The MCL cell line Z138 was purchased from ATCC (Manassas, VA, USA). Both untreated and resistant cell lines were cultured in R10 medium (RPMI 1640 (HyClone Laboratories, Logan, UT, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% 2 mM L-glutamine (Invitrogen)).
Cytarabine (147–94-4, Pfizer, New York, NY, USA) was aliquoted and stored at 4 °C with bulk concentrations of 411 mM. SCREEN-WELL® Epigenetics library (BML-2836, Enzo LifeSciences Inc., Farmingdale, NY, USA) and Chemotherapeutic Agent Library (L1500, Selleck, Munich, Germany) were stored at − 80 °C until use. Substances used for proliferation studies including bortezomib (2204S, Cell Signaling Technologies), lenalidomide (PCID-216326 Santa Cruz Biotechnology, USA), apicidin (A8851, Sigma Aldrich), belinostat (PXD101), M-344 (M5820, Sigma Aldrich), oxamflatin (O3139, Sigma Aldrich, St. Louis, MO, USA), scriptaid (S7817, Sigma Aldrich), trichostatin A (T1952, Sigma Aldrich) and vorinostat (SAHA MK0683, Selleck) were dissolved in DMSO (Sigma Aldrich), aliquoted and stored at − 80 °C until use.
Establishment of resistant sub-clones
The first resistant sub-clone defined as Z138 Cytarabine Resistant (Z138-CytR) was established by continuous exposure of wild type Z138 Cytarabine Naïve Sensitive cells (Z138-CytNS) to increasing concentrations (0.005–0.3 μM) of cytarabine. Using this model, we could identify the approximate time to resistance development, and utilize this information for developing a novel time-controlled cytarabine resistant model, described below.
Z138-CytNS, with viability above 85%, were exposed to 0.005 μM cytarabine and kept at log phase (1–2 × 106 cells/ml). Concentrations were increased two- or ten-fold, and samples for immunoblotting were taken when viability reached above 85%. When reaching a concentration of 0.2 μM cytarabine, cells were expanded and frozen as a cell biobank, a sub-clone called Z138 Cytarabine Exposed Sensitive (Z138-CytES). This cell biobank could then be used for further cytarabine exposure experiments and the establishment of a Cytarabine Resistant 21 days (Z138-CytR21) sub-clone.
Effect of cytarabine on sensitive and resistant cell lines
Cells were seeded in a 48 well plate and incubated with 0, 0.5, 5 and 50 μM of cytarabine at 37 °C (5% CO2) for 24–96 h. Duplicates from each concentration were counted in an automatic cell counter (Countess™, Invitrogen) at each time point, and trypan blue exclusion method was used to monitor viability.
Assessment of cell proliferation by [methyl-14C]-thymidine incorporation
Cells were seeded in a Cytostar-T 96 well plate (Perkin Elmer, Waltham, MA, USA) and cultured for up to 72 h in presence of 0.5 μCi/ml [methyl-14C]-thymidine (PerkinElmer). Cell proliferation was measured at indicated time-points using a Wallac 1450 MicroBeta liquid scintillation counter (Perkin Elmer). Prior to all measurements, cells were centrifuged to allow contact to the scintillation liquid.
Re-introduction of dCK into dCK negative resistant cells
To assess the importance of dCK in relation to resistance, the protein was transiently re-introduced into resistant cells. The Amaxa protocol (Amaxa Biosystems Cologne, Germany) for nucleofection of suspension cell lines was followed, using program CM-138 and Cell Line Nucleofector Solution SF (Amaxa Biosystems). For the re-introduction experiments, 2.5 × 106 cells were mixed with 2 μg of OmicsLink™Expression Clone for dCK (EX-C0081-M46, vector information can be found in Additional file 1) in each reaction and a GFP vector was used as a positive control (both from GeneCopoeia, Germantown, MD, USA).
Gene expression analysis
Triplicate cultures of cells were harvested at different time-points and lysed in TRIzol (Invitrogen). Preparation of tRNA was performed as previously described . Gene expression was assessed using Affymetrix Human Transcriptome Array 2.0 (HTA 2.0; Affymetrix Inc., Santa Clara, CA, USA), and acquired data was pre- processed at the SCIBLU Genomics Centre (Lund University, Sweden) involving quality control and normalization, using the Expression Console software (Affymetrix Inc). Normalized and log2 transformed data was imported into Qlucore Omics Explorer 3.0 (Qlucore AB, Lund, Sweden) for statistical analysis. For confirmation of mRNA expression in different samples, TaqMan probe-based RT-PCR was performed, using TaqMan® Fast Universal PCR Master Mix (Applied Biosystem, Waltham, MA, USA) and the TaqMan assay Hs01040726_m1 (dCK, Applied Biosystem) and Hs00162150_m1 (SPIB, Applied Biosystem). 18S (Hs99999901_s1, Applied Biosystem) was used as reference gene. All data were analyzed using the 7500 software v2.0.5 (Applied Biosystem). Functional annotation of individual genes was obtained from NCBI/Gene (http://www.ncbi.nlm.nih.gov/gene), GeneCards (http://www.genecards.org/) or UniProt (http://www.uniprot.org/).
Library preparation, hybridization capture and MPS sequencing
DNA from Z138-CytNS, Z138-CytES and Z138-CytR cells were purified using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) and thereafter quantified using the Qubit system (Life Technologies, Carlsbad, CA, USA). Two μg of DNA were fragmented using the Covaris S2 Ultrasonicator (Covaris, Woburn, MA, USA) and DNA fragments from 64 target genes, including TP53 were captured using SureselectXT Custom 3–5.9 Mb library kit (Agilent Technology, Santa Clara, CA, USA). Before capture, eight samples were pooled, and the molarity of the pooled library was determined based on and DNA fragment size distribution measured on a Bioanalyzer (Agilent) and concentration measured by Qubit. Sequencing was performed on the Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) with 2 × 101 bp paired end reads.
