In this study we evaluated 6 established osteosarcoma cell lines (U2OS, MG-63, OS252, SAOS2, 143B, MNNG). U2OS, MG-63, SAOS2, 143B, and MNNG were obtained from ATCC. OS252 was a gift from Richard Gorlick at MD Anderson43. The identities of the lines were confirmed using STR analysis. Cells were maintained in DMEM + 15% FBS and antibiotics. Basic growth characteristics and kinetics including doubling time and log-phase were determined at several starting cell concentrations.
Cell viability assays
Screening methodology, described in prior publications24,44, was utilized to explore monoculture and co-culture growth characteristics under environmental stress or in the presence of chemotherapeutic agents. Cell viability was quantified using Cell-Titer Glo 2.0 or Real Time Glo (CT-Glo, RT-Glo, Promega, Madison, WI, USA). Following addition of CT-Glo to cell cultures, cells were agitated for 30 min on a shaker at room temperature. Luminescence was measured using a Cytation 3 plate reader (Bio-Tek Instruments) at room temperature or at 37 °C for CT-Glo or RT-Glo, respectively. Raw data were transferred to Microsoft Excel workbooks, where subsequent background subtraction and normalization analyses were conducted.
High throughput viability assays (2D)
Cells (2.7 × 103) were seeded onto 384-well plates, with subsequent additions of drug treatments (when applicable) and CT-Glo, using a Precision XS liquid handling station (Bio-Tek Instruments, Winooski, VT, USA). Cells were grown 24 h prior to the addition of any chemical agents and were subjected to drug or vehicle control for 72 h before being assayed with CT-Glo. All drug screens were completed in 2D using cell monolayers. For high throughput characterization of cell growth across multiple cell lines concurrently, several plates for each cell line were seeded with a series of cell concentrations ranging from 1,800 to 47,000 cells/cm2 (based on surface area, 1:1.5 serial dilution). Viability was quantified every 24 h until growth curves plateaued at around 96 or 120 h. Each experimental or control condition was split into a minimum of 4 technical replicates. Biological replicates were averaged following normalization.
RNA-seq, expression analysis, and SNV analysis
Cell lines at 80% confluence were subjected to RNA-sequencing for gene expression analysis. Total RNA was prepared from each osteosarcoma cell line using the mirVana miRNA Isolation Kit (Cat# AM1560, Invitrogen, Thermo Fisher) and RNA-sequencing libraries were prepared using the Nugen Ovation Human FFPE RNA-seq Multiplex System according to the manufacturer’s protocol (Tecan Genomics, Inc, Redwood City, CA). Library size and quality were assessed using the Agilent BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) and sequencing was performed using the NextSeq 500 v2 sequencer (150 cycles) to generate a targeted 80 million 75-base paired-end reads. Raw sequence data were demultiplexed using the Illumina bcl2fastq2 software (Illumina, Inc., San Diego, CA, USA). Paired-end RNAseq reads were preprocessed for quality assessments and adapter trimming and aligned to the human reference genome hs37d5 using TopHat v2.0.13 default setting45. Gene-level expression was quantified using HTSeq v0.6.146 based on the RefSeq gene model downloaded from USCS Table Browser. Normalized read counts were obtained via calculating library size factors and scaling raw read counts using the Bioconductor R package DESeq2 v.1.6.347. microRNA-Sequencing was performed using the Illumina TruSeq Small RNA Sample Preparation Kit, which enables the sequencing of small RNAs between 17 and 35 nucleotides. Briefly, universal RNA adapters were ligated to 1 ug of total RNA, which was used to generate single-strand cDNA following the manufacturer’s protocol (Illumina, Inc., San Diego, CA). Following PCR amplification and gel purification, the final libraries were reviewed on an Agilent BioAnalyzer DNA 1000 chip and were then sequenced on the Illumina NextSeq 500 sequencer to generate approximately 10 million sequencing reads per sample.
To detect driver mutations in the RNAseq data, sequence reads were aligned to the reference human genome (hs37d5) with STAR and duplicate identification, insertion/deletion realignment, split and trim, quality score recalibration, and variant identification were performed with the Picard toolkit (http://broadinstitute.github.io/picard/) and Genome Analysis ToolKit (GATK, Broad Institute, Cambridge, MA, USA)48,49. Sequence variants were annotated to determine genic context (i.e., non-synonymous, missense, splicing) using ANNOVAR50. Additional contextual information was incorporated, including allele frequency in other studies such as 1000 Genomes, the NHLBI Exome Sequence Project, in silico functional impact predictions, and observed impacts from databases like ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/) and the Collection of Somatic Mutations in Cancer (COSMIC).
