Establishment and characterization of a novel childhood acute lymphoblastic leukemia cell line, HXEX-ALL1, with chromosome 9p and 17p deletions

Although contemporary chemotherapy has improved the cure rate of childhood acute lymphoblastic leukemia (ALL) to nearly 90%, relapsed/refractory ALL is still a leading cause of tumor-related death in children. To clarify the underlying mechanisms of relapsed/refractory childhood ALL, researchers urgently need to establish novel cell models from patients with relapsed ALL after treatment with contemporary chemotherapy. Cell culture technique was used to establish the HXEX-ALL1 cell line from primary B cell precursor ALL (BCP-ALL) cells. Molecular and cellular biological techniques including flow cytometry, polymerase chain reaction (PCR), short tandem repeat (STR) analysis, conventional cytogenetics, and chromosomal microarray analysis (CMA) were used to characterize the HXEX-ALL1 cell line. Nude mice were used for xenograft studies. A stable ALL cell line, HXEX-ALL1, derived from a 6-year-old boy of Han nationality with BCP-ALL at the second relapse, was established and maintained in culture for more than 18 months. The HXEX-ALL1 cell line was authenticated as being derived from primary leukemia cells based on morphologic, immunophenotypic, cytogenetic and STR analyses and demonstrated tumorigenicity in nude mice. WGS data showed that there were 27,006 novel single nucleotide polymorphisms (SNPs) and 193,951 novel insertion/deletions (InDels) in HXEX-ALL1 cells. Compared with the other BCP-ALL cell lines in use, the HXEX-ALL1 cells have a special karyotype represented by trisomy 8 and 9p and 17p deletions with a multidrug resistance phenotype, especially highly resistant to asparaginase. The HXEX-ALL1 cell line may prove to be a useful model for the study of relapsed/refractory childhood ALL, particularly for the researches on asparaginase resistance.


Background
Cell culture has been performed for more than a century [1][2][3]. Cell lines established from tumors are the most commonly used models in tumor research and have enabled a better understanding of the molecular mechanisms of different tumors and provided the means to develop effective treatment strategies [4]. In addition to primary patient materials and animal models, tumor cell lines are one of the three major models for cancer research [4]. The first human cell line was HeLa, established in 1951 by Gey [5]. In 1955, the first human leukemia cell line was derived from a patient with acute myeloid leukemia (AML) by Osgood and Burke [6]. Unfortunately, this cell line soon became contaminated with HeLa cells [7]. Then, the Raji cell line was established as the first leukemia-lymphoma cell line in 1963 by Pulvertaft [8]. Currently, 637 characterized leukemia-lymphoma cell lines are in use [3]. The leading source of leukemia-lymphoma cell lines is Japan (40%), followed by the USA (26%), and only 4 leukemia-lymphoma cell lines have originated in China [3]. Additionally, there are no pediatric leukemia cell lines from China in the ATCC and DSMZ cell banks.
Acute lymphoblastic leukemia (ALL), a malignancy originating from a B or T cell progenitor, is the most common leukemia in children, accounting for approximately 80% of all pediatric leukemia [9]. Fortunately, due to better risk stratification and the advent of multidrug risk-adapted chemotherapy regimens, improved central nervous system prophylaxis and better supportive care, the cure rates are approximately 90% for pediatric patients with ALL [9][10][11][12][13][14]. For patients enrolled in the CCLG-ALL 2008 protocol in China, the 5-year survival rate was 85.3% [15]. However, 10-15% of patients still relapsed and had a dismal prognosis [14,16]. Due to the high morbidity of ALL, relapsed/refractory ALL is still the leading cause of tumor-related death in children [14,16]. Thus, it is very important to clarify the underlying mechanisms of relapsed/refractory ALL. However, to the best of our knowledge, there are no leukemia cell lines derived from pediatric patients relapsed after treatment with contemporary chemotherapy.
In this study, we reported a novel B cell precursor ALL (BCP-ALL) cell line called HXEX-ALL1, which was established from the Bone marrow (BM) of a 6-year boy with BCP-ALL during his second relapse in our hospital. The cell line was authenticated as being derived from primary leukemia cells and displayed tumorigenicity in nude mice. The cell line had a Complex karyotype (CK; with ≥ 3 structural chromosomal abnormalities) with trisomy 8 and 9p and 17p deletions, and displayed a multi-drug resistant phenotype with highly resistant to l-asparaginase (L-Asp). The phenotypic, genetic and functional properties of this cell line were described following the guidelines for the characterization and publication of human malignant hematopoietic cell lines [17].

