The HXEX-ALL1 cell line was derived from a 6-year-old Chinese boy of Han ancestry with BCP-ALL. The patient was admitted to West China Second University Hospital (Chengdu) in 2016 because of podalgia and hemorrhage under the skin. Physical examination upon admission revealed pale lips and enlarged superficial lymph nodes. Complete blood count revealed a white blood cell count of 22.9 × 109/l with 60% blast cells, hemoglobin level of 105 g/l, and platelet count of 52 × 109/l. BM examination revealed hypercellular marrow with 92% blasts that were negative for peroxidase staining. The primary leukemia cells were positive for CD10, CD19, CD22, cCD79 and HLA-DR, partially positive for CD5, and negative for CD20, sIgM, cIgM, CD2, CD3, CD7, cCD3, CD13, CD33, CD117 and CD34 and were thus categorized as the common B subtype according to the EGIL classification . G-banding analysis of the BM revealed the karyotype 47, XY, +8, del(9p22), del(17p12). FISH analysis demonstrated negative expression of MLL, BCR-ABL, ETV6-RUNX1 and PDGFRB fusion genes. Multiple real-time polymerase chain reaction (RT-PCR) analyses indicated negativity for the following fusion genes: MLL-AF4, MLL-AF6, MLL-AF10, TEL-AML1, MLL-ENL, BCR-ABL P210, BCR-ABL P190, SIL-TAL, E2A-HLF, CALM-AF10, HOX11, HOX11L2, SET-CAN, TEL-ABL1, TLS-ERG, NPM-ALK and E2A-PBX1. The patient received chemotherapy according to the Chinese Childhood Cancer Group ALL 2015 (CCCG-ALL-2015) protocol. The regimen included dexamethasone (DEX), pegaspargase (Peg-Asp), vincristine (VCR), daunorubicin (DNR), cyclophosphamide (CTX), cytarabine (Ara-c), mercaptopurine (6-MP), and methotrexate (MTX). After 19 days of chemotherapy, the proportion of blasts in the BM was reduced to 1%, demonstrating complete remission (CR) and negative minimal residual disease (MRD) (< 0.01%). The patient was classified into a low-risk group. However, he experienced BM relapse after 4 months, and re-induction of chemotherapy led to another CR 1 month later. The re-induction chemotherapy regimen included DEX, mitoxantrone (MTZ), vindesine (VDS), Peg-Asp, MTX, etoposide (VP-16), and Ara-c. Unfortunately, the patient experienced a second BM relapse in 3 months, and this time, chemotherapy did not lead to a CR.
Primary BM cells were obtained from the patient at the second relapse of ALL. The patient provided informed consent. Mononuclear cells were isolated and separated by Ficoll-Hypaque centrifugation and cultured in a 6-well plate (Corning Inc., Corning, NY, USA) at a density of 6 × 106/ml in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 20% fetal bovine serum (FBS; Thermo, Grand Island, NY, USA) and 10 ng/ml rhIL-3 at 37 °C with 5% carbon dioxide (CO2). The medium was replaced every 3–5 days depending on the cell growth rate to maintain the cells at a density of 1–3×106/ml. The cells were examined daily under an inverted microscope, and the cell number was determined every 3 days with a standard hematocytometer using trypan blue dye exclusion. After 3 weeks of lag phase, the cell number dramatically increased, and the cell density was adjusted to 0.5–2×106/ml. After 5 weeks, rhIL-3 was omitted from the complete medium, and the FBS concentration was reduced to 10% in the complete culture medium after 7 weeks. For subcloning, 1 × 105 cells were seeded in MethoCult GFH4434 (Sigma, St. Louis, MO, USA) medium in six-well culture plates and incubated for 7–10 days. Colonies were extracted and cultured in RPMI-1640 medium. In this study, other leukemia-lymphoma cell lines, such as NALM-6, CCRF-CEM, and Raji, were purchased from Shanghai Institute Cell Resources Bank. All cell lines were maintained in RPMI 1640 supplemented with 10% FBS, at 37 °C in a humidified 5% CO2 in-air atmosphere.
Cell morphologic assay
Morphological characteristics of live cultured cells were observed under an inverted microscope (Olympus, Tokyo, Japan). Smears of BM and HXEX-ALL1 cells were stained with Wright-Giemsa and observed under an optical microscope (Olympus). Cell ultrastructures were observed under a transmission electron microscope (JEOL Ltd., Tokyo, Japan).
For the detection of the immunophenotype of the patient sample and HXEX-ALL1 cells, we used antibodies against the following targets: CD34, HLA-DR, CD38, CD117, CD56, CD19, CD20, CD79α, cCD79α, CD10, cIgM, sIgM, TdT, CD7, CD3, cCD3, CD5, CD4, CD8, CD2, MPO, CD33, CD13, CD11b, CD64, CD36, CD14, CD15, CD71, CD61, CD41, CD65, and CD45 (Becton–Dickinson Inc., Franklin Lakes, NJ, USA). Positivity for the antigens was determined using a FACSCalibur flow cytometer (Becton–Dickinson Inc.).
