Melanoma is the most aggressive, deadliest form of skin cancer. Combined BRAF-MEK inhibitor (BRAFi/MEKi) therapy was a breakthrough in the treatment of melanoma with BRAFV600-mutations. However, many patients frequently develop drug resistance to the combinatory treatment. The aim of our study was to characterize the molecular background behind acquired resistance to BRAFi/MEKi-s. After the successful development of resistant cell lines, we investigated the invasion properties, changes in gene and protein expressions, as well as the effect of the “drug holiday” of the resistant cell lines. Drug-resistant melanoma cells had a higher invasive potential and acquired a spindle-like structure, and many cancer-related proteins were overexpressed in the resistant cells. Furthermore, transcriptome analysis revealed that differentially expressed genes are functionally linked to a variety of biological functions that may lead to resistance to the inhibitors. These results may offer valuable insight into further understanding of BRAFi/MEKi resistance, as well as to the development of therapeutic tools to overcome drug resistance.V600E/KBRAF mutant melanoma. However, acquired resistance largely limits the clinical efficacy of this drug combination. Identifying resistance mechanisms is essential to reach long-term, durable responses. During this study, we developed six melanoma cell lines with acquired resistance for BRAFi/MEKi treatment and defined the molecular alterations associated with drug resistance. We observed that the invasion of three resistant cell lines increased significantly compared to the sensitive cells. RNA-sequencing analysis revealed differentially expressed genes that were functionally linked to a variety of biological functions including epithelial-mesenchymal transition, the ROS pathway, and KRAS-signalling. Using proteome profiler array, several differentially expressed proteins were detected, which clustered into a unique pattern. Galectin showed increased expression in four resistant cell lines, being the highest in the WM1617E+BRes cells. We also observed that the resistant cells behaved differently after the withdrawal of the inhibitors, five were not drug addicted at all and did not exhibit significantly increased lethality; however, the viability of one resistant cell line (WM1617E+BRes) decreased significantly. We have selected three resistant cell lines to investigate the protein expression changes after drug withdrawal. The expression patterns of CapG, Enolase 2, and osteopontin were similar in the resistant cells after ten days of “drug holiday”, but the Snail protein was only expressed in the WM1617E+BRes cells, which showed a drug-dependent phenotype, and this might be associated with drug addiction. Our results highlight that melanoma cells use several types of resistance mechanisms involving the altered expression of different proteins to bypass drug treatment.Combination treatment using BRAF/MEK inhibitors is a promising therapy for patients with advanced  BRAF oncogene are the most widespread genetic alterations observed in melanoma, with an incidence of up to 40–60% [V600EBRAFV. This mutation enhances the kinase activity of mitogen-activated protein kinase (MAPK) signalling ~500-fold and controls important cellular functions involving cell proliferation, differentiation, migration, survival, and angiogenesis [V600E/KBRAF mutations in 2018 [NRAS, MAP2K1, and MAP2K2 mutations, V600EBRAF gene mutation or amplification, and different mutations in genes in the PI3K pathway [Despite several advancements in the treatment of patients with malignant melanoma, this disease is still one of the major causes of skin cancer-associated death . Activato 40–60% ,3. The m in 2018 . This in in 2018 ,10. Many pathway . Studies pathway .V600EBRAF mutant melanoma cell line models. Sequentially, we investigated resistance-associated genomic and proteomic alterations in melanoma cells. To achieve our goal, we first established six melanoma cell lines that became resistant after continuous treatment with BRAFi/MEKi. We compared the differences in cell proliferation, protein, and mRNA expression between the drug-sensitive and drug-resistant melanoma cell lines and studied the consequence of the “drug holiday” on cell proliferation in the resistant cells. Proteome profiler analysis and RNA sequencing (RNA-Seq) transcriptome profiling analysis were performed to describe the possible alterations leading to the development of acquired resistance during encorafenib plus binimetinib treatment.Our aim during the current study was to explore the molecular alterations associated with the treatment of the ENCO+ BINI inhibitor combination in Melanoma cell lines were obtained from the Coriell Institute for Medical Research . All cell lines were cultured under the same conditions as described before . The BRA2 at 37 °C). The concentration of the drug combination at the beginning of treatment was 1 nM for each cell line and increased up to 200 nM. To reach the maximum concentration of the drug combination, the time window varied between three and five