Cell line culture
Kasumi-1 and KG-1α cell lines were stored in our own laboratory.Kasumi-1 was cultured in RPMI-1640 (HyCloneTM, Logan, UT, USA) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (1 × P/S) and 10% fetal bovine serum (FBS) (Hy Clone TM, Logan, UT, USA).KG-1α was cultured in IMDM (HyClone TM, Logan, UT, USA) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (1 × P/S) and 10% fetal bovine serum (FBS) (Hy Clone TM, Logan, UT, USA).
Patients and donor samples
Primary samples from bone marrow of AML patients (n = 9) and peripheral blood of healthy donors (n = 8) were collected in Department of Hematology, First Affiliated Hospital of Xiamen University after patients were informed consent. This study was carried out in accordance with the Declaration of Helsinki and approved by the Ethics Review Board of First Affiliated Hospital of Xiamen University, Clinical characteristics of patients with AML are summarized in Table 1. Mononuclear cells were isolated by density gradient centrifugation using Ficol (BD, Franklin Lakes, NJ, USA) and supplemented with RPMI1640 and 10% FBS for short culture.
Cell viability assay
Cell proliferation was determined by a CCK-8 kit (MCE, Shanghai, China). Briefly, cells (3 × 104 cells/well) were plated into 96-well plates containing 100 μl of growth medium and then treated with designated doses of anlotinib for 48h and 72h. CCK-8 agent was added and incubated for 2–4 h in an incubator after treatment and the absorbance at 450 nm were read by a VERSA max microplate reader (Molecular Devices, Sunnyvale, CA, USA). All experiments were repeated three times and performed in triplicate in each experiment.
Flow cytometry detection of apoptosis
Apoptosis of LSC or LSC-like cells was detected by flow cytometry after staining of Annexin V and PI following the manufacturer's instruction. In brief, cells exposed to different treatments were harvested and washed with ice-cold PBS (Gibco, USA) and 1 × Annexin V binding buffer and then subjected to Annexin V-FITC/PI staining. Flow cytometry analysis was performed within 1h after staining. Primary samples with spontaneous apoptosis over 30% without treatment were excluded. The specific apoptosis was adopted to adjust the variation in basal levels of spontaneous cell death following the formula: (% apoptosis in treated cells − % apoptosis in untreated cells)/(1 − % apoptosis in untreated cells) to analysis of the potential relationship between patients' characteristics and ex vivo efficacy of anlotinib,
Western blot analysis
Cells with 2 × 105/well were treated with anlotinib for 12 h. Apoptosis of cells were tested to control apoptotic rates no more than 10% before immunoblot assays. Hereafter cells mentioned above were lysed in RIPA solution containing protease inhibitor cocktails. The protein level of each sample was determined by a BCA protein Assay (Pierce, Thermo Scientific, USA). protein (20 μg/lane) was separated by SDS-PAGE, and transferred to a PVDF membrane (Millipore, UK), blocked by 5% non-fat milk, and incubated with primary antibodies included anti-c-KIT(18691-1-AP, proteintech,USA) and antibodies from Cell Signaling Technology, Danvers, MA, USA including anti-JAK-2(3230), anti-Jak-2 (3230),anti-Phospho-Jak-2(8082),anti-Stat3(30835), anti-Stat3(9339S), anti-Phospho-Stat3(4113S), anti-Stat5(94205S), anti-Phospho-Stat5(4322S,), anti-Bcl-2(4223S), anti-Bcl-xl(2764S), anti-BAX(5023S), anti- Phospho- c-KIT (3073S), and anti-GAPDH(D16H11). After incubation with HRP-conjugated secondary antibodies, the signals were detected using an enhanced ECL substrate (GE Healthcare, Chicago, USA) and visualized using the Amersham Imager 600 (AI600, GE Healthcare, Chicago, USA).
Mice models
All animal experiments were approved by the Animal Center of Xiamen University (Xiamen, China). Kasumi-1 cells (6×106) were subcutaneously injected into 6- weeks-old BALB/c male nude mice (Animal Center of Xiamen University, Xiamen, China). The tumor volume (V) was calculated using V=L*W2/2 (L: tumor length, and W: tumor width) twice a week. After tumor volume reached 50 mm3, mice were randomly divided into 2 groups (n =5/group) by oral gavage of vehicle or anlotinib (6mg/kg/d) for 1 week respectively. All mice were sacrificed after anlotinib exposure for 7 days.
Human AML xenograft experiments
Pre-treated bone marrow mononuclear cells of AML patients with anlotinib (2.5μM) or vechicle for 12h respectively in vitro. To exclude the impact of apoptotic cells on engraftment of LSCs, we detected apoptosis of the two groups. The equal accounts of alive cells were injected into tail vein of NOD-Prkdc-/-IL2rg -/-(NSG) mice (purchased from DMO ltd., Beijing, China) after 1Gy irradiation. In order to confirm the engraftment, the peripheral blood will be stained with human CD45.At the end of this experiment, the NSG mice will be culled to analyze the tumor burden with human CD45-FITC (clone HI30, Biolegend) and murine CD45-BV421 antibodies (clone 30- F11, Biolegend) as well as CD34-APC( clone 581,Biolegend ).
Statistical analysis
The value was expressed as the mean ± standard deviation (S.E.M) for at least three independent experiments. The Student t-test or Wilcoxon and Mann-Whitney tests were applied in two-group comparison according to distribution of the data. Multiple-group comparisons were conducted using the One-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. P<0.05 was considered statistically significant. All statistical analyses were performed using SPSS 22.0 software (La Jolla, CA).