A major challenge in the treatment of platinum-resistant high-grade serous ovarian cancer (HGSOC) is lack of effective therapies. Much of ongoing research on drug candidates relies on HGSOC cell lines that are poorly documented. The goal of this study was to screen for effective, state-of-the-art drug candidates using primary HGSOC cells. In addition, our aim was to dissect the inhibitory activities of Wee1 inhibitor adavosertib on primary and conventional HGSOC cell lines.A comprehensive drug sensitivity and resistance testing (DSRT) on 306 drug compounds was performed on three patient-derived genetically unique HGSOC cell lines and two commonly used ovarian cancer cell lines. The effect of adavosertib on the cell lines was tested in several assays, including cell-cycle analysis, apoptosis induction, proliferation, wound healing, DNA damage, and effect on nuclear integrity.Several compounds exerted cytotoxic activity toward all cell lines, when tested in both adherent and spheroid conditions. In further cytotoxicity tests, adavosertib exerted the most consistent cytotoxic activity. Adavosertib affected cell-cycle control in patient-derived and conventional HGSOC cells, inducing G2/M accumulation and reducing cyclin B1 levels. It induced apoptosis and inhibited proliferation and migration in all cell lines. Furthermore, the DNA damage marker γH2AX and the number of abnormal cell nuclei were clearly increased following adavosertib treatment. Based on the homologous recombination (HR) signature and functional HR assays of the cell lines, the effects of adavosertib were independent of the cells' HR status.Our study indicates that Wee1 inhibitor adavosertib affects several critical functions related to proliferation, cell cycle and division, apoptosis, and invasion. Importantly, the effects are consistent in all tested cell lines, including primary HGSOC cells, and independent of the HR status of the cells. Wee1 inhibition may thus provide treatment opportunities especially for patients, whose cancer has acquired resistance to platinum-based chemotherapy or PARP inhibitors. There is an urgent need for effective therapies for patients with platinum-resistant high-grade serous ovarian cancer (HGSOC). While most HGSOC patients initially respond to the standard first-line platinum–taxane combination chemotherapy, relapse within 18 months is common followed by chemoresistance . ReasonsIn vitro models are essential for identifying effective oncology compounds or drug combinations for any type of cancer, including HGSOC. Until recently, most of the in vitro studies have been conducted using publicly available ovarian cancer cell lines that may not represent the HGSOC subtype and that may have undergone a variety of in vitro alterations during extensive passaging  showed the most consistent inhibitory results. Wee1 kinase plays a crucial role in cell-cycle regulation and DNA damage identification and repair in malignant and non-malignant cells, and its inhibition has shown promising results in early phase clinical trials –14, inclTumor and ascites material and clinical information was collected from consenting patients treated at the Department of Obstetrics and Gynecology, Turku University Hospital, Turku, Finland, as described previously , 15. TheDNA/RNA sequencing and functional assessment of homologous recombination capacity). In addition to these patient-derived cell lines, two conventional HGSOC cell lines were explored: CAOV3  and OVCAR8 .Two patient-derived cell lines were established from ascites (OC002 and M022i), of which OC002 was treatment-naive and M022i was from IDS. One cell line originated from omental metastasis M048i) and was from IDS , and/or cells from the cultures. Germline reference was available for M022i and M048i, fresh frozen tumor tissue or ascites for M048i (N = 4) and OC002 (N = 1), and cultured cells for M022i and M048i. DNA/RNA was extracted from samples with AllPrep DNA/RNA Mini Kit (Qiagen). Sequencing was performed in the BGI (Beijing Genomics Institute) as whole-genome sequencing (WGS) with HiSeq X Ten or with whole exome sequencing (WES) with Agilent SureSelect Human All Exon V5 using HiSeq 2000.To genetically characterize tumors and identify patient-specific TP53 mutations, which were used to verify cell identity in the cultures , we analyzed RNA expression data of the ABC transporters (ATP-binding cassette transporters). The results are provided as reads per kilobase per million (RPKM) values, normalized to GAPDH expression. A heatmap was produced using Log2+1 values at the publicly available web software Heatmapper (heatmapper.ca) .The five cell lines were subjected to high-throughput screening (HTS) with a panel of 306 clinical FDA- and/or EMA-approved and emerging oncology drug compounds Table S4https://breeze.fimm.fi/)  was used , 10. Thiimm.fi/) .® (Promega) cell viability test. To test cells' sensitivity to cisplatin, 5,000 cells/well were plated on a 96-well plate in triplicates. Cisplatin was added in concentrations of 0.01–100 µM, and cell viability was measured after 72 h of incubation. Luminescence was detected with a Victor2 luminometer . The IC50 value of adavosertib for each cell culture was calculated, and a dose of 500 nM was selected to be used for the functional experiments. The IC50 value for cisplatin was calculated using log-transformed data and logarithmic trend line.The cytotoxic effect of adavosertib  was further validated with a CellTiter-GloThe M048i, OC002, CAOV3, and OVCAR8 cells were investigated with a BD LSRFortessa™ flow cytometer . For each measurement, 10,000–30,000 events were assessed. The flow cytometry data were analyzed with Flowing Software 2.5.1 . Duplicates of each sample were tested, and the experiment was repeated a minimum of three times.Early and late apoptoses were detected after 48 and 72 h of treatment with adavosertib with an Annexin V-FITC Apoptosis Detection Kit . Samples were processed according to the kit's protocol.Cell-cycle phases were detected with a Click-iT EdU Flow Cytometry Assay Kit Pacific Blue . Cell-cycle progression was studied in vehicle- and adavosertib-treated cells at time points 24, 48, and 72 h. Samples were collected, and the protocol was performed according to the manufacturers' instructions.Cell proliferation of the HGSOC cell lines was inspected for 72 h at 2-h intervals with an IncuCyte S3 high-content imager . Cells were plated in 96-well plates (Greiner Bio-One) to be 10% confluent and treated with 500 nM adavosertib. Cells treated with vehicle (DMSO) were used as control in all the experiments. Each sample was measured in triplicate, and the experiments were repeated a minimum of three times. Proliferation was measured by confluence area by IncuCyte software (Essen BioScience).Wound healing of the HGSOC cells was measured for 72 h at 2-h intervals with an IncuCyte S3 high-content imager . Experiments were performed on 96-well plates  with adavosertib (500 nM) or vehicle (DMSO). Each sample was measured in triplicate, and the experiments were repeated a minimum of three times. Relative wound density was analyzed by IncuCyte software (Essen BioScience). The wells were precoated with Geltrex (Gibco) for migration experiments and with Matrigel  for invasion experiments. Wells were pretreated for 24 h with adavosertib (500 nM) before wound making with a wound-maker provided with IncuCyte S3 (Essen Bioscience). In the invasion experiments after wound making, the cells were covered with 50 µl Matrigel (2 mg/ml) for 30 min in the incubator; thereafter, adavosertib was added.® TGX™ Precast Protein Gels (Bio-Rad) and transferred to the 0.2-µm PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with 5% BSA  in Tris-buffered saline with 0.05% Tween 20 (TBST) and probed with primary antibodies diluted in the same solution. Primary antibodies used in the Western blotting were rabbit monoclonal anti-Cyclin B1 , rabbit monoclonal anti-Cyclin E1 , mouse anti-PCNA , and mouse monoclonal anti-γH2AX . GAPDH-HRP-conjugated  or mouse monoclonal anti-α-Tubulin  was used as control for protein loading. The secondary antibodies were HRP-conjugated swine anti-rabbit and rabbit anti-mouse immunoglobulins  diluted in a blocking solution. Membranes were washed three times with TBST between antibody incubations. Bound proteins were detected by enhanced chemiluminescence using ChemiDoc™ Gel Imaging System (Bio-Rad), and the signals were quantified using ImageJ.The effect of adavosertib on the cell cycle was investigated in the cell cultures treated for 72 h. Cells were grown in a DMEM/F-12 or RPMI medium and harvested and lysed with a RIPA Buffer supplemented with protease inhibitors. Protein concentrations were measured using a Bio-Rad protein assay kit according to the manufacturer's instructions. Equal amounts of proteins in the Laemmli buffer were separated in 4%–20% polyacrylamide PROTEANCells were grown on glass slides with a Geltrex (Gibco) coating and a modified OCMI or RPMI medium. After 72 h of incubation with 500 nM adavosertib, the cells were fixed for 10 min in 4% paraformaldehyde and washed with PBS. Blocking was performed with 5% BSA and 0.5% Triton X-100 in PBS for 30 min. Slides were incubated at room temperature for 60 min with a primary antibody diluted in a blocking buffer and thereafter for 60 min with a secondary antibody. The slides were washed twice after the antibody incubations with PBS and embedded in a mounting medium containing DAPI for staining nuclei . The following primary antibodies were diluted in the blocking buffer: mouse monoclonal anti-α-Tubulin  and mouse monoclonal anti-γH2AX . Alexa Fluor 555 Donkey anti-Mouse  was used as a secondary antibody together with Alexa Fluor 488-conjugated phalloidin  for actin filament visualization.http://rsbweb.nih.gov/ij/).Nuclear morphology was assessed after 72 h after treatment with vehicle or 500 nM adavosertib by staining the tubulin for cell structure and embedding in mounting medium containing DAPI for the cell nucleus staining. At least 100 cells were counted in each sample, and the nuclei were categorized as normal, abnormal, or multinuclear. Images were taken with a Nikon Eclipse Ni fluorescence microscope, and different channels were merged using ImageJ v1.53a software . P values ≤0.05 were considered statistically significant.The IC50 values for the adavosertib cytotoxicity validation were acquired using a sigmoidal dose–response curve. The differences in proliferation, wound healing, cell cycle, apoptosis, nuclear abnormalities frequencies, and protein levels between vehicle- and adavosertib-treated cells were compared using the two-sided The drug sensitivity testing was performed under two growth conditions, i.e., conventional adherent and spheroid conditions inducing