months. Resistant cells were maintained in complete medium supplemented with a 200 nM inhibitor mixture. Comparisons of the different parameters  between the sensitive and resistant cells were performed at the same passage number. Encorafenib (LGX818) and binimetinib (MEK162) were obtained from Selleck Chemicals LLC . Stock solutions were prepared in dimethyl sulfoxide (DMSO) and stored at −20 °C.Resistant cell lines were generated through long-term high-dose treatment using increasing concentrations of ENCO+BINI in every passage as described by Szász et al. . In brie3 cells/well/100 µL medium) were seeded into 96-well plates for 24 h. On the second day, the cells were treated with a drug combination of ENCO+BINI (1 µM) for 72 h. DMSO (solvent of the inhibitors) was added to the cells as a control. After 72 h, the cell viability was measured using the WST-1 assay . The absorbance of the samples was measured at 440 nm. The control wavelength was set to 650 nm. The viability of the cells was defined by dividing the absorbance of the encorafenib plus binimetinib-treated cells by that of the DMSO-treated control cells (the absorbance of control cells was defined as 100%).Cell proliferation was assessed using the WST-1 test  following the supplier’s protocol. Briefly, before drug treatment, melanoma cells (5 × 103 cells/well/100 µL medium) into a 96-well plate (in triplicate) with culture medium containing 200 nM of ENCO+BINI inhibitors and incubated for 24 h. The next day, cells were switched to growth medium containing DMSO and were cultured for 72 h or were maintained in growth medium containing a 200 nM drug combination of ENCO+BINI for 72 h. After 72 h, 10% of WST-1 reagent was added directly to each well, and the cells were incubated for the next three hours at 37 °C. Absorbance was measured as described above. The absorbance of control cells (cells treated with DMSO) was considered 100%. Relative absorbance was transfigured into the relative percentage of viable cells.A drug holiday experiment was performed similarly as we published earlier . ResistaThe invasive potential of the inhibitor (ENCO+BINI)-sensitive and -resistant melanoma cells was evaluated using BioCoat Matrigel Invasion Chambers  as described before . ShortlyProtein expression analyses were performed as described in detail before . BrieflyTotal RNA preparation was carried out using an RNeasy Mini Kit . RNA concentration was measured using a NanoDrop ND-1000 UV-Vis spectrophotometer. Only samples with ratios >1.8 (measured at 260/280 nm) were included in further analysis. An Agilent 2100 Bioanalyzer was used to evaluate sample quality before RNA sequencing .www.strand-ngs.com; accessed on 14 October 2020) was used for further statistical analysis. Aligned data were normalized using the DESeq algorithm, and then differentially expressed genes were determined by a moderated T-test with Bejamini-Hochberg FDR for multiple testing correction. A p-value 0.05 was considered statistically significant. Library preparations, sequencing, and primary data analysis were performed at Genomic Medicine and Bioinformatics Core Facility of the University of Debrecen, Hungary.RNA-Seq and data analyses were performed as described previously . To obtap-value ≤ 0.05. The RNA-Seq data were deposited into the Gene Expression Omnibus (GEO) repository  under accession number GSE186108.Analysis of differentially expressed genes was carried out as described by Ahn et al. . The folhttps://toppgene.cchmc.org; accessed on 10 January 2021) was applied to find the functional enrichment of genes with at least a 2-fold change difference between the treated (resistant to BRAFi/MEKi) and control groups (sensitive to BRAFi/MEKi) based on GO pathways under default settings and a p-value cut-off of 0.05.To gain mechanistic insight into the gene lists generated from the RNA-Seq data, a functional enrichment analysis was performed to identify the biological pathways more enriched in a gene list than would be expected by chance. The ToppFun tool ToppGene suite .To identify gene sets from the Molecular Signatures Database (MSigDB) v7.2 , GSEA version 4.1.0 was used, which summarizes and represents specific well-defined biological states or processes and displays coherent expression values . Only significantly altered signalling pathways  were included in the analyses. Benjamini-Hochberg adjustment (FDR < 0.05) was applied as a cut-off, and pathways with ≥5 significantly differentially expressed genes were considered and used to define molecular pathways associated with the differentially expressed genes.Significant pathways associated with specific gene expression signatures were identified using the EnrichR web-based application  was used to define the relative mRNA expression of selected genes in five melanoma cell lines  using a Light Cycler 480 Real-Time PCR System . cDNA synthesis was performed applying High-Capacity cDNA Reverse Transcription Kit ) with random primers as described in the supplier’s protocol; 600 ng of total RNA for each reaction was used. SYBR Premix Ex Taq  was applied to carry out the qRT-PCR reaction. GAPDH  