studies using the chick chorioallantoic membrane xenograft models demonstrated that treatment with Lestaurtinib resulted in a significant decrease in endpoint tumor volume and vascularity using power Doppler ultrasound imaging. the cell cycle, indicative of a cytostatic effect. studies using the chick chorioallantoic membrane xenograft models demonstrated that treatment with Lestaurtinib resulted in a significant decrease in endpoint tumor volume and vascularity using power Doppler ultrasound imaging. Overall, this study provides evidence that Lestaurtinib is a potent antiproliferative agent with potential antiangiogenic activity that warrants further investigation as a targeted therapy for ATC. Introduction Thyroid cancer is the most common endocrine malignancy. Well-differentiated thyroid cancers make up the majority of thyroid cancers and have an excellent prognosis. In contrast, anaplastic thyroid cancer (ATC) is a rare type of undifferentiated thyroid cancer that makes up approximately 1% of thyroid cancer cases and is arguably the most lethal human malignancy[3C5]. Patients diagnosed with ATC typically present with a rapidly expanding neck mass resulting in airway and esophageal obstruction, and distant metastases[6,7]. Despite the aggressive use of chemotherapy, radiation and surgical resection, the outcomes for patients with ATC remain dismal, with a mean survival of only 6 months[6,8]. While there have been studies to date with the aim of understanding the molecular pathogenesis of disease, it is evident that ATC is still very poorly understood[9C11]. Presently, there are no effective therapies for patients diagnosed with ATC and therefore, the use of targeted agents directed against specific genetic alterations and signaling pathways remains an attractive cancer treatment strategy. Small-molecule tyrosine kinase inhibitors represent a molecularly-precise method of cancer treatment that can KN-92 phosphate be used to target specific signaling pathways and produce an antiproliferative effect[12,13]. Indeed, kinase inhibitors are undergoing active investigation in every major cancer DKK2 type and have been shown to KN-92 phosphate provide meaningful therapeutic responses in recurrent and metastatic diseases, with increased cure rates when administered concurrently KN-92 phosphate or in the adjuvant setting with surgery or radiation[14C16]. While a small number of targeted agents have been tested in patients with ATC, there are currently KN-92 phosphate no therapies that have been approved for routine treatment of ATC. To begin to fill the gap in our understanding of this disease and how it can be treated, we screened 13 ATC cell lines and identified Lestaurtinib as a highly potent agent with nanomolar potency. Efficacy of Lestaurtinib was further validated both and using the chick chorioallantoic membrane (CAM) xenograft model. Materials and methods Cell lines and culture conditions THJ-11T, -16T, -21T, and -29T were all obtained from Dr. John Copland of the Mayo Clinic. U-Hth7, U-HTh74cl.7, C643, and SW1736 cell lines were obtained from Dr. Nils Erik Heldin (University KN-92 phosphate of Uppsala, Sweden). Cell lines 8505C, ASH3 and KMH2 were all purchased from the Japanese Collection of Research of Bioresources Cell Bank (JCRB). Lastly, BHT-101 and CAL62 were both purchased from the DSMZ Cell Bank. THJ-11T, -16T, -21T, and -29T cell lines were cultured in RPMI 1640 media supplemented with 10% FBS (GIBCO), 1x non-essential amino acids (Wisent), 1 mM sodium pyruvate (Wisent), penicillin (100 g/mL) and streptomycin (100 g/mL) (Invitrogen). U-Hth7, U-HTh74cl.7, C643, SW1736 and 8505C cell lines were cultured in EMEM media supplemented with 10% FBS (GIBCO), penicillin (100 g/mL) and streptomycin (100 g/mL) (Invitrogen). ASH3 and KMH2 cell lines were cultured in a 1:1 mixture of DMEM and RPMI 1640, which was supplemented with 10% heat-inactivated FBS (GIBCO), penicillin (100 g/mL) and streptomycin (100 g/mL) (Invitrogen). BHT-101 and CAL62 cell lines were cultured in DMEM supplemented with 10% heat-inactivated FBS (GIBCO), 1% human serum (Wisent), penicillin (100 g/mL) and streptomycin (100 g/mL) (Invitrogen). Short tandem repeat (STR) profiling of ATC cell lines DNA was extracted from cultured cells using the AllPrep DNA/RNA/Protein kit (Qiagen), using the instructions provided by the manufacturer. A total of 100 ng of DNA per cell line was analyzed by short tandem repeat (STR) profiling at The Center for Applied Genomics (TCAG, Toronto, Canada). Cell lines were genotyped with 16 selected markers (including the 8 Combined DNA Index System (CODIS)) core STR loci, employed by the American Type Culture Collection (ATCC) and confirmed against published information (S1 Table). Drug selection A Beckman BioMek FX liquid handler was used to dispense cells into 384-well culture plates (Corning, NY, USA) at a density of 12,000 cells/ml in a total volume of 50 l/well. Seeded cells were incubated for 24 hours (h) at 37C, 5%.