Stable Instability of Sarcoma Cell Lines Genome Despite Intra-Tumoral Heterogeneity: A Genomic and Transcriptomic Study of Sarcoma Cell Lines

Research Article

Austin J Genet Genomic Res. 2015; 2(2): 1014.

Stable Instability of Sarcoma Cell Lines Genome Despite Intra-Tumoral Heterogeneity: A Genomic and Transcriptomic Study of Sarcoma Cell Lines

Lagarde P1,2, Brulard C¹, Pérot G¹,³, Mauduit O¹, Delespaul L¹, Neuville A1,3, Stoeckle E4, Le Guellec S5, Rochaix P5, Coindre JM1,2,3 and Chibon F1,3*

¹INSERM U916, France

²Bordeaux University, France

³Department of Biopathology, Institut Bergonié, Comprehensive Cancer Centre, France

4Department of Surgery, Institut Bergonié, Comprehensive Cancer Centre, France

5Departement of Pathological Anatomy and Cytology, Oncopole, 1 avenue Irène Joliot-Curie, France

*Corresponding author: Frédéric Chibon, Tumor Genetics - Department of Biopathology and INSERM U916, Genetics and Biology of Sarcomas, Insitut Bergonié, 229 cours de l’Argonne, 33076 Bordeaux, France

Received: October 13, 2015; Accepted: December 04, 2015; Published: December 07, 2015

Abstract

Background: Sarcoma oncogenesis is still poorly understood and functional studies are hampered by the dearth of cell lines representative of diverse sarcoma types. The existing models suffer from two main pitfalls: lack of representativeness of the tumors from which they were derived and lack of information about their evolution over passages. There is therefore a pressing need to generate new cell lines.

Methods: All sarcoma tumors from patients receiving surgery in a large tertiary referral center in France were cultured to establish the corresponding cell lines. We performed comparative genomic and transcriptomic studies of the original tumor and cell lines to evaluate the representativeness of the cell line to the original tumor and evolution over passages.

Results: Pleomorphic sarcomas are genetically heterogeneous. As a consequence, cell lines derived from that kind of tumor developed from selected clones roughly representing the initial tumor. More importantly, our results show that there are no genetic imbalances and transcription modifications along passages.

Conclusion: Even if pleomorphic sarcomas are genetically unstable at the cellular level, they appear to be genetically stable at the multicellular one, and therefore remain representative of the initial tumor even after passages. We established a sarcoma cell line panel gathering 32 cell lines with genomic, transcriptomic and clinical data, which will be significant to understand genomic alterations, sarcoma biology and to manage preclinical studies and clinical trials.

Keywords: Cell lines; Heterogeneity; Instability; Sarcoma

Introduction

Soft Tissue Sarcomas (STS) are a heterogeneous group of mesenchymal tumors that account for 1-2% of all cancers. More than 100 types and subtypes of sarcomas are listed by the World Health Organization (WHO). Metastatic risk depends on histological type and varies from 20% to 60% [1], meaning that an accurate initial diagnosis is essential for patient clinical management.

Based on cytogenetic and Comparative Genomic Hybridization (CGH) data, sarcomas can be divided into two main groups: one characterized by known alterations such as translocation t(11;22) (q24;q12) in Ewing sarcomas [2], t(X;18) in synovialosarcomas [3,4], mutation in Gastrointestinal Stromal Tumors (GIST) [5,6] or amplifications as MDM2/CDK4 in dedifferentiated and welldifferentiated liposarcomas [7,8]; and a second group of sarcomas without known alterations and with complex genetics, including leiomyosarcomas and undifferentiated pleomorphic sarcomas [9-11].

Although considerable genomic and transcriptomic data are currently available for sarcomas [11-14], oncogenesis is still poorly understood. In order to better understand sarcoma biology, to determine the involvement of a gene and to develop a therapeutic target, genomics-guided functional genetic studies are necessary. Yet, functional studies in sarcomas are hampered by the dearth of appropriate models. Only a limited number of human sarcoma cell lines exist, in part because of the rarity of certain diagnoses and resulting scarcity of samples. Moreover, for each of the subtypes with complex genomes, multiple cell lines are needed to represent the diversity of genetic alterations within that subtype. Several large-scale projects now aim to genetically characterize large numbers of human cancer cell lines and screen these against a range of anticancer therapies to correlate drug sensitivity with genetic markers [15]. Among these are the Cancer Cell Line Encyclopedia and the Sanger Cancer Cell Line Project. The Sanger project is assembling approximately 800 cell lines, of which only 10 (1.3%) represent complex soft-tissue sarcomas. As a result, there is a real need to generate cell lines representative of diverse sarcoma types, mainly for the subtypes with complex karyotypes. The creation of a sarcoma cell line panel with genomic and transcriptomic profiles that mirror the diversity observed in their corresponding tumor types would represent a critical step in understanding the influence of heterogeneity on variability of response to targeted therapies [16]. Such a panel could also drive genomics-guided functional genetics, either with arrayed or pooled loss-of-function RNAi screens [17,18], or ‘ORFeome’ approaches [19,20].