stemness features and mimicking the environment of the malignant cells in ascitic fluid. We employed a correlation plot analysis for sDSS to assess the condition-related variations in drug sensitivity Table S6Ten clinically interesting drug compounds from the initial screen were selected for further investigation according to two criteria: literature search and ongoing clinical studies in HGSOC or other cancers Table S8To study whether adavosertib induces apoptosis, HGSOC cells were treated for 48–72 h before labeling with Annexin V. Adavosertib-induced apoptosis was evident in all of the examined cell lines, surprisingly also including M048i, the most resistant cell line in viability tests   was detected with Western blotting to confirm the confluence area-based proliferation result. The amount of PCNA decreased after 72 h of adavosertib treatment in the tested HGSOC lines  in the HGSOC lines  capacity using both genomic Table S2Despite the advances in understanding the molecular background of HGSOC, patients who do not initially respond or acquire resistance to platinum compounds or PARP inhibitors have limited treatment options. Based on the high-throughput screen of HGSOC cell lines, and with a focus on compounds in the clinical development, we identified two compound groups cytotoxic to HGSOC independent of the cell culture method: Wee1 inhibitor  and Hsp90 inhibitors . In a further validation using three patient-derived and two conventionally available HGSOC cell lines, adavosertib provided the best cytotoxicity result. Wee1 has already proven to be a potential target in genomically unstable cancers, including HGSOC, due to its role in cell-cycle control and DNA-damage response (DDR) pathways [reviewed in –28]. OurWe also noticed generally similar effects on the three HRP and two HRD cell lines. Additionally, adavosertib inhibited equally well patient-derived HGSOC cells and the publicly available HGSOC cell lines. One of the patient-derived HGSOC cell lines, M048i, was less responsive than the other cell lines in the high-throughput cytotoxicity assay to most of the 306 drugs including adavosertib for reasons that remain unclear. No significant difference was found in the DNA sequencing or RNA expression data of the multidrug resistance (MDR) ABC transporters (ATP-binding cassette transporters) compared to other cell lines , 30. In via inhibiting CDK1 and delays the mitosis entry of cells with DNA damage. Its inhibition in cells with DNA damage allows these cells to enter mitosis prematurely, which leads to mitotic catastrophe and apoptosis. Wee1 is also involved in regulating the G1/S checkpoint via CDK2 and thereby DNA replication by phosphorylating CDK2-bound cyclin A/E during the S phase  found that adavosertib impairs HR and may work as a combination therapy with PARP inhibitor olaparib in BRCA1/2-mutant leukemias . AnotherOur findings lay the basis for treatment of HRP patients, with whom treatment strategies are scarce. It is especially important to find an effective DNA-damaging agent to be used in combination with adavosertib for the platinum-resistant HGSOC patients. In this regard, gemcitabine has already shown promising results.In conclusion, our study suggests Wee1 inhibitor adavosertib as a candidate compound to treat HGSOC patients independent of the HR status of the tumor.The raw sequencing data are available from the European Genome-Phenome Archive (EGA). RNA data is under accession number EGAD00001006456 and DNA data is available under same DAC (EGAC00001001760).The studies involving human participants were reviewed and approved by Ethics Committee of the Hospital District of Southwest Finland (ETMK): ETMK 53/180/2009 §238 and ETMK 69/180/2010 and The Finnish National Supervisory Authority for Welfare and Health in Finland : DNRO 6550/05.01.06/2010 and STH507A.The patients/participants provided their written informed consent to participate in this study.PR, AA, and OC conceived the project. JH, SG, and AA contributed to the material acquisition. KK and PR established and maintained patient-derived HGSOC cell lines. PR, AS, VH, SaP, and KK designed the experiments. PR, AS, VH, PM, SaP, and SwP carried out the experiments. PR, AS, VH, KH, SaP, YL, and JO analyzed the data. PR, AS, and VH generated the figures. PR prepared the original draft. All authors reviewed and commented on the article. All authors contributed to the article and approved the submitted version.The project has received funding from EU Horizon 2020 (667403), The Academy of Finland (292606), Finnish Cancer Society, Sigrid Juselius Foundation, Finska Läkaresällskapet, and Helsinki University Hospital Research Funds. PR has received research grants from K. Albin Johanssons Stiftelse, Varsinais-Suomen rahasto (85211878), Orion Research Foundation, Lounais-Suomen Syöpäyhdistys, University of Turku Postgraduation Education Unit, and Ida Montinin Säätiö.We thank the DDCB core facility (FIMM HTB unit) supported by the University of Helsinki and Biocenter Finland. We also thank CSC – IT Center for Science Ltd. for computational resources.AA reports receiving honoraria and travel support from GSK and is a member in Nordic Society of Gynecologic Oncology NSGO-CTU Foundation Board. KW reports receiving payment for attending and presenting at meetings organized by Pfizer and Amgen. JH has received funding from EU Horizon 2020 (965193).The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


There is significant interest in developing early passage cell lines with matched normal reference DNA to facilitate a precision medicine approach in assessing drug response. This study aimed to establish early passage cell lines, and perform whole exome sequencing and short tandem repeat profiling on matched normal reference DNA, primary tumour and corresponding cell lines.in vitro study was conducted of patients with primary human papillomavirus positive and human papillomavirus negative tumours.A cell culture based, Four early passage cell lines were established. Two cell lines were human papillomavirus positive, confirmed by sequencing and p16 immunoblotting. Short tandem repeat profiling confirmed that all cell lines were established from their index tumours. Whole exome sequencing revealed that the matched normal reference DNA was critical for accurate mutational analysis: a high rate of false positive mutation calls were excluded (87.6 per cent).Early passage cell lines were successfully established. Patient-matched reference DNA is important for accurate cell line mutational calls. Model systems are therefore increasingly necessary to test the molecular basis of drug activity, which cannot be explored directly in patients.Clinical trials are the ‘gold standard’ for establishing the efficacy of anti-cancer agents. However, only a limited number of hypotheses can be tested because of the high expense, the limited number of patients and the length of time necessary to complete these studies.4 Cell lines do, however, present with several advantages, including the fact that they cost relatively little to maintain, can be easily manipulated in biological assays, are relatively amenable to genetic manipulation and provide rapid results. Indeed, they are the only model system that can be feasibly screened with a large number of agents to thoroughly explore mechanisms of drug sensitivity and resistance.5 In particular, there have been large-scale cell line drug screening efforts to correlate genetic profiles with drug response.8Cancer cell lines are the most widely used model systems in cancer research; however, they are imperfect, as they lack a three-dimensional environment, stromal interactions, tumour-microenvironmental features like hypoxia, and immune system effects.10 They have the potential additional advantage that patient-matched reference DNA and the primary tumour may be available to confirm that the cell line was indeed derived from the source patient and improved the accuracy of the genomic profiling.Although there are well-established high-passage cell lines, patient-matched reference DNA is not available for accurate characterisation. Early passage cell lines are thought to better retain the molecular features of the primary tumours and have a stronger correlation with patient drug response.For these reasons, we describe the establishment and characterisation of four head and neck cancer cell lines, including two human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (SCC) cell lines. We confirmed the origin of all the cell lines through short tandem repeat profiling, and used whole exome sequencing of triplet samples  to compare the genetic landscape of the cell line with the primary tumour and determined the importance of the normal reference DNA for making accurate mutational calls.Patients with head and neck SCC undergoing biopsy or resection were consented for tumour collection with University of Western Ontario Health Sciences Research Ethics Board approval 16579E.Ten millilitres of blood were drawn intra-operatively into heparinised tubes. The diagnosis of viable head and neck SCC was confirmed by frozen section in the operating theatre, and a portion of the remaining specimen was transported immediately in saline for processing. A portion of tumour was stored at −80°C. The remainder of the sample was further rinsed with saline and then minced into as small pieces as possible with a sterile razor blade. The tissue was incubated in 3 ml of Dulbecco's modified Eagle's medium/F12 medium supplemented with 10 per cent fetal bovine serum (Gibco®), penicillin (100 IU/ml) and streptomycin , amphotericin  and collagenase  for 1 hour at 37°C with regular agitation. Cells were washed twice with phosphate buffered saline, resuspended in primary tumour cell growth medium and divided into a six-well dish.The medium was changed every 72 hours, and evaluated by microscopy for epithelial cell adherence and growth. If the cells were able to achieve confluence and clusters of cells appeared epithelial, passaging was attempted; if no yeast or fungus contamination was evident, the cells were transferred to cell line complete growth medium, which consisted of Dulbecco's modified Eagle's medium/F12 medium supplemented with 10 per cent fetal bovine serum and penicillin and streptomycin mixture. This was performed by incubation with 1 ml of trypsin  for 2–5 minutes, followed by re-plating of 100 per cent of the cells into T75 flasks. This process was repeated for cell lines that continued to be able to achieve confluence and continued to have an epithelial appearance of greater than 50 per cent of cells. Representative stocks of each cell line were collected and frozen at −80°C during the passaging process in filter-sterilised freezing medium comprising 85 per