Copy number variation (CNV) and analysis
Copy number variation (CNV) and loss-of-heterozygosity (LOH) status were obtained with the CytoScan HD Assay (Affymetrix, Santa Clara, CA, USA), which was performed on each cell line starting with 250 ng of DNA. DNA was extracted using the Blood and Cell Culture DNA Midi Kit (Qiagen, Hilden, Germany). The CytoScan HD assay uses 750,000 SNP probes and 1.9 million non-polymorphic probes to report genome-wide copy number aberrations at a resolution of 25–50 Kb. In addition, the assay can measure genome-wide LOH, including copy-neutral LOH. The data generated from the assay were normalized, copy number status calculated, and the data reviewed for quality using the Chromosome Analysis Suite (ChAS) v3.0 (Thermo Fisher Scientific, Waltham, MA).
Twelve different conditions were assessed at notated concentrations (10, 50, 90, 100% (or single culture)) with an initial spheroid size of 1000 cells/well (with 3.5% Matrigel, Corning, Inc., Corning, MA). Media containing 4.5 g/L glucose, 2.5 mM L-glutamine, pH 7–7.4 and antibiotics was deemed “normal” media. Subsequent conditions included no glucose (0 g/L), no glutamine, and no glucose or glutamine. Due to prior work and the importance of tumor microenvironmental conditions the lab was interested in varying pH on the cells and the effects of the small molecules tested, As a results we included conditions with high pH (8.0), low pH (6.5), high pH with no glucose, low pH with no glucose, high pH with no glutamine, low pH with no glutamine, high pH with no glucose or glutamine, and finally low pH with no glucose or glutamine media.31 Media was changed every ~ 3 days. Plates were incubated in an Incucyte SX5 where brightfield and fluorescent scans were taken every 24 h.
Drug screening with cells in log phase was conducted using phamocokinetic guided dose levels when available and high-throughput cell viability assays as previously described24. Briefly, we utilize a system to evaluate combinations with the aim of rapidly translating that data into clinical trials24. We selected FDA-approved agents or agents with strong preliminary data for OS, clinically achievable dosages, and tolerable dosing schedules. Treated experimental wells were normalized to untreated wells for a given cell line. In the case of these earlier agents without consistent human or animal drug data, we chose 3 fixed concentrations for each agent. Drugs were then selected based on differential sensitivity to either the 143B or the SAOS2 cell line for further use.
Coculture (3D spheroids)
The 143B and SAOS2 cell lines were transduced with GFP lentivirus (Cat#: PLV-10002-200, Cellomics Technology, Halethorpe, MD, USA) and mCherry lentivirus (Cat#: LVS(VB220407-1474cpn), VectorBuilder, Chicago, IL, USA) respectively, according to manufacturer’s protocol. The GFP/mCherry containing cells were selected with 5 µg/ml of puromycin. Cells were then plated on Greiner 96-well U-bottom, Cellstar® cell-repellent plates (Greiner Bio-One, Kremsmünster, Austria) using 3.5% Matrigel (Corning, Inc., Corning, MA) to form spheroids. Spheroids were characterized with an initial spheroid size of 1,000 cells/well comprised of SAOS2 alone, 143B alone and SOAS2 comprising 10, 50, or 90%, with 143B cells comprising the remainder to 100%.
Spheroids again at notated SAOS2 compositions (10, 50, 90, 100% (or single culture)) with a total cell number of 20,000 cells/well to start with a larger spheroid were treated for 96 h with panobinostat or trametinib or both in normal media or media with no glutamine at clinically achievable levels based on available pharmacokinetics from clinical trials. Cells were then treated for 72 h with Courmermycin-A1 (CA1), a Cdc45-MCM-GINS helicase inhibitor (CMGi) that helps prevent cell recovery after treatment, and then the whole cycle was repeated. Plates were incubated in an Incucyte ZOOM system where brightfield and fluorescent scans were taken every 12 h.