Cell culture
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 × 10 6 /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 (CO 2 ). The medium was replaced every 3-5 days depending on the cell growth rate to maintain the cells at a density of 1-3×10 6 /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 0.5-2×10 6 /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 × 10 5 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% CO 2 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).

G-banding analysis
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% SDSpolyacrylamide 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

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 × 10 5 / 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(N t /N 0 ), where t is the time of continuous culture, N t is the final number of cells, and N 0 is the initial number of cells.

Cell cycle analysis
For each analysis, 10 6 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 DNAsefree 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.

Animal experiments
Cultured 1 × 10 7 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.

Statistical analysis
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.

Establishment of the HXEX-ALL1 cell line
A stable and prominent cell population was observed following 8 weeks of culture of primary BM mononuclear cells. The cells were maintained in fresh medium (RPMI-1640 medium containing 10% FBS) replaced at 2-to 3-days intervals. The obtained cells were designated HXEX-ALL1 and made available. The HXEX-ALL1 cells had been in culture for 18 months. The Doubling time (Td) was 26-32 h. The cells proliferated consistently and were negative for EBV, HCMV, HBV, HCV, HIV, and mycoplasma infection based on PCR. The cells could be frozen under standard conditions using 60% RPMI-1640 medium, 30% FBS and 10% dimethyl sulfoxide (DMSO) and successfully revived after storage in liquid nitrogen, with more than 80% viability. The cells maintained the same properties after freezing and thawing.

Morphological characteristics of HXEX-ALL1 cells
The HXEX-ALL1 cells grew in suspension as single cells or clumps resembled the morphology of lymphoblastic leukemia cells in the patient's BM smear at diagnosis and second relapse (Fig. 1a, b). The cells were usually round in shape, and the cytoplasm was weakly basophilic with occasional vacuoles; most cells contained only one nucleus that was commonly round; the nucleocytoplasmic ratio of these cells was high (Fig. 1c). Mitotic figures were usually observed. Transmission electron microscopy revealed that most cells had a single, large and eccentric nucleus with a fine chromatin network. Few organelles were present in the cytoplasm, except for some mitochondria and ribosomes (Fig. 1d).

Gross chromosomal alterations in HXEX-ALL1 cells
Chromosomal analysis was carried out using the G-banding technique and demonstrated the following karyotype for primary leukemia cells: 47, XY, +8, del(9p22), del(17p12) (Fig. 2a). Because of the failure of G-banding analysis in HXEX-ALL1 cells, CMA was performed after 10 months of culture. The results indicated a gain of chromosome 8, a 38.56-Mb loss of 9p24.3-p13.1 and a 16-Mb loss of 17p13.3-p11 in all HXEX-ALL1 cells (Fig. 2b), consistent with the results of whole genome sequencing. Therefore, the karyotypes of the HXEX-ALL1 cells and primary leukemia cells from the patient were virtually identical. The protein expression levels of p16 ARF, p14 INK4A and Pax5, encoded by the corresponding genes located on chromosome 9p, were low in HXEX-ALL1 cells (Fig. 2c).

Identity verification of HXEX-ALL1 cells
Using a multiplex STR system, which enables the detection of unique DNA fingerprints through the genotyping of 22 STR loci, the primary leukemia cells and HXEX-ALL1 cells were found to share 100% identity. These c Cells were lysed, and extracts were analyzed by western blotting for p16 ARF, p14 INK4A and Pax5. β-Actin was used as an internal control. Bar graphs show the ratio of protein to β-Actin. Experiments were performed in triplicate results confirmed that the genetic source of HXEX-ALL1 cells was, in fact, the patient's leukemia cells (Table 1). The HXEX-ALL1 cell line profile did not match any other cell line profile in the current databases from ATCC, DSMZ or elsewhere.