cent fetal bovine serum and 15 per cent dimethyl sulfoxide.DNA was extracted from cultured cells and matched primary tumours using the AllPrep DNA/RNA Kit , following the instructions provided by the manufacturer. The DNA was extracted from the buffy coat layer separated from whole blood using the QIAamp DNA Blood Mini Kit (Qiagen). Minimum quality requirements for sequencing studies were 1 μg of genomic DNA with an optical density 260/280 ratio of between 1.7 and 1.9.11 The lines were genotyped with a panel of 10 selected markers. The cell line short tandem repeat profiling results were compared with the corresponding primary tumour tissue and blood to confirm the identity of the sample. Cell line DNA and RNA were also tested for the presence of all high-risk HPVs  by real-time polymerase chain reaction, as we have previously described.12For each cell line that was developed, the corresponding matched blood and primary tumour were analysed by short tandem repeat profiling at The Centre for Applied Genomics , as we have previously reported.13 Membranes were exposed to chemiluminescence reagent (Luminata™ Crescendo or Luminata™ Forte (Millipore®)) using a Bio-Rad ChemiDoc™ MP Imaging System.Whole cell lysates were prepared, immunoblotted and analysed as described previously.14Photographs of all cell lines were taken at 4× and 10× magnification. In order to measure cell line doubling time, cells were counted and seeded into T25 flasks. Forty-eight hours later, the cells were collected and counted. Doubling time was calculated using an online calculator.p > 0.05 indicative of both samples having a greater overlap than expected by chance.DNA samples were submitted to The Centre for Applied Genomics for whole exome sequencing. FASTQ files were aligned to GRCh38 using Burrows–Wheeler aligner algorithm with maximal exact matches (version 0.7.15). Duplicates were marked. Indel realignment and recalibration were performed using the Genome Analysis Toolkit (version 3.7.0). For tumours with a matched normal, germline single nucleotide polymorphisms were called using the Genome Analysis Toolkit HaplotypeCaller (version 3.7.0), and filtered for quality and read depth. The SomaticSniper (version 1.0.5.0) genome modelling tool was used to call single nucleotide variants in paired samples (tumour or cell line), with the reference being leukocyte DNA. A hypergeometric test was used to assess the probability of overlap between tumour and cell line samples, with 15Germline single nucleotide polymorphisms were generated using the Genome Analysis Toolkit (version 3.7.0). HaplotypeCaller was then run on the realigned and recalibrated individual binary alignment maps, followed by genotyping, variant selection and hard filtering. Unpaired mutation calling was first performed using the MuTect identification algorithm (version 1.1.7), without supplying the paired normal for the tumour, and the cell line was realigned with recalibrated binary alignment maps for each patient. A panel of ‘normals’ was generated using MuTect (version 1.1.7) from 438 of Cancer Genome Atlas head and neck SCC normal exomes. The single nucleotide variants, unpaired with the panel of ‘normals’, were further filtered to retain only the ‘pass’ calls. The single nucleotide variants that were in the panel of ‘normals’ but were also in the ‘allowlist’  database) were also removed. Single nucleotide variants in the capture regions and in chromosomes chr 1-22, X, Y and M were preserved. Somatic single nucleotide variants were filtered to remove non-functional (intronic or intergenic) variants prior to downstream analyses. Average true positive, false positive and false negative rates were then calculated for the four cell lines. Data visualisation employed the BPG  package.The Journal of Laryngology & Otology website). In all cases, the short tandem repeat profiles matched exactly between the blood and primary tumour DNA; however, there were occasional differences at a single marker between the cell line and the parental tumour samples, likely because of minor drift as the cell line adapted to a plastic environment.This section concerns the establishment of cell lines, confirmation of tissue of origin and HPV status. Short tandem repeat profiling of the patient's blood, the primary tumour and the developed cell line demonstrated a match for the majority of all 10 markers, confirming that the cell lines were indeed derived from the index patient and tumour   . UWO23 cMatched reference DNA was essential for accurate cell line mutation calls. Whole exome sequencing was undertaken for the four tumours and their corresponding cell lines, and compared against the patient's reference leukocyte DNA (patient-matched). This was accomplished using the MuTect algorithm, where matched leukocyte DNA was employed as a reference versus using a panel of ‘normals’ generated from 438 Cancer Genome Atlas head and neck SCC samples (as described in the Methods section); this is in contrast to cell line mutation calls, which were performed against the matched leukocyte DNA. When we performed unmatched analysis, we observed an average of 314.5 true positive single nucleotide variants, 20.5 false negative single nucleotide variants and 2222.75 false positives relative to the gold standard paired analysis . The fal16 single nucleotide variants in NOTCH1, TP53 and CDKN2A genes were identified . While cell lines and primary tumours shared significant numbers of specific mutations, demonstrating that they are related, both contained unique mutations, suggesting that genetic drift occurred in the cell lines as they adapted to culture conditions. It is also worth noting that the mutational calls between normal tissue and primary tumours that are shared are sufficient, but not necessary, for tumour transformation. These would be missed when using reference DNA.7 which have identified known, as well as novel, genetic markers of drug response.7 We specifically reanalysed the head and neck SCC data from both these studies and identified associations between PIK3CA mutations and response to a PI3K inhibitor; however, our analysis was limited by the small number of head and neck SCC lines and the limited number of drugs tested.17There is an urgent need to identify novel chemicals with high activity in head and neck cancers in order to improve outcomes. Cancer cell lines are by far the most widely used model systems in cancer research, as they cost relatively little to maintain and can be easily manipulated in biological assays and feasibly screened with a large number of agents. Potentially, genomic characterisation of these cell lines could allow correlation of molecular features with drug response. Indeed, there have been two large-scale studies involving hundreds of genotyped cell lines of different tissue types,18 However, our original intention was to correlate the cell line genomic profiles with drug response. We characterised our cell line panel with both OncoScan™ copy number arrays and whole exome sequencing, which our team has analysed extensively.25 While copy number arrays perform well without reference normal DNA,26 exome sequencing is prone to frequent false positives and negatives for both primary tumours and cell lines. In an effort to specifically address this, we developed a ‘reference-free somatic variant’ pipeline, which uses an extensive mutational ‘allowlist’ (known pathogenic variants) and ‘denylist’ (likely germline variants based on large population cohorts) as well as additional bioinformatics analysis.27 This pipeline is highly accurate in primary head and neck SCC and prostate tumours; however, despite exhaustive efforts, it does not perform well on cell lines.27 For this reason, we have started to generate a panel of both HPV-positive and HPV-negative cell lines from primary tumours, along with matched blood. To date, we have generated 17 such lines, and we have presented genomic analysis of the first 4 of these here.In order to expand drug discovery and develop additional biomarkers, we previously screened 1433 agents in a panel of 26 head and neck SCC established cell lines, and identified drugs with preferential activity in HPV-positive and HPV-negative cell lines.30 potentially because of additional molecular drift from the primary tumour profile through selection pressure in culture.10 Although beyond the scope of this manuscript with regard to comparison of early versus late passage cell lines, there is some evidence that early passage cell lines may represent superior models for drug screening.30 Thus, panels of genomically characterised early passage cell lines with patient-matched reference DNA, such as our novel University of Western Ontario panel, may be beneficial for the first phase of drug testing.Four early passage cell lines were established, and genomic characterisation was undertakenTwo cell lines were human papillomavirus positive, confirmed by sequencing and P16 immunoblottingShort tandem repeat profiling confirmed that all four cell lines were established from their index tumoursWhole exome sequencing revealed that matched normal reference DNA was critical for accurate mutational analysis: a high rate of false positive mutation calls (87.6 per cent) were excludedAn additional advantage of the cell lines described in this study is that they are early passage cell lines. Established cell lines have the distinct advantage of being broadly available to the cancer research community through established commercial culture collections. This offers the opportunity to reproduce data independently with the same reagents. Nevertheless, the correlation of established cell line drug responses with patient tumour responses appears to be modest,31 However, while numerous HPV-negative head and neck SCC cell lines exist, there is a paucity of HPV-positive lines.32 The ideal HPV-positive cell lines would be derived from non-smoking patients with a treatment-naïve oropharyngeal cancer; however, almost all lines, including our own, are derived from smokers, radiation failures (UWO37) or non-oropharyngeal primary sites .32 Only recently has an ideal cell line been described.33 Further culture efforts are needed to generate additional models to facilitate drug discovery.The HPV-positive cell line models are of great interest to the head and neck research community in light of the epidemic of HPV-related oropharyngeal cancer.We describe the establishment and characterisation of four head and neck SCC early passage cell lines with patient-matched reference DNA. These cell lines will act as a resource for drug discovery, as they can be genomically characterised and may be more accurate than late passage models. Development of additional early passage lines, particularly HPV-positive models, is needed.