was used as a reference gene, and the Livak method (2^-ddCT equation) was applied for the qRT-PCR data analyses [p-value < 0.05 was considered statistically significant. Data are presented as the average ± standard deviation (±SD) of at least three independent experiments. Error bars on the figures represent ±SD.Statistical analysis was performed using SPSS  and Graph Pad Prism 9  software. Pearson’s correlation coefficient was calculated to correlate the RNA-seq and qRT-PCR data. The cellular parameters were statistically analyzed using the Mann–Whitney–Wilcoxon test. Only a V600EBRAF mutation, one the Q61LNRAS mutation (WM1366), and one (WM3211) was wild type for both genes. The viability of melanoma cells was investigated using 1 µM drug mixture for 72 h. The combined BRAFi/MEKi treatment resulted in a significant (p < 0.05) decrease in cell viability in cell lines carrying the V600EBRAF mutation (We investigated the growth-inhibitory effect of the BRAFi/MEKi combination treatment on nine melanoma cell lines. Seven cell lines carried the mutation .Q61LNRAS mutant (WM1366) or BRAF/NRAS wild-type (WM3211) melanoma cell line.The most significant decrease (more than 30%) was observed in five cell lines . WM793B cells were sensitive to the treatment, but the viability of these cells decreased by less than 10%. In contrast, there was no significant decrease in cell viability in the To establish resistant cell line variants during the ENCO+BINI treatment, the cells were treated continuously with increasing concentrations of the drug combination for 3–5 months, starting at 1 nM and increasing with every passage up to 200 nM. The morphology of the drug-sensitive cells differed from that of the ENCO+BINI-resistant cells, and the resistant cells mainly displayed an elongated phenotype .E+BRes, WM278E+BRes, and WM902BE+BRes) showed significantly enhanced invasive properties compared to their corresponding sensitive cell lines . Three drug-resistant cell lines (WM983All lines .E+BRes and WM1617E+BRes) did not change significantly. However, the WM793BE+BRes cells showed significantly decreased invasive potential compared to the sensitive cells.The invasive properties of two metastasis-derived resistant cell lines (WM983BOncology arrays (Proteome Profiler Human XL Oncology Array) were used to detect protein expression differences between BRAFi/MEKi-sensitive and -resistant melanoma cell lines. The array contained 84 cancer-related proteins. These analyses revealed numerous differentially expressed proteins in the resistant cell lines compared to their sensitive counterparts. However, we did not notice a similar expression pattern among the tested resistant cell lines . Proteins with detectable differences (>10%) in at least one cell line revealed 17 differentially expressed proteins in the resistant cell lines; data are summarized in E+BRes cells, including Survivin, Osteopontin, Amphiregulin, EGFR, FGF, and HO-1. Interestingly the expression of Galectin increased in four cell lines , being the highest in the WM1617E+BRes cells, but the expressions of other proteins were inconsistent.A number of differentially expressed proteins (n = 12) were detected in the WM983ABecause acquired resistance can lead to drug dependency, we removed the drug mixture from the cell cultures and replaced it with the drug solvent (DMSO). Unexpectedly, we did not observe significantly decreased proliferation or cell death after 72 h of “drug holiday” in five cell lines  compared to the control cell lines that were treated continuously with 200 nM ENCO+BINI, indicating that these cell lines are not addictive to the drug combination .E+BRes, WM278E+BRes, and WM793BE+BRes) exhibited significantly increased cell proliferation after 72 h of “drug holiday”, which indicates that these cells still experienced drug pressure. In contrast, the WM1617E+BRes cell line behaved differently and showed significantly reduced cell proliferation  and cultured the cells in the absence of the drug combination for 3 and 10 days and then measured the cell viability and protein expression changes and compared these to the control (cells were continuously treated with the inhibitor mixture).To determine whether drug dependence could be developed in resistant cells, we selected three cell lines  as we used before. After several days of the “drug holiday”, we detected seven proteins , Survivin, and Vimentin) whose expressions changed in all resistant cell lines following drug withdrawal .E+BRes and WM983BE+BRes cell lines. The vimentin protein expression did not change after 10 days of drug removal in any cell line. The expression of survivin was variable in the different resistant cells. It should be noted that in addition to the co-expressed proteins, several other proteins were also highly expressed: Cathepsin S, EGFR, Endoglin CXCL8, and CCL20 were detected in the WM983BE+BRes cells, and HIF-1α, Endoglin, P53, and Snail were detected in the WM1617E+BRes cells  in the resistant cell lines . Seven of the ten genes examined  exhibited the same direction of gene expression in the parental and resistant