Chromosomes were prepared by a standard method and analyzed by the G-banding technique. Karyotype was determined according to the International System for Human Cytogenetic Nomenclature (ISCN, 2013).
Chromosomal microarray analysis
Genomic DNA was extracted with a Qiagen DNeasy Blood Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instructions. Chromosomal microarray analysis (CMA) was performed using Affymetrix CytoScan HD arrays (Affymetrix Inc., Santa Clara, CA, USA). The data were collected and analyzed using the Affymetrix GeneChip Microarray Instrument System (Affymetrix Inc.).
Western blotting analysis
Western blotting analysis was performed on lysates obtained from HXEX-ALL1, NALM-6, CCRF-CEM, and Raji cells. Proteins were separated by 15% SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (0.22 μm, Millipore, Billerica, MA, USA). Proteins were visualized by incubation with ECL plus reagent (Millipore). All experiments were independently carried out at least 3 times. The level of β-actin protein was used as a control for the amount of protein loaded onto each lane.
Ig and TCR arrangement analysis
Immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements analysis were detected by PCR. Genomic DNA was analyzed using multiplex primers designed against IgVH-A (FR1-JH), IgVH-B (FR2-JH), IgVH-C (FR3-JH), IgDH-A (DH1-6-JH), IgDH-B (DH7-JH), Igκ (Vκ-Jκ), Igλ (Vλ-Jλ), TCRB A, TCRB B, TCRB C, TCRG A, TCRG B and TCRD. The PCR mixture included the GoTaq Green Master Mix (Promega, Madison, WI, USA), primer mix and genomic DNA. PCR products were visualized using agarose gels stained with SYBR Green I.
Short tandem repeat analysis
The identity of the HXEX-ALL1 cell line was checked using short tandem repeat (STR) analysis against a BM sample taken from the patient. DNA was prepared from whole BM and HXEX-ALL1 cells using a Qiagen DNeasy Blood Kit (Qiagen) according to the manufacturer’s instructions. The following 22 highly polymorphic STR loci were tested by multiplex PCR: Amelogenin, CSF1PO, D13S317, D16S539, D5S818, D7S820, TH01, TPOX, vWA, Penta E, Penta D, D2S441, D2S1338, D3S1358, D6S1043, D8S1179, D10S1248, D12S391, D18S51, D19S433, D21S11 and FGA.
Whole genome sequencing analysis
The whole genome sequencing (WGS) was conducted according to the BGISEQ-500 protocol. Clean reads were aligned to the human reference genome (GRCh37/HG19) using the Burrows-Wheeler Aligner (BWA).
Cell growth assay
Cells were cultured in a 6-well round-bottom plastic culture plates (Corning Inc., Corning, NY, USA) at 6 × 105/ml in RPMI-1640 medium with 10% FBS and grown for 8 days. Viable cells were counted using trypan blue (Sigma) staining every day. Td was calculated for cells in exponential growth with the following equation: Td (h) = t×lg2/lg(Nt/N0), where t is the time of continuous culture, Nt is the final number of cells, and N0 is the initial number of cells.
Cell viability and in vitro drug sensitivity assay
Cell viability was evaluated by the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The chemotherapeutic drugs, Dex, L-Asp, VCR, DNR, Ara-C and MTX, were purchased from Sigma. Cells were cultured in the presence of 6 different concentrations of Dex (0.0001–0.1 μM or 0.01–5 μM), L-Asp (0.001–1 U/ml or 0.01–500 U/ml), VCR (0.1–0.6 nM or 0.2–1.2 nM), DNR (1–50 nM), Ara-C (5–100 nM or 50–800 nM) and MTX (2–20 nM or 5–50 nM) for 48 h, respectively, followed by assessment of cell viability by MTT assay. Drug sensitivity was assessed by the IC50, drug concentration that inhibits 50% of cell viability. The IC50 was calculated by linear interpolation.
Cell cycle analysis
For each analysis, 106 cells were harvested and fixed overnight in 70% ethanol at 4 °C. The cells were then washed and stained with 5 μg/ml PI in the presence of DNAse-free RNAse (Sigma). After 30 min at room temperature, the cells were analyzed via flow cytometry (Beckman Coulter Inc., Miami, FL, USA) with the acquisition of 30,000 events.
Cultured 1 × 107 HXEX-ALL1 cells were subcutaneously injected into the right flanks of 6-week-old female BALB/c (nu/nu) nude mice, with 0.2 ml of PBS injected into the left flanks as the control (n = 6). Tumor size was measured by calipers every 2 days. The approximate tumor volume was calculated using the equation V = (length × width × depth)/2. All animal care was in compliance with the guidelines established by the internal Institutional Animal Care and Use Committee and Ethics Committee of Sichuan University. After the mice were euthanized, the tumor mass was excised, fixed in 10% formalin, and routinely processed for paraffin embedding. Five-millimeter-thick sections were obtained and prepared for standard histopathological examination.
All assays were performed in triplicate, and the data were expressed as the mean value ± SD. One-way ANOVA was used to compare two groups. A P-value < 0.05 was considered to be significant.