Stratification of malignancy is valuable for cancer treatment. Both optical redox imaging (ORI) indices and nuclear-to-cytoplasmic volume/area ratio (N:C ratio) have been investigated to differentiate between cancers with varying aggressiveness, but these two methods have not been directly compared. The redox status in the cell nucleus has not been studied by ORI, and it remains unknown whether nuclear ORI indices add new biological information.We sought to compare the capacity of whole-cell and subcellular ORI indices and N:C ratio to differentiate between breast cancer subtypes with varying aggressiveness and between mitotic and nonmitotic cells.ORI indices for whole cell, cytoplasm, and nucleus as well as the N:C area ratio were generated for two triple-negative (more aggressive) and two receptor-positive (less aggressive) breast cancer cell lines by fluorescence microscopy.We found positive correlations between nuclear and cytoplasmic ORI indices within individual cells. On average, a nuclear redox status was found to be more oxidized than cytoplasm in triple-negative cells but not in receptor-positive cells. Whole-cell and subcellular ORI indices distinguished between the receptor statuses better than the N:C ratio. However, N:C ratio was a better differentiator between nonmitotic and mitotic triple-negative cells.Subcellular ORI analysis differentiates breast cancer subtypes with varying aggressiveness better than N:C area ratio. There is an unmet need for biomarkers that can detect malignant or aggressive tumors to aid in the selection of treatment strategies. Optical metabolic metrics have been under development in (pre)cancer imaging based on the endogenous fluorescence of oxidized flavoproteins  and reduced nicotinamide adenine dinucleotide (NADH).in vitro. It was found that increased ORR correlated with higher invasive potential,in vitro. Although most of the Fp and NADH signals come from the mitochondria, these previous analyses were performed with intensity thresholding and global averaging of the whole cell without partitioning into subcellular compartments. However, it is known that the nucleus also contains FAD and NADH and emits low levels of Fp and NADH signals.–In particular, ORR has been utilized to predict the invasive potential or aggressiveness of breast cancer cells Therefore, a major goal of this study is to examine the optical redox imaging (ORI) index differences between the whole cell, cytoplasm, and nucleus and to determine whether the compartmental ORI indices can differentiate between breast cancer cell subtypes exhibiting varying levels of aggressiveness.–We also aimed to compare the utility of ORI indices and nuclear-to-cytoplasmic area (N:C) ratio to differentiate between breast cancer cell types. The N:C ratio based on the ratio of cross-sectional areas has been investigated as an indicator of malignancy in several types of cancers.2We examined four breast cancer cell lines, i.e., HCC1806 (triple-negative breast cancer A), MDA-MB-231 (triple-negative breast cancer B), MCF7 , and BT474 . Their classifications were determined by previous studies.L-glutamine .Breast cancer cells were cultured in RPMI 1640 Medium  supplemented with 10% fetal bovine serum and maintained at 37°C with 5% To take live cell optical redox images, cell dishes were maintained in an environmental chamber surrounding the entire microscope for temperature control set at 37°C and imaged with the Zeiss Axio Observer 7 wide-field microscope , available in the Cell and Developmental Biology Microscopy Core Facility at the University of Pennsylvania. The scope is equipped with a Colibri 7 light-emitting diode (LED) light source and a Zeiss Axiocam 702 monochrome complementary metal oxide semiconductor (CMOS) camera. All imaging was conducted with an objective of Supplemental Material]. The temporal power drifts within 100 days were Supplemental Material). The fluorescence signals of all solution samples were found to be stable without significant time-dependent drifts [Figs. S1(c) and S1(d) and Table S2 in the Supplemental Material]. The PBS signals had CVs within 2% reflecting the excellent stability of the microscope system. The nonsignificant trends of signal decay of NADH and FAD solutions, 7% and 4%, respectively, within 100 days probably indicate a slight chemical decay during the long-term storage in a Supplemental Material). The fluorescence CVs of both NADH and FAD solutions were found to be within 4%. In comparison, the biological sample variations are usually up to 10% to 20% or more. In short, our Zeiss microscope system is very stable for the period of 3 months, and its calibration with standard references is not necessary for the purpose of this study.To demonstrate that the microscope remains stable with no significant system drift during this period, the illumination power of four LED light sources  was measured by a power meter and found to be very stable with the coefficient of variations (CVs) within 1% for all wavelengths and levels of intensities . Fifteen mitotic cells were analyzed per dish for both MDA-MB-231 and HCC1806. The same analysis was not performed for BT474 and MCF7 as they have doubling times of bwarea. The cytoplasmic area was calculated based on the subtraction of the nuclear area from the area of the entire cell.Mitotic cells were chosen randomly and visually based on nuclear morphology displaying apparent division in the later stages of cell mitosis, such as telophase [Figs. S2(d)–S2(f) in the We performed ORI and N:C ratio analyses using a dish-based analysis or cell-based analysis. Dish-based analysis treated each dish as an independent replicate  to run one-way Brown–Forsythe analysis of variance tests followed by Dunnett’s T3 tests to correct for multiple comparisons with 33.1Supplemental Material). For the receptor-positive group, BT474 and MCF7 lines experienced no significant differences between compartments for all redox indices, except that the nuclear compartment of MCF7 had a significant decrease of 28% in NADH compared to the cytoplasm . The pooSupplemental Material). These results suggested that NADH redox status  exhibits a connection between nuclear and cytosolic compartments, which is common across the four breast cancer cell lines. In contrast, for Fp across the four cell lines, there were no significant correlations between nuclear Fp and cytoplasmic Fp and between nuclear Fp and whole-cell Fp, and the positive correlation between cytoplasmic Fp and whole-cell Fp was also marginal . Thus, nuclear Fp may correlate with cytosolic Fp within individual cells of each cell line . Fp signals exhibited no significant difference among the four cell lines in the whole cell and nucleus except a lower cytoplasmic Fp was found in HCC1806 than in MDA-MB-231 cells. However, there were more significant differences in NADH among the four cell lines, with BT474 having the highest value in all compartments. Furthermore, the receptor-positive BT474 and MCF7 lines had or tended to have higher NADH intensity than TNBC lines. Across all compartments, significantly higher redox ratios were observed for HCC1806 and MDA-MB-231 compared to BT474, with an increasing trend compared to MCF7. Figure S5 in the Supplemental Material demonstrates that a cell-based analysis leads to more significant differences between cell lines across all redox indices in all three cell regions due to increased sample size  demonstrating the consistency between the two analyses.We examined the differences in the redox indices between cell lines within each compartment using the dish-based analysis . With the pooled analysis of the two cell lines, the redox indices still showed no significant differences in any compartments, but the N:C ratio exhibited a significant increase in mitotic cells when compared to nonmitotic cells [Using visible nuclear division as the criterion for mitosis, we investigated the redox and N:C ratio differences between nonmitotic and mitotic cells for the two TNBC cell lines. For both redox indices and N:C area ratio, no significant differences between mitotic and nonmitotic cells were found using the dish-based analysis for MDA-MB-231 or HCC1806 cells . However, there was a significant increase in the N:C ratio of mitotic cells compared to nonmitotic cells for both MDA-MB-231 and HCC1806 lines. Pooling HCC1806 and MDA-MB-231 data for cell-based analysis generated consistent results with the dish-based analysis, i.e., no significant changes in redox indices and a significant increase in the N:C ratio of mitotic cells compared to nonmitotic cells  . Thus, t44.1,Previous ORI studies utilizing intrinsic fluorescence imaging focus on whole cell or cytoplasmic analysis of fluorescence indices. Quite often, nuclear compartment was removed by thresholding,In addition, there are significant linear correlations of NADH or redox ratios between the nucleus and cytoplasm  and 4 ac4.2,–The nuclear compartmental redox status has not been investigated for the purpose of cancer biomarker development. With manual segmentation of clearly visible nuclei, we observed a more oxidized nuclear redox status compared to the cytoplasm in the more aggressive triple-negative cells, but nuclear and cytoplasmic redox ratios were similar in the less aggressive receptor-positive cells . We also4.3,The N:C ratio has been used to differentiate between normal, precancerous, and malignant tissues for both cervical and colon cancers.The cell cycle is an important cellular process in cancer proliferation. Oxidative phosphorylation is induced early in the G1 phase of the cell cycle and remains active during the entire cell cycle.When pooling data from the two TNBC cell lines, the N:C ratio marked an increase in mitotic cells compared to nonmitotic cells . These r4.4Supplemental Material), with variations and temporal drifts smaller or even negligible compared to the variability of biological samples . The statistically significant results demonstrate that 2D imaging acquired through a We acknowledge that our widefield microscopy approach in this study is limited to two-dimensional (2D) imaging and its spatial resolution may not be ideal for imaging the subcellular details compared to three-dimensional imaging by confocal fluorescence microscopy, which can be utilized to confirm the results of this study in the future. Nevertheless, a widefield microscope has the advantages of accessibility and simplicity with imaging and data analysis and thus can be more cost- and time-effective for certain purposes. Our data in this study demonstrated sufficiently high SNR (,,In this study, nuclear segmentation was manually performed only on selected cells with clear nuclear boundaries. Despite its shortcomings as an operator-dependent approach subject to artifacts, manual segmentation is a regular analysis approach used in the literature for optical imaging studies of cell cultures.,We also acknowledge that cellular NADPH, expected to be approximately fivefold to tenfold smaller than NADH level,5To the best of our knowledge, this is the first study utilizing ORI to characterize and compare the redox status between nuclear and cytoplasmic compartments of cancer cells and compare the ORI indices to the nuclear-cytoplasmic ratio in their potentials as biomarkers for breast cancer aggressiveness. A more oxidized nuclear redox status relative to the cytoplasm is associated with the more aggressive TNBC subtype. There are strong correlations between nuclear and cytoplasmic compartments for the ORI indices for all cell lines, suggesting a redox connection between the subcellular compartments. Optical redox indices of the whole cell, cytoplasm, and nucleus can differentiate between triple-negative and receptor-positive breast cancer groups with different aggressiveness. The N:C ratio does not differentiate between receptor statuses as well as the ORI indices do ; however, it is a better differentiator between nonmitotic and mitotic TNBC cells than the ORI indices. These results indicate the advantage of ORI over the N:C ratio in differentiating breast cancer subtypes with varying aggressiveness.Click here for additional data file.