cell lines.The top 30 downregulated genes are listed in p ≤ 0.05). The gene sets that correlated with drug resistance are displayed in Using GSEA, we found several ontology gene sets that were significantly enriched, including regulation of cell proliferation, biological adhesion, and regulation of cell death, response to drug, vasculature development, regulation of cell development, and regulation of chromosome organization  enriched GO terms grouped by biological processes (n = 194), molecular functions (n = 9), and cellular components (n = 6); these included cell adhesion, cell migration, axon guidance, response to drug, regulation of the MAPK cascade, MAP kinase tyrosine/serine/threonine phosphatase activity, and the extracellular matrix (ECM)  .To further understand the involved pathways and biological functions during the development of resistance, we performed pathway analysis using MSigDB. Based on this analysis, we observed that the differentially expressed genes were associated with a wide range of molecular pathways. The significantly altered pathways (those including at least five upregulated genes) were functionally associated with the following: ATF-2 transcription factor network, AP-1 transcription factor network, EMT, TNF-alpha signalling via NF-kB, and reactive oxygen species (ROS) pathway . On the To further understand how the identified molecular pathways link to each other, a visual pathway network analysis was carried out . The interaction between key significant KEGG pathways was displayed. The approach was established on the notion that two pathways are linked if they share a certain fraction of genes . The majV600BRAF mutations in malignant melanoma, the development of new drugs, including effective small molecule inhibitors of the MAPK signalling pathway, antibodies targeting immune checkpoint inhibitors including cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death (PD)-1, and PD-ligand 1 (PD-L1), has expanded dramatically [V600BRAF mutation-positive advanced stage melanoma [Since the discovery of the atically ,18. Recemelanoma . Howevermelanoma . ENCO+BImelanoma . These nmelanoma , we alsoE+BRes) was different. Similar to our observations, morphological changes were also noted by Dratkiewicz et al., and Szász et al. [E+BRes), the resistant cell lines had higher invasive potential than the original, sensitive cell lines. Additionally, using transcriptome analysis, we observed that numerous invasive markers, including MMP2, were substantially elevated  in the resistant cell lines [During the development of the BRAFi/MEKi-resistant cell lines, we observed morphological changes of the resistant cells. The phenotype of these cells was dramatically changed compared to the BRAFi/MEKi-sensitive cells. Resistant cells were elongated and spindle shaped, and the morphology of only one cell line , followed by the WW902BE+BRes cell line (75%). In the other two cell lines, the expression differences were less than 10%. Galectin-3 is involved in many different biological functions, including cell adhesion, cell activation, the cell cycle, apoptosis, cell growth, and differentiation as reported by Mourad-Zeidan et al. [E+BRes and WM1617E+BRes), its expression increased, and in all other cell lines, its expression decreased. Furthermore, Enolase 2 (Gamma-enolase) and CapG were expressed in at least two cell lines with a greater than 10% difference, and the expression of these proteins varied greatly across cell lines. Interestingly, Enolase 2 and CapG are used as tumour markers in the diagnosis and prognosis of several cancer types, and both proteins have been associated with cell proliferation, invasion, migration, and metastatic capacity in several types of cancer, including melanoma [Several molecular changes associated with drug resistance have previously been found; these include overexpression of EGFR, PDGFR, HGF, IGF, CRAF, COT, and MITF and downregulation of STAG2 or STAG3 ,17. Durin et al. . Galectin et al. . The antn et al. . Enolasemelanoma ,33. The E+BRes and WM902E+BRes) in which galectin-3 was also highly expressed. The enhanced expression of the Vimentin protein in resistant cell lines was consistent with the findings of Molnár et al. [EMT is a complex mechanism that enables tumour cells to switch from the epithelial to the mesenchymal phenotype, and this transition allows cells to migrate from the primary site . Vimentir et al. .E+BRes); in these cells, drug withdrawal was associated with significantly decreased cell proliferation, indicating that the cells became addicted to targeted therapy, exposing potential therapeutic vulnerabilities and highlighting that intermittent dosing could potentially be beneficial for therapeutic gains for melanoma patients [E+BRes and WM983BE+BRes) responded with elevated cell proliferation during the “drug holiday”. On the other hand, drug withdrawal resulted in protein expression changes in all three cell lines. Using the proteome profiler, we identified seven proteins  with altered expression after the removal of the inhibitors. Some of these proteins are well-known players in