To address this issue, we cultured sarcoma tumor cells from every patient treated by surgery at the Institut Bergonié, a large tertiary referral center in France. Among the 32 established cell lines, we performed genomic and transcriptomic studies on seven of them to evaluate the representativeness of the original tumor and the evolution over more than 50 passages.

Materials and Methods

Ethics statement

The samples used in this study were part of the Biological Resources Center of Bergonie Cancer Institute (CRB-IB). In accordance with the French Public Health Code (articles L. 1243-4 and R. 1243-61), the CRB-IB received the agreement from the French authorities to deliver samples for scientific research (number AC-2008-812, on February 2011). These samples were obtained from regular patient care and requalified for research. Patients provided written informed consent approved by the Committee of Protection of Individuals.

Establishing a cell line

Following surgical resection, fresh tumor tissue was minced with scissors and then digested with 200 IU/ml type II collagenase (Roche) in serum-free RPMI 1640 with glutamax, supplemented with antibiotics (Gibco BRL, Life Technologies) overnight. After digestion, isolated cells and pieces were washed and seeded in a 25cm2 plastic flask containing culture medium, and maintained in a humidified atmosphere of 5% CO2 at air temperature of 37oC. The culture medium was composed of a RPMI 1640 with glutamax (Gibco BRL, Life Technologies, Cergy Pontoise, France) supplemented with 10% fetal calf serum and 1% antibiotics (penicilline /streptomycine, Gibco BRL, Life Technologies, Cergy Pontoise, France). All cell lines were tested for mycoplasma by Polymerase Chain Reaction (PCR) (Sigma; Look Out Mycoplasma PCR Detection Kit) according to manufacturer’s recommendations.

Cell sorting by flow cytometry

Cells from a culture flask were collected into a conical tube and centrifuged at 400g for 5 min. The supernatant was discarded and the pellet was resuspended in medium (cell culture medium Or Phosphate-Buffered Saline [PBS] with 1% bovine serum albumin). Cells were counted and resuspended at an appropriate concentration, in the range of 106–107 per mL.

The pellet was resuspended in 5 mM EDTA (ethylene-diaminetetra- acetic-acid). Cells were isolated by FACS Aria [BD Biosciences, San Jose, CA], every cell isolated was placed in a well of 96-wells plate.

Nucleic acid isolation

DNA from the cell lines and from snap-frozen tumors was isolated for Comparative Genomic Hybridization (CGH). Genomic DNA was isolated with a standard phenol-chloroform extraction protocol after Rnase treatment. Total RNA for gene expression studies was extracted from cell lines (before passage 20 and after passage 30) and from frozen tumor samples with TRIzol reagent (Gibco BRL, Life Technologies) and purified with the RNeasy Min Elute Cleanup Kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s procedures. We checked RNA quality on an Agilent 2100 bioanalyzer (Agilent Technologies, Massy, France) according to the manufacturer’s recommendations.

Array-CGH analysis

DNA was hybridized to 8 x 60K whole-Genome Agilent Arrays (G4450A) according to the manufacturer’s protocol. The ADM-2 algorithm of Agilent Genomic Workbench Lite Edition 6.5.0.18 was used to identify DNA copy number anomalies at the probe level. A low-level copy number gain was defined as a log 2 ratio >0.25 and a copy number loss was defined as a log 2 ratio <-0.25. A high-level gain or amplification was defined as a log 2 ratio >1.5 and a homozygous deletion was suspected when the ratio was < -1.

Gene expression profiling

Gene expression analysis was carried out using Agilent Whole human 44K Genome Oligo Array (Agilent Technologies) according to the manufacturer’s protocol. All microarrays were simultaneously normalized using the Quantile algorithm. T-tests were performed using Gene Spring (Agilent Technologies) and P-values were adjusted using the Benjamini-Hochberg procedure. The P-value and fold change cut-off for gene selection were 0.001 and 3, respectively. Gene Ontology (GO) analysis was performed to establish statistical enrichment in GO terms using Genespring (Agilent Technologies, Massy France).

Statistical analysis

Genomic normalized data files were formatted to obtain 55077 unique probes and in case of duplicate probes the mean value was retained. Pearson’s correlations and graphs were established with R software version 2.14.1. For transcriptomic normalized data files, we excluded controlling probes, and selected unique probes for each gene by maximum Inter Quartile Range (IQR) on R environment. Overall, 30995 probes were used to perform Hierarchical Ascending Classification (HAC) with the Ward method using the “cluster” R library. The agglomerative coefficient is 0.54 for tumor/cell line analysis and 0.61 for early cell lines/late cell lines analysis.

Results

From 2009 to 2012, surgery specimens of malignant mesenchymal tumors of every patient treated at Bergonié Institute were cultured and 32 cell lines were established from 134 tumor samples submitted to cell culture (24%; Supplementary Table S1).

To test whether sarcoma cell lines were representative of their matching tumor and whether their genomic and transcriptomic profile was stable during passages, seven cell lines (Table 1) were characterized at early and late passages and compared to their matching tumor.