Proliferation of HXEX-ALL1 cells
We determined the proliferation for 8 days at different cell Population doubling levels (PDLs) of HXEX-ALL1 cells (Fig. 3a). The results revealed that HXEX-ALL1 cells stably proliferated in RPMI-1640 medium containing 10% FBS with a population Td of 26-32 h. The percentage of cells in G 0 /G 1 , G 2 /M and S phase were similar without significant differences (P > 0.05) at different PDLs (Fig. 3b), suggesting that the HXEX-ALL1 cells could stably maintain the cell division cycle during continuous culture in vitro.

WGS data of HXEX-ALL1 cells
The WGS analysis resulted in a mean of 2,359,620,422 clean reads. There were 3,357,771 single nucleotide polymorphisms (SNPs), 847,741 insertion/deletions (InDels), 3093 copy number variations (CNVs) and 6896 structural variations (SVs). Among them, 27,006 SNPs and 193,951 InDels were novel when compared with dbSNP and the 1000 Genomes Project databases.

Discussion
Improvements in the prognosis of childhood ALL represent one of the most successful stories in modern medicine. Moreover, this improvement has taken place not only in developed countries but also in developing countries because conventional chemotherapy is inexpensive and readily available worldwide [12][13][14]. The major reason for the improvement in prognosis is the reduction in the risk of relapse as a result of risk-directed chemotherapy [12][13][14]. However, the management of relapsed and refractory ALL is still difficult due to chemoresistance. Despite efforts to intensify re-induction strategies, including allogenic hematopoietic stem cell transplantation, the Overall survival (OS) rates for patients with relapsed ALL remain between 25 and 40%, highlighting the need for researches on new promising drugs, such as immunotherapeutic agents [19]. However, the mechanisms underlying ALL progression, relapse and resistance remain elusive. Thus, the establishment of new cell lines derived from patients who are refractory to contemporary chemotherapy would facilitate the elucidation of the biological properties of relapsed and refractory ALL and development of novel therapeutic strategies. However, the establishment of a hematopoietic malignancy cell line from the BM is still challenging.  19:113 Currently, there are 637 characterized leukemia-lymphoma cell lines, and among them only 4 are from China. The majority of these cell lines were established from 1977 to 1997 [3]. Contemporary chemotherapeutic protocols have improved the cure rate of pediatric ALL to nearly 90% in the last 2 decades [9][10][11][12][13][14]. Nevertheless, to the best of our knowledge, there are no cell lines derived from patients who are resistant to contemporary chemotherapeutic protocols, highlighting the need for novel cell lines derived from relapsed and refractory patients. Fortunately, we established a novel cell line, HXEX-ALL1, from a Chinese boy of Han ancestry with a second relapse of BCP-ALL. All major features of the HXEX-ALL1 cell line are summarized in Table 2. As authenticated by the morphologic, immunologic, cytogenetic and STR analyses, the HXEX-ALL1 cells are derived from the patient's primary leukemia cells. Thus far, the HXEX-ALL1 cell line has been continuously grown in culture for over 18 months (over 400 PDL) with a Td of 26-32 h and has maintained stable proliferation and cytogenetic features. Therefore, the HXEX-ALL1 cells are representative of patient leukemia cells. More importantly, compared with the other BCP-ALL cell lines in use [20,21], the HXEX-ALL1 cells have a special karyotype represented by trisomy 8 and 9p and 17p deletions and display a multidrug resistant phenotype with highly resistant to L-Asp. Moreover, WGS data showed that there were 27,006 novel SNPs and 193,951 novel InDels in HXEX-ALL1 cells.