drug resistance [The effect of continuous versus intermittent dosing of drugs is highly controversial regarding patient survival; only limited data are available about which molecular pathways are dominant in this phenomenon ,36,37. Tpatients  and can patients . In contsistance ,41,42,43CXCL12, COL5A1, ALPK2, ABCC3, CHST15, RP11-326A19.5, LAMA5, SAMD11, RP11-54O7.3, and HHI PL2. Pathway analysis revealed that the upregulated genes were significantly associated with the ATF-2 transcription factor network, EMT, and ROS. The ECM component COL5A1 has already been determined to be associated with BRAFi resistance in melanoma [ALPK2 is involved in cancer by regulating the cell cycle and DNA repair [HHIPL2-has been linked to Hedgehog signalling in gastric cancer [SAMD11 is associated with radioresistance in oesophageal cancer cells [Several studies using human melanoma cell lines have reported differentially expressed genes between BRAFi-sensitive and BRAFi-resistant cells ,44. Howemelanoma . The othA repair , and HHIc cancer . Moreoveer cells . The ATPer cells . SimilarDMRT2, MRGPRX4, TRIM51, CTD-2207A17.1, RP4-718J7.4, VEPH1, RP11-459E5.1, GJB1, ART3, and FABP7. Pathway analysis discovered that the downregulated genes were associated with KRAS and IL-2/STAT5 signalling. These genes have earlier been described to play crucial roles in cell migration and metastasis formation, and all have been implicated in tumorigenesis through different pathways; for example, DMRT and FABP7 are involved in EMT [VEPH1 suppresses vascularization by inhibiting AKT activation [VEPH1 expression suppresses EMT and invasion through TGF signalling pathway inhibition [The top 10 downregulated genes were d in EMT ,51, and tivation . Additiohibition . SimilarRP11-326A19.5, RP11-459E5.1, and RP4-718J7.4. The RP11-326A19.5 transcript was upregulated in the developed resistant cell lines, whereas RP11-459E5.1 and RP4-718J7.4 were downregulated. Additionally, it was reported previously that some lncRNAs (RP11-326A19.5 and RP11-459E5.1) are functionally involved in tumorigenesis and drug responses [RP4-718J7.4 is associated with inflammation and antibiotic resistance [Long non-coding RNAs (lncRNAs) regulate numerous biological processes in cancers through different molecular mechanisms and can be used as potential markers for monitoring therapeutic responses . Emerginesponses ,56. Furtsistance ,58. HoweOur current data offer the first insight into differentially expressed genes and provide protein expression patterns that are associated with a BRAFi/MEKi-resistant phenotype in melanoma cell lines. Our findings facilitate a more thorough understanding of the development of the complex mechanisms leading to acquired resistance during combined treatment in BRAF-mutant melanoma. However, further studies are needed to identify the key molecules and signalling pathways responsible for therapeutic escape during BRAFi/MEKi treatment and to prevent the initiation of acquired drug resistance in melanoma.


Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Among the key challenges in developing effective therapeutics is the poor translation of preclinical models used in the drug discovery pipeline. This leaves drug attrition rates and costs at an unacceptably high level. Previous work has highlighted the discrepancies in therapeutic response between current in vitro and in vivo models. To address this, we conducted a comparison study to differentiate the carboplatin chemotherapy response across four different model systems including 2D monolayers, 3D spheroids, 3D ex vivo tumors and mouse xenograft models. We used six previously characterized EOC cell lines of varying chemosensitivity and performed viability assays for each model. In vivo results from the mouse model correlated with 2D response in 3/6 cell lines while they correlated with 3D spheroids and the ex vivo model in 4/6 and 5/5 cell lines, respectively. Our results emphasize the variability in therapeutic response across models and demonstrate that the carboplatin response in EOC cell lines cultured in a 3D ex vivo model correlates best with the in vivo response. These results highlight a more feasible, reliable, and cost-effective preclinical model with the highest translational potential for drug screening and prediction studies in EOC. Most women are diagnosed at late metastatic stages III-IV, for which only 30.2% will survive 5 years after diagnosis2. Although these patients initially respond to first-line treatment (combination of cytoreductive surgery and platinum-based chemotherapy), most patients will eventually recur and develop resistance3. Despite the appeal for personalized medicine, no biomarkers have been clinically accepted to accurately predict first-line therapeutic response. To date, carboplatin chemosensitivity remains the main predictor of EOC clinical prognosis4.Epithelial ovarian carcinoma (EOC) is the most lethal gynecological cancer. In 2019, 22,530 women were diagnosed with EOC in the United States and 13,980 died of the disease5. The current drug discovery pipeline is dependent on 2D cell culture model systems