Dear Editor,1. Therefore, genes essential in cancer but not normal cells are good therapeutic targets. However, while genetic dependencies for many cancer cell lines have been defined through genetic screens, much less was known for normal cells. To overcome this problem, the DepMap project has developed a method to identify genes selectively essential in a subset of cancer cell lines, making them less likely to be in core pathways critical for normal cells. Based on genome-wide CRISPR-based fitness screen data from ~1000 human cancer cell lines4, DepMap calculates CERES scores to measure the effect of gene knockout on cell fitness . Next, DepMap calculates a Normality Likelihood Ratio Test (NormLRT) score for each gene. A higher NormLRT score indicates that the distribution of this gene’s CERES scores across cancer cell lines is more deviated from a normal distribution, and the gene may be more selectively essential. Identification of selectively essential genes will not only deepen our understanding of cancer biology but also guide cancer drug development. However, manually sorting out which of them is understudied and warrants investigations is time-consuming and probably impractical.An ideal cancer therapy drug should kill cancer cells while displaying limited toxicities toward normal cells4, we collected 347 potential selectively essential genes and ranked them by PubMed publication count5. In our ranking system, the top nine most understudied genes are C11orf53, C3orf38, TMEM164, ZNF511, KCNK13, BEST3, CYB561A3, C12orf49, and COLCA2  reported previouslyCA2 Fig. . In fact7. However, the mechanistic details of how POU2F3 drives the expression of the tuft cell-specific program remain unclear. Based on our analysis, we hypothesized that C11orf53/COLCA2 and POU2F3 may work in the same pathway to regulate the growth of tuft cell-like SCLC.We subsequently focused on C11orf53 and COLCA2 because of the significant CERES scores in their dependent cell lines where they are highly expressed, suggesting strong growth phenotypes Fig. . Remarka9. Since copy number increase was observed at C11orf53 locus in NCI-H526 cells  as a co-activator for POU2F3 and demonstrate that their physical interaction is critical for the growth of SCLC.Our co-activator hypothesis predicts that C11orf53 and COLCA2 physically interact with POU2F3. Indeed, co-immunoprecipitation detected physical interactions between COLCA2 and POU2F3 Fig. . FurtherP-value < 0.05) showed similar changes upon POU2F3 knockout  upon COLCA2/POU2F3 knockout 15 and both C11orf53 (renamed as POU2AF2/OCA-T1) and COLCA2 (renamed as POU2AF3/OCA-T2)14 in SCLC, and our conclusion is essentially the same as those reached by these investigators. We noticed that in addition to SCLC, COLCA2 is also essential in a multiple myeloma cell line, JJN-3, where POU2F1 instead of POU2F3, is highly expressed and essential  functions as a co-activator for POU2F3 to drive the transcriptional program important for tuft cell-like SCLC. The highly selective nature of these co-activators in cancer coupled with the reports that ial Fig. , suggestial Fig. . How theial Fig. , suggestSupplementary information


Tumor regression was induced at doses less than 5 mg/kg, while maximal activity with complete cures were observed starting at 10 mg/kg. In MCL Mino and Jeko-1 xenografts, STRO-001 starting at 3 mg/kg significantly prolonged survival or induced tumor regression, respectively, leading to tumor eradication in both models. In summary, high CD74 expression levels in tumors, nanomolar cellular potency, and significant anti-tumor in DLBCL and MCL xenograft models support the ongoing clinical study of STRO-001 in patients with B-cell NHL.Overexpression of CD74, a type II transmembrane glycoprotein involved in MHC class II antigen presentation, has been reported in many B-cell non-Hodgkin lymphomas (NHLs) and in multiple myeloma (MM). STRO-001 is a site-specific, predominantly single-species antibody-drug conjugate (ADC) that targets CD74 and has demonstrated efficacy in xenograft models of MM and tolerability in non-human primates. Here we report results of preclinical studies designed to elucidate the potential role of STRO-001 in B-cell NHL. STRO-001 displayed nanomolar and sub-nanomolar cytotoxicity in 88% (15/17) of cancer cell lines tested. STRO-001 showed potent cytotoxicity on proliferating B cells while limited cytotoxicity was observed on naïve human B cells. A linear dose-response relationship was demonstrated  The addition of rituximab to regimens used to treat B-cell non-Hodgkin lymphomas (NHLs) marked a milestone in the treatment of hematologic malignancies , 2. SincCD74 is a type II transmembrane glycoprotein primarily involved in MHC class II antigen presentation, with additional roles in B-cell maturation, T-cell responses, and macrophage migration inhibitory factor-induced signaling . CD74 exWe previously reported the cell-free synthesis of STRO-001, a site-specific, predominantly single-species anti-CD74 ADC incorporating a non-cleavable linker-maytansine warhead with a drug-antibody ratio (DAR) of 2 . STRO-00D = 1.75 nM) and cynomolgus monkey (KD = 2.70 nM) CD74 ECD (extra-cellular domains), but not to mouse CD74 ECD. SP7219 bound with high affinities to CHO cells transfected with human (KD = 0.93 nM) and cynomolgus monkey (KD = 1.1 nM) CD74. Conjugation to the non-cleavable linker-maytansine warhead did not affect the binding affinity of SP7219 since STRO-001 retained high binding affinity to CHO cells expressing human (KD = 1.1 nM) and monkey CD74 (KD = 1.0 nM) (In SPR (surface plasmon resonance) based Biacore binding assay, the anti-CD74 antibody SP7219 bound to recombinant human (K 1.0 nM) . Neither 1.0 nM) .CD74 surface expression was determined in 9 GCB-like DLBCL cell lines, 4 ABC-like DLBCL cell lines, and 4 MCL cell lines. Among the 13 DLBCL cell lines, CD74 copy number varied from high levels  to below the lower limit of detection . Among t2 = 0.41) toward increased STRO-001 potency with higher CD74 receptor number was observed but appeared to be weak. Notably, nanomolar potency was observed for STRO-001 in 2 of the 3 cell lines with low CD74 expression level below detection limit , anti-CD74 antibody SP7219 was conjugated to DBCO-Alexa 647 to generate SP9241, which was used to stain cells isolated from bone marrow aspirates for 5 healthy human and 5 healthy cynomolgus monkey donors. An anti-GFP antibody produced by cell free Xpress+ system was also conjugated to DBCO-Alexa 647 and used as an isotype control (SP9367). The B cells, T cells, NK cells, monocytes, myelocytes, neutrophils, granulocytes and stem cells from human and cynomolgus monkey BMA were labeled with species specific commercial antibodies, respectively (Supplementary Tables 2 and 3). Relative expression of CD74 was expressed as MFI ratio of SP9241 over SP9367 on different immune cell populations. CD74 expression on human B cells was 1.4–2.0-fold higher relative to background, while other cell types did not show CD74 expression above GFP background (Supplementary Table 4). Relative CD74 expression was found to be 2.0–7.3-fold higher on cynomolgus monkey B cells, while other cell types did not show CD74 expression above GFP background (Supplementary Table 5).in vitro was associated with upregulation of cell surface markers CD86 and HLA-DR, as well as increased CD74 expression, after 5 days of treatment . Expansion of B cell populations reatment . B cell r 5 days .The cytotoxic activity of STRO-001 toward naïve B cells and proliferating B cells was determined using a FACS based cell viability assay after 6 days of treatment. in vivo anti-tumor activity of STRO-001 was evaluated in NHL models including DLBCL and MCL subcutaneous and disseminated tumor xenografts. Two DLBCL cell lines, SU-DHL-6 and U2932, were chosen based on moderate to low CD74 expression levels (p = 0.053) with 87% TGI and 4 out of 6 complete responders. Maximal activity was observed at 20 mg/kg administered either once-weekly or twice-weekly, led to tumor regression  with all animals (7/7) in both groups remaining tumor free through the end of the study (The n levels  and reprn levels . Althougn levels . At 10 mhe study .p < 0.01)   and 5B.  < 0.01)  and 4D.3, mean tumor sizes of animals in all three STRO-001 dosing groups were significantly smaller than those in the vehicle control group (p < 0.0001)  . Signs o 0.0001) . At 10 m 0.0001)  and 5B.In the disseminated Mino model, STRO-001 was administered starting on day 14 post-tumor inoculation. The survival endpoint criteria included body weight change loss >20% and clinical signs of morbidity. Mean survival in the vehicle group was 81.3 days, while treatment with STRO-001 at all the doses tested significantly prolonged survival up to 54 days relative to the vehicle control . Almost in vitro expansion. Taken together, these findings provide a strong rationale for potently and selectively targeting of CD74 in patients with B-NHL.In considering novel cancer therapies, the selection of an appropriate target depends on its sufficient expression in or on targeted cells, with limited expression elsewhere. The present study investigated CD74 expression in B-NHL cell lines and patient tumor samples. Biotinylated SP7219, the antibody portion of the ADC STRO-001, which displayed high affinity for human CD74, was used to determine expression levels by immunohistochemistry. We found CD74 expression in 88% 15/17) of DLBCL and MCL cell lines, as well as near-ubiquitous expression in DBCLC, FL and MCL tissue microarrays. These results are consistent with a previous study using a limited number of patient biopsies /17 of DL. Other iWe previously reported a pharmacodynamics/safety study of STRO-001 in cynomolgus monkey that identified bone marrow toxicity as dose-limiting in this species . At an iSTRO-001 displayed nanomolar to sub-nanomolar potency in 88% (15/17) of cell lines, including both GCB-like and ABC-like DLBCL cell lines as well as MCL cell lines. Among these, CD74 surface expression varied considerably. Interestingly, although a weak correlation between CD74 expression and STRO-001 cytotoxicity was noted, the high internalization rate of CD74 may drive robust activity in spite of low target expression as observed in several cell lines. And as expected, the antibody-only portion of STRO-001, SP7219, did not show any cytotoxicity on any of the cell lines tested. Similarly, SP7219 has been shown to lack cytotoxicity in multiple myeloma cell lines  and no ein vitro expanded B cells. Also of note, the major and minor catabolites of STRO-001 containing the maytansinoid warhead, SC246 and SC401, were >15-fold less cytotoxic in MM cell lines, suggesting that the clinical safety profile of STRO-001 may benefit from the consequent minimization of post-catabolic non-specific toxicity (bystander effects) /Winitial) × 100 where Wintial is the weight on the first day of treatment (day 0 post-treatment). Significant toxicity was defined as a >20% decrease in animal weight, at which point affected animals were euthanized. All graphs are presented as mean or individual values ± standard error of measurement (SEM). All results were reproduced in at least 2 independent studies, and representative data from one experiment is shown.In all models, STRO-001 and vehicle control (PBS) were administered intravenously (IV) at the intervals and doses indicated. Body weight change (BWC) was calculated relative to weight on the first day of treatment using the formula BWC = ([Wp < 0.05) was considered statistically significant. Annotations for p-values are: *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001; ns: not significant.Statistical analyses of final tumor sizes were performed using one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test using Prism software (version 6). When Brown-Forsythe and Bartlett’s tests showed significant data heterogeneity, non-parametric analysis was subsequently performed using Kruskal-Wallis test with Dunn’s multiple comparisons test. A probability of <5% (