Analysis of sequencing data
Picard Extract IlluminaBarcodes and IlluminaBasecallsToSam (https://broadinstitute.github.io/picard/) was used for format conversion and demultiplexing of raw Illumina sequencing data and sequence reads were aligned to the human reference genome hs37d5ss (1000 genome with decoy sequences) using Novoalign (http://www.novocraft.com). Picard MarkDuplicates were used to identify and exclude PCR duplicates in subsequent analyses and quality scores were recalibrated and indels realigned using the Genome Analysis Tool Kit (GATK) . GATK UnifiedGenotyper with a call confidence cutoff of 10 were used to identify genetic variants and genotypes. Variants were annotated for their effect on protein coding transcripts using snpEff and Annovar using RefSeq reference transcripts. Bases of coding exons and 20 bp of adjacent introns were covered by at least 30 reads and variants affecting coding exons and 20 bp of adjacent introns were evaluated for pathogenicity.
Silencing of SPIB in the resistant cell line
Cell Line Nucleofector Solution SF was used with program CM-138 following the Amaxa protocol for suspension cell lines (Amaxa Biosystems). Cells were mixed with 1000 nM of siRNA (Ambion, Austin, TX, USA) or a scrambled sequence. GFP-producing plasmid was used as control for the transfections (Amaxa Biosystems).
Effect of bortezomib on resistance development
Based on the set up presented above, Z138-CytES cells were co-treated with 0.3 μM of cytarabine and 0.001 or 0.01 μM of bortezomib during the 21 days expected for resistance to develop. A positive control with only 0.3 μM of cytarabine was grown in parallel (as visualized in Fig. 10c). Z138-CytNS cells and Z138-CytR cells were subjected to the same treatment during the same period. Lysates for immunoblotting were sampled continuously during the treatment period and after completed treatment. After 21 days of treatment, proliferation in cytarabine containing medium was assessed by [methyl-14C]-thymidine incorporation as previously described.
Cells were harvested for western blot and lysed on ice with lysis buffer (1% NP40 (Sigma Aldrich) in PBS supplemented with 1× complete protease inhibitor (Roche Applied Sciences, Indianapolis, IN, USA)) for 30 min followed by centrifugation at 4 °C, 1300 rpm for additional 30 min. Supernatants consisting of protein lysates were collected and protein concentrations were measured using a bicinchoninic acid kit (Sigma Aldrich). For western blot, 25 μg protein was loaded on a Bis-Tris gel (Life Technologies, Carlsbad, USA). Following electrophoresis using an XCell Surelock Mini- Cell system (Life Technologies), the proteins were immediately blotted onto a PVDF membrane using program P3 on the iBlot Dry Blotting System (Life Technologies). The membranes were then blocked for 60 min with 5% milk in PBS, before incubation with primary antibodies targeting dCK (TA502698, OriGene Technologies, Rockville, MD, USA), ENT1 (ab 11,337–1-AP, Proteintech, Chicago, IL, USA), SPIB (Cell Signaling), NF- κB (D14E12, Cell Signaling), IκBα (44D4, Cell Signaling) and/or GAPDH (G8795, Sigma Aldrich). Horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin (P0260, Dako, Glostrup, Denmark) and swine anti-rabbit immunoglobulin (P0217, Dako) were used as secondary antibodies. Protein levels were visualized in a ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) with SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific). Quantification of the results was performed using the Image Lab software (Version 5.2.1, Bio-Rad Laboratories).
Screening of compound libraries and validation of selected drugs
Z138-CytNS or Z138-CytR cells were seeded in a Cytostar-T 96 well plate as previously described, and treated with different concentrations of chemotherapeutic and epigenetic drugs. Non-treated cells re-suspended in R10 medium were considered as R10 controls, and DMSO (0.01%) treated cells as vehicle controls. Proliferation was measured 0, 24 and 48 h after addition of chemotherapeutic and/or epigenetic drugs. To evaluate the additive effect of epigenetic drugs to cytarabine, 50 μM cytarabine was added 6 h after pre-incubation of cells with epigenetic compounds.
Patients, cohorts and treatment protocols
Materials from patients included in the Nordic Lymphoma Group MCL2 and MCL3 trials at hospitals in Sweden, Finland, Norway and Denmark, were selected for TMA construction as previously described . The treatment protocols for MCL2 and MCL3 both include high-dose cytarabine, rituximab and ASCT as previously described .
Immunohistochemistry staining and digital scoring
Immunohistochemistry was performed as previously described . The sections were stained for dCK (TA502698; OriGene Technologies) and visually analysed using a Nikon ECLIPSE 80i microscope (Nikon Instruments Inc., Melville, NY, USA) at a magnification of 20× (Plan Flour 20× DIC M/N2, Nikon) with a numeric aperture of 0.5. Images were captured using a Nikon DS-U2/L2 USB (Nikon) camera, and NIS Elements BR 3.10 (Nikon) as acquisition software. For digital scoring, dCK stained slides were scanned at an absolute magnification of 20× (resolution of 0.493 μm per pixel) and digitally scored using HALO™ (Indica Labs, Corrales, NM, USA). Positive areas (tumour) and negative areas (stroma) were separated and quantified based on a pattern recognition algorithm in the HALO platform. Image analysis based on RGB (red, green, blue) spectra was used to detect all cells by counterstaining with hematoxylin (blue). All analysis settings including thresholds set for weak, intermediate and strong nuclei staining were maintained throughout the whole study (Additional file 2: Table S1).