that are devoid of the inherent complexity of their original tumors, which are better captured by in vivo models. In particular, 2D cultures put into question the proper representation of tumor heterogeneity due to cell selective pressures and remain devoid of immune cells and a tumor microenvironment (TME) including the extracellular matrix (ECM), tumor cell-stromal cell interactions, and additional important components6. While 2D cell models remain as the primary method of in vitro preclinical testing, attrition rates of anti-cancer drugs continue to be high and EOC survival remains low. To address this, more reliable and practical preclinical models are needed to study drug response and putative biomarkers.Significant research efforts have been invested in the discovery of new cancer treatments, with limited focus on the actual experimental models used to test new agents7. To bridge the gap between the shortcomings of 2D models and animal experimentation, there has been a shift to further develop 3D cell culture systems. Overall, 3D spheroids better mimic the structural cell–cell interactions and the chemical nutrient and oxygen gradients10 featured in different cell layers: proliferative, quiescent, and necrotic11. In the setting of EOC, this model is particularly relevant given the physiologic development of ascites  that contain clusters of cells (spheroids)12. Additionally, tumor-derived ex vivo models may better mimic the tumor heterogeneity by preserving original TME characteristics, allowing the study of interactions with cancer-associated cells.An ideal preclinical model for drug response has optimal physiologic relevance and downstream analysis compatibility, can be translated in a clinically relevant timeframe, and is affordable13. This calls into question the precision of EOC preclinical models, and it is currently unclear whether these in vitro models or an ex vivo model reliably reflects the in vivo gold standard response. To better understand the carboplatin response, we investigated two different in vitro systems  and one ex vivo system (3D micro-dissected tumors14) and compared them to the in vivo therapeutic response (xenograft mouse model)  for the same cell line and that this variation did not follow the same trend across cell lines19. Cell lines originated from patient tumors (TOV) or ascites (OV). OV4485 and OV4453 are BRCA1 and BRCA2 mutated cell lines, respectively16. Cells were cultured in complete OSE medium  supplemented with 10% fetal bovine serum , 2.5 ug/mL of amphotericin B  and 50 µg/mL of gentamicin . Cells were cultured under conditions previously determined17. OV90, TOV21G, TOV112D and OV1946 cells were maintained at 21% O2 and 5% CO2 at 37 °C. OV4485 and OV4453 were kept at 7% O2 and 5% CO2 at 37 °C. Experiments were carried out with cells between passage 70 to 80 (at ~ 90% confluence). Mycoplasma testing and short tandem repeat (STR) analysis were performed for all cell lines.Six EOC cell lines were selected for their different carboplatin sensitivities 21 were used to establish xenograft tumors with cell lines. A 200 µL suspension of 1 × 106 cells in 100 µL cold Dulbecco’s PBS  with 100 µL of Matrigel® Matrix  was injected subcutaneously in the flank of each mouse for the TOV112D, TOV21G and OV90 cells, while 5 × 106 cells were injected for the OV1946, OV4453 and OV4485 cells. Eight mice were used for the control (vehicle) group and for each of the three carboplatin treatment groups per cell line , twice weekly. Tumors were measured with calipers 2–3 times weekly. To alleviate the known negative side effects of carboplatin treatment, anti-nausea medications (1 mg/kg of maropitant and 0.8 mg/kg of ondansetron) were given one hour before the chemotherapy dose and at 24- and 48-h following treatment. Mice were sacrificed at the end of treatment period or if ethical limits were attained through an intraperitoneal injection of euthanyl  at a dose of 400 mg/kg (concentration of 240 mg/ml). Tumors were collected, measured and were formalin-fixed and paraffin-embedded (FFPE). FFPE tumor blocks were cut into 4 µm sections for histological hematoxylin & eosin (H&E) staining.All animal procedures were performed in accordance with the guidelines for the Care and Use of Laboratory Animals of the CRCHUM as well as the recommendations in the ARRIVE guidelines. This study was approved by the Comité institutionnel de protection des animaux . NOD.Cg-Rag123. For all cell lines, 2,000–2,500 cells in 100 µL of complete OSE medium were seeded in each well. Plates were centrifuged at 1,000 rpm for 5 min at room temperature. Spheroids were allowed to form over 48 h in their respective incubation conditions  microplates ions see , generat24. Briefly, tumors were sliced into 1 cm thick sections by a scalpel and placed on the McIlwain™ tissue chopper to obtain 350 µm thick slices. Tumor slices were placed in Hank’s balanced salt solution  supplemented with 10% FBS, 2.5 µg/mL of amphotericin B (Wisent) and 50 µg/mL of gentamicin (Wisent). A biopsy punch of 500 µm  pierced tumor slices to produce sphere-like MDTs, which were placed