Triple negative breast cancer (TNBC) remains a therapeutic challenge due to the lack of targetable genetic alterations and the frequent development of resistance to the standard cisplatin-based chemotherapies. Here, we have taken a systems biology approach to investigate kinase signal transduction networks that are involved in TNBC resistance to cisplatin. Treating a panel of cisplatin-sensitive and cisplatin-resistant TNBC cell lines with a panel of kinase inhibitors allowed us to reconstruct two kinase signalling networks that characterise sensitive and resistant cells. The analysis of these networks suggested that the activation of the PI3K/AKT signalling pathway is critical for cisplatin resistance. Experimental validation of the computational model predictions confirmed that TNBC cell lines with activated PI3K/AKT signalling are sensitive to combinations of cisplatin and PI3K/AKT pathway inhibitors. Thus, our results reveal a new therapeutic approach that is based on identifying targeted therapies that synergise with conventional chemotherapies. Triple negative breast cancer (TNBC) represents between 10–15% of all breast cancers and can be defined histologically as a subtype of breast cancer that lacks expression of the three key receptors: estrogen, progesterone, and HER2 . The resIn clinical practice, chemotherapies including platinum-based drugs continue to be the mainstay of standard treatments for TNBC. The use of chemotherapy in TNBC is supported by clinical evidence that demonstrates the benefit of treating breast cancer patients with cisplatin . HoweverIn this study, we aimed to reconcile these approaches by testing how cisplatin-based chemotherapy interacts with signal transduction pathways in order to identify targeted therapies that can overcome resistance to cisplatin. Most targeted therapies, such as kinase inhibitors, target components of signal transduction pathways. There is also evidence that DNA-damaging drugs synergize with tyrosine kinase inhibitors (TKIs) that inhibit EGFR to kill TNBC cells . Therefo2.TNBC cell lines BT20, HCC1143, MDA-MB-157, MDA-MB-231, MDA-MB-468, MFM-223, and the immortalized mammary epithelial cells MCF10A cells were obtained from the American Tissue Culture Collection . TNBC cell lines CAL120, CAL851, and HDQP1 were obtained from the German Tissue Repository DMSZ . All cell lines were tested for mycoplasma and authenticated by short tandem repeat (STR) typing. The HCC1143, MDA-MB-231, and MDA-MB-468 cells were cultured in RPMI  containing 10% fetal calf serum ; the HDQP1 cells were cultured in DMEM (Sigma-Aldrich) containing 10% FCS; the CAL120 and CAL851 cells were cultured in DMEM (Sigma-Aldrich) containing 10% FCS, 1 mM sodium pyruvate, and 2 mM glutamine (Life Technologies); the BT20 cells were cultured in DMEM-HAM F12 (Sigma-Aldrich) containing 10% FCS; the MDA-MB-157 cells were cultured in Leibovitz L15 (Sigma-Aldrich) containing 10% FCS. MCF10A cells were cultured in MEGM with 0.5 mL insulin, 0.5 mL hydrocortisone, 0.5 mL rhEGF and 2 mL BPE. Cells were incubated at 37 °C and 5% COIpatasertib (S2808), TAK-632 (S7291), PF-00562271 (S2672), Dorsomorphin (S7840), CHIR-98014 (S2745), Everolimus (S1120), PF-4708671 (S2163), and Stattic (S7024) were purchased from SelleckChem, whilst AICAR (Acadesine) (NA03552) and QNZ (EVP4593) (1237744-13-6) were purchased from Carbosynth, and U0126 (V1121) was purchased from Promega.QNZ (EVP4593), Ipatasertib, Stattic, CHIR-98014, PF-4708671, AICAR (Acadesine), PF-00562271, and U0126 were made up to 10 mM stocks, whilst Dorsomorphin (7.4 mM) and Everolimus (10μM) were also reconstituted in DMSO to the stock concentrations listed. Cisplatin (Selleckchem Cat #S1166 50 mg) was prepared in a stock solution in DMF at a concentration 12 mg/mL (39.99 mM).3 cells/well for MDA-MB-157 cells, 4 × 103 cells/well for CAL851 cells, and 3 × 103 cells/well for the other cell lines were seeded in 96-well plates. Following overnight incubation at 37 °C, drugs were added at the indicated concentrations and incubated for 5 days at 37 °C. Cell proliferation was determined using the acid phosphatase assay, as described previously [50 values) and combination index (CI) values were determined using the Chou–Talalay equation on CalcuSyn software. Loewe synergy was calculated using Combifit Analysis ™ [A total of 5 × 106 cells per well whilst CAL851, MCF10A, and HDQP1 cells were seeded at 0.3 × 106 cells per well, and MFM-223, MDA-MB-468, and MDAMB-157 cells were seeded at 0.5 × 106 cells per well in 6 well plates. Cell density was chosen to reflect 75–80% confluence prior to starting treatment. Afterward, cells had the attached media removed and replaced with serum-free medium at least 16 h prior to treatment with inhibitors.On day 0 of the experiment, MDA-MB-231 cells were seeded at 0.45 × 10On day 1, cell lines were treated with the relevant drug as per Protein quantification was performed on samples using the bicinchoninic acid assay (Pierce Biotechnology) and subsequently stored at −80 °C until required.Samples were prepared for RPPA analysis by adjusting protein concentrations to 1 μg/μL and by the addition of 4× SDS Sample buffer  +1/10 Bond breaker TCEP solution . Samples were heated to 96 °C for 5 min and the prepared sample was collected and stored at −80 °C until further analysis.Baseline and on-treatment determination of proteins/phosphorylated proteins in the panel of 6 TNBC cell lines and the MCF10A cell line were determined by RPPA as described previously . The anthttps://github.com/dirkfey/TNBC-RPPA accessed on 15 February 2022).Network inference was performed for each cell line using the corresponding perturbation data by applying the BMRA algorithm described previously, and available as MATLAB code . The priHierarchical clustering using Euclidean distance and average linkage was performed using the MATLAB R2019b clustergram function.p-value < 0.05 was considered significant.Spearman’s rank correlation was conducted using the PRISM software package, and a 50 to cisplatin in the immortalised normal breast epithelial cell line MCF10A (5.25 ± 0.55 µM). Taking their IC50 as a guide to avoid harming normal epithelial tissue, we categorised our panel of TNBC cell lines as cisplatin-sensitive if they had an IC50 of < 5 µM, or cisplatin-resistant if they had an IC50 to cisplatin of >5 µM. We also classified cisplatin response in our panel of TNBC cell lines based on their molecular subtype, as determined by Lehmann et al. [We tested the antiproliferative effects of cisplatin in a panel of eight TNBC cell lines representing the main subtypes of TNBC . To defiWe utilised a systems biology approach with reverse protein array data to determine inferred networks that predict inhibitor sensitivity. We performed RPPA analysis on a panel of seven TNBC cell lines that included three cisplatin-resistant and three cisplatin-sensitive cell lines and the normal MCF10A cell line. We tested a panel of 11 inhibitors that target nodes of pathways found altered in breast cancer, including the AMPK, STAT, NFkB, PI3K/AKT, and MAPK/ERK pathways in each cell line. The RPPA contained a panel of 72 proteins, which was enriched for members of the PI3K/AKT and MAPK pathway to determine differences in protein expression and phosphorylation between untreated and inhibitor-treated cell lines.First, we wanted to understand if there were any drug-specific response patterns across the cell lines. Thus, we clustered the treatment-induced fold changes for each drug across the seven cell lines . A few oNext, we identified the cell-line specific interaction networks from these data using Bayesian molecular response analysis (BMRA) . BMRA usp-value < 0.05, two-sample students t-test) in the resistant versus sensitive network were (1) markedly increased inhibition of p38 MAPK by AKT, (2) change from activation to inhibition of CHK1 by GSK3B, (3) change from activation to inhibition of PTEN by AMPK, (4) a loss of negative feedback from AKT to PI3K, and (5) a change from inhibition to activation of PDK1 by FAK  should be active in the basal state. Thus, we assessed the networks’ basal activity states in the unperturbed condition . Severaln = 299), AKT1 mRNA expression was downregulated compared to both HER2 positive n = 127) and hormone receptor positive patients , but not PIK3CA (p = 0.032 > 0.01) and PDPK1 (p = 0.14), significantly associated with poor relapse-free survival in triple negative patients  is upregulated in resistant cell lines, (ii) features several crosstalk and feedbacks that are more active in resistant cell lines, (iii) overrides DNA damage checkpoints by inhibiting CHK1, (iv) enhances survival by suppressing p38 activity, and (v) promotes cell proliferation by inactivating the cell-cycle inhibitor p27kip .n = 237) showed that TP53 mutations occur in 81.9% of TNBC patients whilst PIK3CA mutations occur in 14.8% TNBC patients. Further, we determined that PIK3CA and TP53 mutations are neither mutually exclusive nor do they significantly co-occur in TNBC patients [The results of this computational analysis reverberate with biological findings. PTEN and PI3KCA  are often mutated in TNBC resulting in the constitutive activation of the PI3K-AKT pathway . Howeverpatients ,18, sugg50s ranging from 0.11 ± 0.02 μM in MFM-223 cells to 7.34 ± 3.40 μM in HDQ-P1 cells. We correlated sensitivity of the S6-kinase inhibitor with cisplatin and found no significant correlation . We further assessed the GSK3B inhibitor CHIR-98014 in our panel of TNBC cell lines, but found limited antiproliferative response with only one cell line achieving an IC50 at 1 μM (MDAMB-468). Lastly, we tested the PI3K (αδ specific) inhibitor taselisib and the AKT inhibitor ipatasertib in a panel of TNBC cell lines. As expected, cell lines with PIK3CA mutations were most sensitive to the PI3K/AKT inhibitors.Therefore, we focused our investigation on possible links between TP53 and PIK3CA mutational status and cisplatin response in TNBC cell lines, which were treated with cisplatin and a PI3K/AKT pathway inhibitor. For this, we tested inhibitors for