Table 1 STR analysis of HXEX-ALL1 cells, primary leukemia cells and cells from the tumor mass
Gross chromosomal alterations are a hallmark of ALL [22]. Cytogenetic risk is used to classify patients into low-, intermediate-or high-risk groups. As defined by the suggestion of the Subspecialty Group of Pediatric Hematology, of Chinese Medical Association (2014), patients with a Philadelphia (Ph) chromosome (encoding the BCR-ABL1 fusion gene) and MLL rearrangements are classified into the high-risk group, patients with E2A-PBX1 rearrangements in the intermediate-risk group, and other patients into the low-risk group [23]. The list of genetic alterations in childhood ALL that are associated with the The growth curve of subcutaneous tumors in nude mice (n = 3, age 5-6 weeks). All animal procedures were carried out in accordance with the guidelines established by the internal Institutional Animal Care and Use Committee and Ethics Committee guidelines of Sichuan University. c HE staining of the tumor showed that the tumor mass was composed of leukemia cells and blood vessels. Original magnification: ×200 magnification risk of relapse continues to grow. Ph chromosome, BCR-ABL1 like with IZKF1 and JAK2 alterations, MLL rearrangements, hypodiploidy (< 44 chromosomes or DNA index < 0.81), and iAMP are assigned as unfavorable characteristics [24]. In contrast, hyperdiploidy with trisomy 4 and 10 and ETV6-RUNX1 fusion are designated favorable genetic factors [24]. The results of G-banding analysis in the patient showed that the primary leukemia cells had 3 gross structural chromosomal abnormalities, namely, trisomy 8, and 9p and 17p deletions, but no unfavorable characteristics at diagnosis. The patient was classified into the low-risk group at initial CR. Unfortunately, the patient experienced relapse 4 months after the CR and achieved a second CR after re-induction chemotherapy but then relapsed again after 3 months.
A CK with ≥ 3 structural chromosomal abnormalities is not listed among the criteria of risk classification for childhood ALL. However, a CK with ≥ 4 structural chromosomal abnormalities is predictive of poor outcomes in adult AML patients without favorable or adverse aberrations [25]. A study of 428 adult patients with Ph-negative ALL demonstrated that a CK is an adverse prognostic factor independent of the MRD status, but the definition of CK should include ≥ 5 chromosomal abnormalities [26]. The MRC UKALL XII/ECOG E2993 study enrolled 1522 adult patients with ALL and demonstrated that patients with a CK comprising ≥ 5 chromosomal abnormalities had an inferior prognosis [27]. A study of 79 pediatric patients with ALL indicated that a CK with ≥ 3 chromosomal abnormalities should be considered a poor prognostic factor in childhood ALL [28]. Nevertheless, the poor prognosis associated with CK remains controversial; some other studies have indicated that a CK does not have a significant impact on either OS or event-free survival in ALL [29][30][31].
Additionally, deletion of 17p, a segment containing the TP53 gene, is an independent risk factor in adult patients with AML and chronic lymphocytic leukemia (CLL) [25,32,33]. In adult ALL, abnormal 17 alterations including 17p deletions may be a risk factor in T cell precursor ALL (TCP-ALL) [34,35] but not in BCP-ALL [27]. In childhood BCP-ALL, 17p deletions predict a poor prognosis and a higher rate of relapse in patients without abnormalities in ETV6-RUNX1 or hyperdiploidy [36].