in HBSS supplemented with antibiotics without serum until the loading procedure. The loading, trapping and culturing of MDTs were performed as described in our previous work14.The micro-dissection procedure was adapted from previously published work14, which were cut into 4 µm sections for histological H&E staining. Specimen size shrink after this processing technique as previously reported14.MDTs were fixed with 10% formalin  after carboplatin treatment and recovery periods, including respective controls. Untreated spheroids were similarly fixed after 48 and 96 h of formation. All specimens were further processed through the previously published paraffin-embedding lithography procedure to create micro-dissected tissue microarray (MDTMA) blocksBased on pilot toxicity studies, carboplatin  treatment in the xenograft models was given once weekly by intraperitoneal (IP) injections at either 25, 50, and 75 mg/kg for up to six cycles. The carboplatin vehicle, 0.9% NaCl solution, was used for controls.27. A 24-h recovery was chosen based on published in vitro studies demonstrating the effect of chemotherapy only after its removal28 and to mimic the physiologic metabolism of the drug. The optimal concentration range was determined for each cell line for minimal growth inhibition and for an effect well below the 50% threshold.Optimization studies with spheroids determined the carboplatin treatment range. Thereafter, spheroids were treated by adding 100 µL of three different concentrations of carboplatin , whereas the control received 100 µL of complete OSE medium. Spheroids were treated for 24 h followed by a 24-h recovery period, based on literature suggesting that 24 h of drug exposure is required to penetrate the spheroid50 values from monolayers and 3D spheroids. Two carboplatin regimens were tested for MDTs: a 10-h treatment induction followed by a 14-h recovery, and a 16-h induction followed by a 24-h recovery.MDTs obtained from untreated xenograft tumors of our cell lines were treated with 6–7 different carboplatin concentrations based on the IC50 values for carboplatin for OV4453, OV4485, TOV112D and OV90 were previously determined by clonogenic survival assay19. Carboplatin sensitivity for the OV1946 and TOV21G cell lines was determined in this study using the same clonogenic assay16. Briefly, cells were seeded in a 6-well plate at a volume of 1 mL/well and at a density that allowed the formation of individual colonies . Cells were allowed to adhere for 16 h in 5% CO2 at 37 °C. Then an additional 1 mL of medium containing carboplatin  was added in each well and cells were incubated for 24 h. After this period, medium was completely removed and replaced with fresh OSE complete medium. When colonies became visible at 2X magnification, plates were fixed with cold methanol and stained with a solution of 0.5% blue methylene  in 50% methanol. Colonies were counted under a stereomicroscope and reported as percent of control. IC50 values were determined using Graph Pad Prism 6 . Each individual experiment was performed in duplicate and repeated three times.The IC50 values. Each experimental analysis was performed in duplicate and repeated three times.After treatment and recovery, 10 spheroids (one replicate) were pooled per condition and dissociated with trypsin–EDTA (0.05%) for 30–45 s to obtain single-cell suspensions. Two replicates were done per experiment. Single cells were labelled using the LIVE-DEAD™ Fixable Aqua Dead Cell Stain Kit  stain at 1:100 dilution. After an incubation of 15 min at room temperature, stained cells were analyzed by flow cytometer, LSR-Fortessa , using 405 nm excitation, and fluorescence emission was monitored at 525 nm. Data were analyzed using FlowJo , identifying dead cells (stained) and live cells (non-stained). Normalized live and dead cell rates were plotted using GraphPad Prism 6 (GraphPad Software Inc.) to generate dose–response inhibition curves with respective ICFFPE xenograft blocks were cut into 4 µm sections and placed on Fisherbrand superfrost plus microscope slides . MDTMA blocks (MDT and spheroids) were sliced into 4 µm thick slices and placed on Matsunami TOMO® hydrophilic adhesion slides . Treatment response in xenograft and MDT experiments was assessed by IF, and viability in untreated spheroids was assessed by IHC. Anti-Ki-67 antibody (cell proliferation) and DAPI (nuclei detection) were used for xenografts and MDTs. Anti-human mitochondria and anti-cytokeratin 8/18  antibodies were additionally used for MDTs. Lastly, antibodies for IHC in spheroid experiments included anti-Ki-67 and anti-cleaved caspase-3 .IF/IHC slides were stained using the BenchMark XT automated stainer . TOMO slides were incubated at 60˚C for 20 min before staining. For IF staining, antigen retrieval was carried out with Cell Conditioning 1 solution (VMSI) for 60 min. Mouse anti-Ki-67 (1:500) antibody , mouse anti-CK8 (1:200) antibody , mouse anti-CK18 (1:200) antibody , and mouse anti-human mitochondria (1:2500) antibody  were automatically dispensed. Slides were incubated at 37 °C for 60 min. Secondary antibodies (dilution 