multiple points along the PI3K/AKT/S6-kinase axis that were identified from our systems biology analysis . The S6-In our TNBC cell-line panel, we demonstrated that the proliferation of cisplatin responsive TNBC cell lines with or without TP53 mutations could be decreased by inhibiting key nodes of the PI3K/AKT S6 kinase pathway. We therefore determined the antiproliferative effects of combining a PI3K inhibitor (taselisib) or the AKT inhibitor ipatasertib with cisplatin in a panel of cisplatin-sensitive/resistant TNBC cell lines . The resWhen we analysed the synergism to PI3K inhibitor and cisplatin in the remaining cisplatin-resistant cell lines, we found that the combination showed limited synergy in cell lines that harboured TP53 mutation but were PIK3CA wildtype (WT) . We also found that the CAL-51 cell line which is cisplatin sensitive and PIK3CA WT but harbours a TP53 mutation showed reduced synergy to the combination of cisplatin and a PI3K inhibitor. Finally, the BT20 cell line which is cisplatin sensitive but has mutations in both PIK3CA and TP53 exhibited strong synergism to the combination of cisplatin and PI3K inhibition. However, the strongest synergism between cisplatin and PI3K was observed in MFM223 cells. To test whether this synergy extends down the pathway, we combined an AKT inhibitor with cisplatin, also observing a strong synergy B.Overall, our analysis suggests that cisplatin-resistant TNBC cell lines can be resensitised to cisplatin by combination therapies with either a PI3K inhibitor or AKT inhibitor, if they also harbour mutations in PIKC3A and TP53.Chemotherapies that target DNA including anthracycline- and platinum-based drugs contribute towards the standard treatments for many cancers . In TNBCDespite these advances, cisplatin’s efficacy to treat cancer can be limited due to either acquired or intrinsic resistance—a clinical observation also observed in TNBC . ResistaWe utilised RPPA data and applied BMRA to reconstruct signalling pathways and their rewiring in cisplatin-sensitive and cisplatin-resistant cell lines. It is important to acknowledge that the TKIs used in this study—similar to all kinase inhibitors—may have off-target effects. However, it is not currently possible to determine every single off-target effect of specific TKIs . TherefoThe BMRA analysis reconstructed two consensus networks, one from cisplatin-sensitive and the other from cisplatin-resistant cell lines showing that a large number of connections were rewired either qualitatively or quantitatively. Looking at how the drug-resistant network could be converted into a sensitive configuration initially suggested that cisplatin-resistant TNBC cell lines should have greater sensitivity to inhibition of either p70S6-kinase or GSK3B. However, in our panel of TNBC cell-line sensitivity to either p70S6-kinase (M2698) or GSK3B (CHIR-98104) did not correlate with cisplatin resistance. We showed that the MFM223 cell line which is cisplatin resistant is the most sensitive to M2698, and previous studies also displayed M2698 effectively inhibited in vivo TNBC tumour growth. However this antitumour effect could be associated with cell lines that harboured PIK3CA mutations , such as50s < 1 μM. Again, the PIK3CA mutant MFM223 cells had the greatest sensitivity to both taselisib (PI3Kα/δ) and ipaterisib (AKTi).It is known that PTEN and PI3KCA  are often mutated in TNBC, resulting in the constitutive activation of the PI3K-AKT pathway . The frep-AKT in cancer cells also increased [Several studies have targeted the PIK3/AKT pathway in TNBC ,30 with ncreased . HoweverTNBC features frequent TP53 mutations, and previous studies have linked TP53 mutation status with increased activity of the PIK3CA gene and downstream PI3K-AKT signalling . AstanehTherefore, to confirm that targeting TNBC cell lines that harbour either or both TP53 and PIK3CA mutations is an effective treatment for cisplatin-resistant TNBC, we tested combinations of either PI3Ki or AKTi with cisplatin. We found that TNBC cell lines responded synergistically to the combination; however, the MFM223 cell lines which harbour a dual PIK3CA TP53 mutation and were cisplatin resistant responded best to both combinations.Further assessing why this combination of drugs has robust anticancer effects in cisplatin-resistant TNBC cell lines; studies have associated that cell lines with PIK3CA mutations are associated with chemotherapy resistance . SpecifiIn conclusion, our results demonstrate that TP53- and PIK3CA-mutant TNBC cell lines with innate resistance to cisplatin are sensitive to drug combinations of cisplatin and PI3K/AKT pathway inhibitors, representing a potential therapeutic approach that warrants further investigation.


ERCC2 rs13181 and rs1799793, ERCC1 rs11615) were identified on both DNA and RNA derived libraries in association with CDDP resistance. A change of the GSTP1 rs1695 polymorphism from AA to AG genotype was observed in the RNA of all six CDDP-resistant variants. These SNPs emerged to be causally associated with CDDP resistance in HGOS cells.Cisplatin (CDDP) is a drug for high-grade osteosarcoma (HGOS) treatment. Several germline pharmacogenetic studies have revealed associations between single nucleotide polymorphisms (SNPs) and CDDP-based therapy response or CDDP-related toxicity in patients with HGOS. Whether these variants could play a biological role in HGOS cells has not been studied so far. The aim of this study was to explore 28 SNPs of 14 genes in 6 CDDP-resistant and 12 drug-sensitive human HGOS cell lines. An innovative multimodal targeted next generation sequencing (mmNGS) approach with custom primers designed for the most commonly reported SNPs of genes belonging to DNA repair, CDDP transport or detoxification, or associated with CDPP-related toxicity was applied. The mmNGS approach was validated by TaqMan genotyping assays and emerged to be an innovative, reliable tool to detect genetic polymorphisms at both the DNA and RNA level. Allele changes in three SNPs ( High-grade osteosarcoma (HGOS), the most common malignant tumor of bone, is treated by surgery and systemic neo-adjuvant multidrug chemotherapy ,2. CisplPharmacogenetic studies have revealed several single nucleotide polymorphisms (SNPs) of genes belonging to either DNA repair, drug transport, folate metabolism, and detoxification pathways to be associated with therapy-related parameters in HGOS, as survival and drug response, or development of drug-associated toxicity . The genIn the last decade, pharmacogenomic approaches have been increasingly applied to the study of HGOS providing a series of interesting insights related to genetic polymorphisms, which may be causally related to drug resistance or susceptibility to develop treatment-related adverse toxicities ,6. HowevThis analysis was performed by using an innovative multimodal targeted next generation sequencing (mmNGS) approach that allowed for the contemporary study of the selected SNPs on both DNA- and RNA-derived libraries. Data obtained by mmNGS on DNA-derived libraries were validated by TaqMan genotyping. RNA expression level of the 14 genes in CDDP-resistant variants compared to their parental cell lines was also determined. Heatmap analysis was performed, including all CDDP-resistant and drug-sensitive cell lines.Data obtained for the 28 SNPs on DNA-derived libraries by the custom mmNGS approach were validated by TaqMan genotyping in 24/28 SNPs. Variants with an allele frequency greater than 3% were considered reliable. Those SNPs that were homozygous wild-type for the reference allele were not reported in By comparing the data obtained from sequencing and genotyping, we found that for 11/18 (61%) cell lines data obtained by both techniques matched in 100% of the SNPs, whereas in 4/18 (22%) cell lines the match ranged from 90 to 93%, and was below 90% in 3/18 (17%) cell lines. The fact that 39% of the cell lines did not show a complete match could be explained by the presence of different subpopulations within the same cell line.ABCC2 rs17222723, ACYP2 rs1872328, TPMT rs12201199, rs1142345, and rs1800460 the homozygous wild-type genotype was identified in all 18 cell lines.Interestingly, for five SNPs, ERCC2 gene, (rs13181 and rs1799793), which exhibited a genotype change in relation to the acquisition of CDDP resistance , while in the variant with the highest resistance level (U-2OS/CDDP4µg) the genotype of the polymorphism shifted to homozygous wild-type (TT).In the CDDP-sensitive, parental cell line and in the two variants with the lower level of CDDP resistance (U-2OS/CDDP300 and U-2OS/CDDP1µg), the genotype of ERCC2 rs13181 in U-2OS/CDDP1µg variant for which mmNGS reported GT but TaqMan genotyping a TT genotype. This apparent discordance may be due to the different sensitivity of the techniques and the presence of subpopulations with TT and GT genotypes. However, these data indicate that in these cells the transition toward a TT genotype is associated with development of CDDP resistance.The data obtained for these SNPs by TaqMan genotyping and mmNGS were concordant for all cell lines except for ERCC2 rs1799793, the sensitive cell line and the two U-2OS/CDDP300 and U-2OS/CDDP1µg resistant cell lines showed a heterozygous variant genotype CT, which became homozygous (CC) in the variant with the highest resistance level  GAT, was detected.All genotype variations identified at the DNA level and described above were also identified on the RNA level, indicating that these changes had been selected and maintained during development of CDDP resistance. Differently, the ibraries . The genGSTP1 rs1695 variant allele was identified by the CLC GWB in all three CDDP-resistant U-2OS variants , which estimates the fold-change in RNA expression, for each CDDP-resistant variant compared to its drug-sensitive parental cell line is graphically shown in ABCC2, ABCC3, ACYP2, COMT, ERCC2, and XRCC3 emerged to be increased more than 2-fold compared to the parental U-2OS cell line, whereas four genes, ATM, ATR, TP53, and XPA were downregulated in CDDP-resistant variants. Considering all three CDDP-resistant variants together, the differential gene expression tool of the CLC GWB identified the downregulation of ATM, ATR, and TP53 as significant with a Bonferroni corrected p-value < 0.05.In U2OS-derived CDDP-resistant variants, six genes, ABCB1, ABCC2 and XRCC3 in all three variants, whereas ACYP2, COMT and ERCC2 only in Saos-2/CDDP300, the variant with the lowest resistance level. CDDP-resistant variants also presented downregulation of ATM, ATR, GSTP1, TPMT, and XPA genes. Evaluating all three CDDP-resistant variants together, a significant difference after Bonferroni correction with a p-value < 0.05 was identified for upregulation of ABCB1 and downregulation of TPMT and XPA.In Saos-2-derived CDDP-resistant variants, six genes were increased more than 2-fold: Similarities between CDDP-resistant and CDDP-sensitive cell lines were assessed by using the heatmap tool of the CLC GWB, including all 14 genes . Two maiThe 10 drug-sensitive cell lines formed the second