Deletion of 9p is a common chromosomal alteration detected since 1983 [37]. Chromosome 9p contains numerous tumor-associated genes, such as PAX5 at 9p13.2, CDKN2A, CDKN2B, MTAP, IFN, MLLT3, PTP-LAD2 at 9p21.3, and JAK2 at 9p24.1 [38,39]. However, the impact of 9p deletions on ALL outcomes is contradictory. Deletion of 9p is associated with favorable prognosis in adult patients with Ph-negative ALL [27] and unfavorable prognosis in patients with Ph-positive ALL [40]. In childhood ALL, some studies have shown that 9p deletions have no association with outcomes [36,41,42]. In contrast, other studies have indicated that 9p deletions predict poor prognoses and risk of relapse in both BCPand TCP-ALL [43][44][45][46]. A recent study on relapsed childhood ALL indicated that 9p deletions are associated with relapse [47]. In the refined risk classification of childhood ALL, favorable genetic features include ETV6-RUNX1, hyperdiploidy, lack of deletion of IKZF1, CNKN2A/B, PAR1, BTG1, EBF1, PAX5, ETV6 or RB1, isolated deletions affecting ETV6/PAX5/BTG1, and deletions of ETV6 with an additional deletion of BTG1/PAX5/CDKN2A/B; other genetic features are classified as risk factors [48]. PAX5 and CDKN2A/B genes are located on chromosome 9p. The protein expression levels of Pax5, p16 ARF and p14 INK4A, encoded by PAX5 and CDKN2A/B genes, were low in HXEX-ALL1 cells compared with the NALM-6 and Raji cells. Most recently, novel subtypes of ALL based on diverse PAX5 alternation, including PAX-5alt, PAX5 p.Pro80Arg, or PAX5-plus, were reported [49][50][51]. PAX5-plus patients, with PAX5 p.Pro80Arg as hotspot and biallelic genomic alterations, had a favorable treatment outcome in trials of population-based German study cohorts [50]. In contrast, the outcome for PAX5 p.Pro80Arg in children treated on St. Jude Total Therapy protocols was unfavorable [49]. PAX5alt patients, with homozygous or heterozygous PAX5 alterations, including rearrangements, sequence mutations and focal intragenic amplifications, had an intermediate outcome in the Children's Oncology Group AALL0232 study [49]. In this study, PAX5 p.Pro80Arg patients had an intermediate outcome also [49]. Therefore, 9p deletion might play a complex and paradoxical role in relapsed ALL patients.
Trisomy 8 is reported in 10-15% of adult AML patients, 9-12% of adult ALL, and 2-10% of childhood ALL, mainly as a part of hyperdiploid karyotypes or CKs with structural abnormalities [25,26,52,53]. The prognosis of AML patients with trisomy 8 depends on whether it occurs as an isolated abnormality or is accompanied by other aberrations [54]. Trisomy 8 alone had no significant impact on the prognosis of adult ALL [27]. However, the relationship between trisomy 8 and prognosis in childhood ALL is still unknown [54]. Increased attention should thus be paid to the impact of CK, trisomy 8, 9p and 17p deletions on the prognosis of ALL.
Furthermore, accompanying with the special karyotype, HXEX-ALL1 cell line shows a multidrug resistant phenotype with resistant to DNR, MTX, Ara-c, and L-Asp. The outstanding characteristic of HXEX-ALL1 is that the IC 50 to L-Asp was higher than 100 nM after 48 h treatment, and were 409.8-fold more resistant than NALM-6 cells. Over the past 50 years, Asp has become a key chemotherapeutic agent for ALL and has led to a substantial improvement in cure rates, especially in children [55,56]. Asp resistance plays a main role in the treatment failure of ALL [56]. The underlying mechanism of ALL cells resistant to Asp remains unclear. However, the researches on Asp resistance are far fewer than the researches on glucocorticoid resistance. One important reason is that there are some glucocorticoid resistant ALL cell model, but few Asp resistant model.

Conclusions
To the best of our knowledge, HXEX-ALL1 is the first BCP-ALL cell line derived from a pediatric patient who relapsed after receiving appropriate contemporary chemotherapy. The novel BCP-ALL cell line resembles the primary leukemia cells at diagnosis. Moreover, the novel cell line has 27,006 novel SNPs and 193,951 novel InDels. Compared with the other BCP-ALL cell lines in use, the HXEX-ALL1 cells have a special karyotype represented by trisomy 8 and 9p and 17p deletions with multi-drug resistant phenotype, especially highly resistant to L-Asp. Thus, HXEX-ALL1 may be a valuable cell model for further investigation on the mechanisms and potential targeted therapy of relapsed/ refractory ALL, particularly for those developed a dominantly resistant to Asp. Meanwhile, the HXEX-ALL1 cell line represents a useful model to explore the impact of 9p and 17p deletions on the pathogenesis and prognosis of ALL and to develop new target drugs.