Notch2 signal is required for the maintenance of canine hemangiosarcoma cancer stem cell-like cells

Abstract

BackgroundHemangiosarcoma (HSA) is a malignant tumor derived from endothelial cells which usually shows poor prognosis due to its high invasiveness, metastatic rate and severe hemorrhage from tumor ruptures. Since the pathogenesis of HSA is not yet complete, further understanding of its molecular basis is required.ResultsHere, we identified Notch2 signal as a key factor in maintaining canine HSA cancer stem cell (CSC)-like cells. We first cultured HSA cell lines in adherent serum-free condition and confirmed their CSC-like characteristics. Notch signal was upregulated in the CSC-like cells and Notch signal inhibition by a γ-secretase inhibitor significantly repressed their growth. Notch2, a Notch receptor, was highly expressed in the CSC-like cells. Constitutive activation of Notch2 increased clonogenicity and number of cells which were able to survive in serum-free condition. In contrast, inhibition of Notch2 activity showed opposite effects. These results suggest that Notch2 is an important factor for maintaining HSA CSC-like cells. Neoplastic cells in clinical cases also express Notch2 higher than endothelial cells in the normal blood vessels in the same slides.ConclusionThis study provides foundation for further stem cell research in HSA and can provide a way to develop effective treatments to CSCs of endothelial tumors.Electronic supplementary materialThe online version of this article (10.1186/s12917-018-1624-8) contains supplementary material, which is available to authorized users.

KDM2B promotes cell viability by enhancing DNA damage response in canine hemangiosarcoma

“…The HSA cell lines were donated by Dr. Sakai (Gifu University) 36 and cultured as described previously 37 . CnAOEC was purchased from Cell Applications (CA, USA), and ISO-HAS-B was received from Cell Resource Center for Biomedical Research Cell Bank in Tohoku University (Sendai, Japan) 38 .…”

“…qRT-PCR was performed with StepOne™ Real Time System (Thermo Fisher Scientific). The oligos for qRT-PCR were designed as described elsewhere 37,43 , and listed within Supplementary Table 2.…”

High drug efflux pump capacity and low DNA damage response induce doxorubicin resistance in canine hemangiosarcoma cell lines

“…They were cultured with Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich, MO, USA) containing 10% fetal bovine serum (FBS; Biowest, UT, USA), 100 unit/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, MA, USA) at 37°C with 5% CO2. To establish doxorubicin-resistant (DR) HSA cell lines, five hundred thousand cells were seeded in 10 cm cell culture dish (Falcon, NC, USA) and cultured in medium containing 10 nM doxorubicin (Wako, Osaka, Japan) dissolved in DMSO (Kanto Chemical Co., Inc., Tokyo, Japan) based on our previous work (Aoshima et al, 2018). The medium was replaced to the fresh one every 3 days until the surviving cells restarted proliferating and became 80-90% confluent.…”

“…Samples were prepared, separated and transferred to PVDF membranes as described previously (Aoshima et al, 2018). Membranes were either incubated with anti-ABCB1 antibody (#ab170904, 1:2,500, abcam, Cambridge, UK), anti-ABCG2 antibody (#ab3380, 1:1,000, abcam), anti-ATM antibody (#NB100-104, 1:1,000, Novus biologicals, CO, USA), anti-cleaved caspase-3 (Asp175) antibody (#9661, 1:1,000, Cell signaling, MA, USA) (Penzo-Méndez et al 2015) or anti-actin antibody clone C4 (#MAB1501, 1:10,000, Merck Millipore) for overnight at 4°C.…”

“…Primers and samples were prepared as described previously (Aoshima et al, 2018). qPCR was performed using the primers listed in Table 1.…”