main cluster, which was mostly separated from that of CDDP-resistant variants.As also shown in In this study, a custom mmNGS approach has been used to study 28 SNPs of 14 genes, contemporarily on the DNA and RNA level, in 6 CDDP-resistant and 12 CDDP-sensitive human HGOS cell lines. To our knowledge, this innovative approach has not been used so far for pharmacogenomic studies.The successful validation of the DNA variant calling by TaqMan genotyping confirmed that this approach is an appropriate method to study even rare SNPs. Compared to genotyping by single TaqMan assays, the custom mmNGS approach is faster and also offers the possibility to identify additional SNPs mapping to the target region. Moreover, small targeted panels allow the pooling of higher sample numbers compared to whole-genome NGS. Another advantage of the mmNGS approach is the low amount of starting material that is required for library preparation, which facilitates the application of this method to tumor tissue samples. In addition, the simultaneous analysis of SNPs on the DNA and RNA level, as well as the possibility to estimate the level of RNA expression associated with the polymorphic gene status allow for a direct correlation between the genotype status with the biological function of each SNP.The frequencies of variant alleles per cell line ranged from 9 (found in IOR/OS15 and MG-63) to 22 (detected in IOR/SARG), confirming the heterogeneity and high genetic instability of HGOS.ACYP2 rs1872328 [ABCC2 rs17222723 [TPMT rs12201199, rs1142345, and rs1800460 [ABCC2 rs717620 and GSTP1 rs1695, were found as homozygous wild-type or heterozygous but not as homozygous variant. These findings suggest that the variant allele of these seven SNPs could be of biological disadvantage in HGOS tumor cells. Differently, the two SNPs of TP53, rs1042522 and rs1642785, were identified either in a homozygous wild-type or variant but not heterozygous status.Particular genotype distributions were found for 9 SNPs. Five SNPs that had been reported in association with CDDP-related ototoxicity, s1800460 ,11, wereGSTP1 rs1695, ERCC2 rs13181, ERCC2 rs1799793, and ERCC1 rs11615.The most relevant SNPs that emerged in this study to be associated with the development of CDDP resistance were GSTP1 rs1695 was the only SNP for which the genotype changes found in the RNA-derived libraries differed from those revealed in DNA-derived libraries. Interestingly, in all five drug-sensitive cell lines with the heterozygous genotype in the DNA , the genotype status in the RNA was homozygous wild-type. The presence of the variant also in the RNA of all six CDDP-resistant cell lines, with the consequent amino acid change Ile105Val, strongly suggests that the AG genotype is associated with reduced CDDP response. These findings further support the previously demonstrated relevance of GSTP1 enhanced enzymatic activity in these CDDP-resistant HGOS cell lines [The ll lines . The phaThis observation is concordant with the data reported in almost all germline studies. A significant association between AG+GG genotypes and poor histological response as well as decreased event-free and overall survival was observed in five studies ,14,15,16GSTP1 rs1695 was excluded from further analyses in the study by Goricar and co-workers because the genotype frequencies of rs1695 were not in Hardy Weinberg Equilibrium [Interestingly, ilibrium . Since tERCC2 rs1799793 and rs13181 and the synonymous ERCC1 rs11615 at the DNA and RNA level. All three SNPs have been reported to be associated with survival and toxicity, but the data are quite discordant [Genotype changes in relation to CDDP resistance were found for the two non-synonymous SNPs scordant ,27,28,29ERCC2 rs1799793 GG genotype was reported in association with poor event-free survival compared to the GA +AA genotypes and ERCC2 rs13181 AA with poor response to chemotherapy compared to AC+CC [However, the ERCC2 rs13181 and ERCC2 rs1799793 SNPs were associated with survival [In one germline study performed on 130 patients with osteosarcoma treated with neoadjuvant cisplatin-based therapy in combination with doxorubicin, methotrexate, and ifosfamide the survival . The autERCC2 rs1799793 and rs13181 on the protein structure and stability have recently been elucidated [The functional consequences of ucidated ,31. MoleERCC2 rs13181 variant caused the loss of C-terminal alpha-helix and beta-sheet [In addition, the ta-sheet . AlthougERCC1 rs11615, which changed to the homozygous wild-type genotype status in the Saos-2 CDDP-resistant variant with the highest level of resistance, two germline studies reported similar evidence being better survival associated with the TT compared to the CC genotype [For genotype ,25. Howegenotype ,21,22,23ATM and ATR. It has been shown that cancer cells that are deficient in one DNA repair pathway can activate other functional repair pathways, which underlines the importance to study not only one of them for treatment optimization [Differential gene expression analysis identified dysregulations in CDDP-resistant variants compared to their parental cell lines suggesting that development of CDDP resistance influences not only genes of the NER pathway, which is known to be mainly responsible for the removal of CDDP-associated DNA adducts, but also genes belonging to other DNA repair mechanisms, such as mization .ATM and ATR observed in Saos-2/CDDP-resistant variants is a relevant finding that needs to be further explored, since inhibitors against ATR have already been used in clinical trials in other cancers [ACYP2 and COMT, although not significant, warrants attention because SNPs of these two genes had been described to be associated with ototoxicity after CDDP treatment [The biological consequence of the significant downregulation of  cancers . On the reatment ,9,10,11.In conclusion, the mmNGS approach emerged to be an innovative, reliable tool to detect genetic polymorphisms at both DNA and RNA level, allowing for the identification of genetic changes causally related to CDDP resistance in HGOS cells. Once further validated in tumor samples series, these SNPs could be useful to identify patients with reduced sensitivity to CDDP-based therapy and/or increased susceptibility to CDDP-related adverse toxicities.The study was performed on a panel of 12 drug-sensitive human HGOS cell lines: U-2OS, Saos-2, MG-63, and HOS  and IOR/OS9, IOR/OS10, IOR/OS14, IOR/OS15, IOR/OS18, IOR/OS20, IOR/MOS, IOR/SARG, which were established from tumor specimens at the Laboratory of Experimental Oncology of the Orthopaedic Rizzoli Institute .The panel of 6 CDDP-resistant variants derived either from U-2OS  or Saos-2  CDDP-sensitive cell lines, as previously reported . ResistaCDDP sensitivity of each cell line was expressed as IC50 (drug concentration resulting in 50% inhibition of cell growth after 96 h of in vitro treatment). The fold-increase in CDDP resistance of each variant was determined by comparing its IC50 value with that of its corresponding parental cell line and, as previously described, ranged from 4.0- to 62.5-fold for U-2OS variants and from 7.4- to 112.1-fold for Saos-2 variants [2 at 37 °C. Drug resistant variants were continuously cultured in the presence of the CDDP concentrations used for their selection. Cell pellets were prepared according to standard procedures when cells were confluent, snap-frozen, and stored at −80 °C.All cell lines were cultured in Iscove’s modified Dulbecco’s medium (IMDM) added with 10% fetal bovine serum  and maintained in a humified atmosphere with 5% CODNA fingerprint analyses were performed for all cell lines using 17 polymorphic short tandem repeat sequences confirming their identity.DNA and RNA were simultaneously isolated and purified from the same pellet obtained from each cell line by using the AllPrep DNA/RNA mini kit  according to the manufacturers’ instructions. During this process, the DNA and RNA were isolated from the entire sample by passing the lysate first to the AllPrep DNA spin column to isolate high molecular weight total genomic DNA and through the AllPrep RNA spin column to isolate total RNA.A DNA and RNA quality check was performed for all samples by spectrophotometry . All RNA samples were run on a 2100 Bioanalyzer system  using the RNA 6000 kit .Library preparation was performed according to the QIAseq Multimodal Panel handbook v06/2020  for small panels. The primers for the libraries derived from DNA were designed for 28 SNPs of 14 genes related to DNA repair, CDDP transport and detoxification, and TP53 . For theDNA- and RNA-derived libraries were prepared for all 18 cell lines. Prior to library preparation, the nucleic acid concentrations were determined fluorometrically by Qubit high-sensitivity assays on a Qubit reader version 4.0 .For library preparation, the input amount was 40 ng of DNA and 100 ng of RNA. All libraries were run on a 2100 Bioanalyzer system  using the High Sensitivity DNA kit  to check the profile of the samples. The fragment lengths of all libraries ranged between 400 and 600 base pairs, as expected according to the protocol.In order to provide an accurate quantification of the amplifiable libraries, the QIAseq Library Quant Assay kit  was performed on a real-time PCR system  for all of them.For sequencing, libraries were diluted to 1.2 pM, pooled together and analyzed by paired-end sequencing on a NextSeq 500 instrument  using a mid-output reagent kit v2.5 (300 cycles) with a custom sequencing primer provided with the library preparation kit.All bioinformatic analyses were performed using the CLC GWB software  v22.04. FastQ files were downloaded from the BaseSpace cloud  and imported in the CLC GWB .For the detection of DNA variants and gene expression, the FastQ files were analyzed using the Biomedical Genomics Analysis plugin running the Qiaseq Multimodal Analysis workflow.The DNA and RNA reads were aligned to the human genome hg38 reference sequence and filtered using a coverage of 100× and a variant allele frequency (VAF) higher than 3%.For RNA variant calling a custom workflow was provided by the Qiagen bioinformatics support. This workflow worked with UMIs, mapped the reads on the human genome hg38 and filtered with specific parameters for rare RNA variant calling.p-value < 0.05 were considered significant.For differential gene expression analysis between the groups of drug-resistant variants and their respective parental cell line, the differential expression tools of the CLC GWB were used and changes with a Bonferroni corrected For hierarchical clustering analysis the tool for creating heatmaps of the CLC GWB was used with Euclidean distance and complete linkage.In total, 24 of the 28 selected polymorphisms were validated by real-time genotyping PCR . TaqMan The genotyping experiments were performed according to standard protocols using 10 ng DNA as input material using the VIIA 7 DX realtime PCR system  and the results were analyzed with the TaqMan Genotyper software , which generated allelic discrimination cluster plots to determine the genotype of each SNP.