Alabama 35233.
    E-mail address: [email protected] (D.M. Carmona Matos).
    malignancies de-differentiate and lose capacity for radioactive iodine uptake. Further de-differentiation limits therapeutic options and leads to poor prognosis.3 Therefore, alternative treatments for these nonresponsive patients are needed.
    Many carcinomas have been shown to express somatostatin re-ceptors (SSTRs). Of these, SSTR2 is overexpressed commonly in en-docrine tumors.4 Furthermore, thyroid carcinomas are known to express SSTR2 and SSTR5. The presence of SSTRs in tumors pro-vides the possibility of diagnostic imaging and therapy with radi-olabeled SST analogs such as the FDA-approved analog octreotide and lutetium Lu-177 dotatate.5,6 Thyroid tumors are known to ex-press SSTRs distinctly depending on whether they are medullary thyroid cancer tumors or nonmedullary tumors: papillary thyroid cancer (PTC), follicular thyroid cancer (FTC), or anaplastic thyroid cancer (ATC).7,8
    FTC is a malignant epithelial tumor that accounts for 10%–32% of differentiated thyroid carcinomas.1,9 Although it is uncommon, FTC has a greater rate of distant metastasis (29%) and can ulti-mately lead to death (17%) compared to PTC (9% and 8%).10 ATC is the least common of all thyroid carcinomas (<1%). Still, ATC ac-counts for about 40% of thyroid cancer deaths. The mean survival rate of ATC is less than 6 months after diagnosis with a 5-year sur-vival rate of 0–25%. Current aggressive, multimodal therapies fail to improve this survival rate.11 Roughly 50% of patients exhibit metas-tasis to lungs (80%), bones (6%–15%), and CFTRinh-172 (5%–13%). There-fore, there is a clear need for novel therapies for these aggressive thyroid cancers. The limited and conflicting data on SSTR expres-sion for FTC and ATC hinders the application of somatostatin ana-logues as a potential treatment. Our study seeks to characterize SSTR expression in FTC and ATC and to assess the effects of so-matostatin analogs in these thyroid carcinomas.
    Materials and methods
    Cell culture and reagents
    Hth7 cells, an ATC cell line, were obtained from Dr Re-becca Schweppe (University of Colorado, Denver, CO). FTC-236, a metastatic FTC cell line, and 8505C, an ATC cell line, were pur-chased from Sigma Life Science/European Collection of Cell Cul-tures (Sigma-Aldrich, St. Louis, MO). All cell lines were authen-ticated and confirmed with their unique identity by DNA profil-ing.11 Hth7 cells were maintained in glutamine (+) RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA) with 10% fe-tal bovine serum. FTC-236 cells were maintained in glutamine (+), DMEM:F-12 medium (Invitrogen Life Technologies) with 10% fe-tal bovine serum, 10 μg/ml insulin, and 10 mIU/ml of human thy-roid stimulating hormone (Sigma-Aldrich). 8505C cells were main-tained in glutamine (+) EMEM medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum and 1% non-essential amino acids (Sigma-Aldrich). All media were supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin. All cell lines were grown in a humidified environment of 5% CO2 at 37°C.
    Octreotide (OCT), an SSTR2 specific agonist, was purchased from Sigma-Aldrich. Pasireotride (SOM230), an SSTR5, SSTR3>>SSTR2 agonist, and KE-108, a pansomatostatin receptor agonist SSTR1– SSTR5, were purchased from Biomatik USA (Wilmington, DE). OCT, SOM230, and KE-108 were dissolved in dimethyl sulfoxide and stored at −20°C as 50mM stock solutions.
    Cellular proliferation assay
    FTC-236, Hth7, and 8505C were treated with OCT, SOM230, and KE-108 in varying doses to determine the half-maximal in-hibitory concentration (IC50) of each compound using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as-say. Each cell line was plated in quadruplicate on 96-well plates at a density of 5,000 cells per well. Plated cells were incubated overnight to allow cell attachment; treatment medium was pre-pared in serial dilutions for various concentrations of OCT (0–200 μM), SOM230 (0–200 μM), and KE-108 (0–128 μM). Each quadru-plicate of cells was exposed to only 1 compound at a time. Quad Cell viability was measured 48 h after treatment. On the day of measurement, treatment medium was removed before cells were incubated at standard conditions in 25 μL of serum-free RPMI-1640 containing 0.5 mg/ml −1 MTT for 3 h. To dissolve the MTT formazan, 75 μL of dimethyl sulfoxide was added to each well and mixed thoroughly. Absorbance was measured at 562 nm us-ing a spectrophotometer (μQuant; Bio-Tek Instruments, Winooski,  VT). The IC50 value was calculated by GraphPad Prism 6 (GraphPad Software, La Jolla, CA).