1:250) including Alexa 488  and TRITC  were added at room temperature. For IHC staining, antigen retrieval was carried out automatically with Cell Conditioning 1 solution for 30 min (CC3) and 60 min (Ki-67). Rabbit anti-CC3 (1:200) antibody  and mouse ant-Ki-67 (1:500) were automatically dispensed followed by horseradish peroxidase secondary antibody. Counterstaining was achieved with hematoxylin and bluing reagent (VMSI). All sections were scanned with an Olympus BX61 microscope using 20 × 0.75 NA objective with a resolution of 0.3225 μm .http://visiopharm.com).Stained tumor sections were quantified using VisiomorphDP software version 2020.08  detection of core surface area through DAPI, 2) detection and calculation of total epithelial area within the core through Alexa-488, 3) identification of nuclei of each epithelial cell and calculating total nuclei area through DAPI, and 4) identification of Ki-67 positive nuclei and calculating total positive area for each stain through TRITC.50 of 3D models was calculated by transforming all concentrations into logarithms, normalizing the response, and performing nonlinear regression analysis (dose–response inhibition equation – variable slope). P values < 0.05 were considered significant. All statistical analyses were done using GraphPad Prism version 6 .Values are expressed as the means ± standard error of the mean (SEM) from at least three independent experiments in the case of 3D spheroids. We used eight tumors per condition per cell line for the xenograft model and 15 MDTs per condition per cell line. Comparison between multiple groups (different carboplatin concentrations) was determined by one-way ANOVA comparison test. The IC19 and represent the diverse range of EOC subtypes  and response to carboplatin treatment  and resistant cell lines have IC50 values above 10 µM (TOV112D and OV90). Cell lines with IC50 values in between are categorized as intermediate (OV1946 and OV4485). This same 2D carboplatin sensitivity criteria have been used previously by others29.The selected EOC cell lines have been extensively characterized in vivo tumor growth. OV1946 and OV4453 were categorized as sensitive, demonstrating tumor volumes that were significantly lower than the controls at time of sacrifice for all three carboplatin doses (highly responsive). OV90 and OV4485 showed intermediate responses with a significant decrease in tumor volumes at the two highest doses but no response to the lower dose . TOV21G and TOV112D were resistant as they showed no statistical difference at even the highest dose (unresponsive). IF with Ki-67 was quantified from collected xenografts after carboplatin treatment  in patients 30. Therefore, cell lines with IC50 values higher than 250 µM were considered resistant. On the other hand, response to doses below 100 µM were considered sensitive based on previous reports of carboplatin treatment of 3D ovarian cancer models32. Response between the two cut-offs were considered intermediate. Using these criteria, OV1946 was categorized as sensitive, OV90 as intermediate, and TOV21G, TOV112D, OV4453 and OV4485 as resistant. These results show a positive correlation with the in vivo response for 4/6 EOC cell lines , TOV21G and TOV112D were resistant (IC50 > 250 µM), and OV4485 was intermediate. These findings were in complete agreement with the in vivo chemosensitivity results (5/5 EOC cell lines) and provided the best correlation compared to the other two in vitro models  and in vivo responses. Interestingly, we observed some notable differences between 2D culture and in vivo carboplatin responses. In the case of TOV21G, both its clear cell histology and microsatellite instability35 supports the in vivo response of a platinum-resistant cell line. However, 2D culture experiments have consistently shown this cell line as carboplatin sensitive (Supplementary Table 37). For OV90 and OV1946, an increased sensitivity to carboplatin is seen in mice. This may be due to their histological high-grade serous subtype of which the majority of patients respond to first-line platinum treatment3. Indeed, none of our high-grade serous cell lines showed in vivo resistance to carboplatin, but showed sensitivity in the 2D or 3D spheroid models.The mainstay of preclinical studies remains cell line-based and the in vivo response from animal models is often used as the gold standard in preclinical testing of novel therapies/combinations. To our knowledge, only one study38. Increasing in model complexity is our ex vivo tumor model of MDTs that not only offers a 3D structure, but also includes mouse-infiltrating stromal cells which may impact the tumor response to a therapeutic agent. In vivo models further increase model complexity by incorporating important elements such as drug metabolism, influence of endogenous hormones and mammalian physiology39. In general, our study suggests that the relative carboplatin response of our 3D models was in line with in vivo results. However, two cell lines, OV4453 and OV4485, did not have concordant results as 3D spheroids and demonstrated higher carboplatin resistance in our spheroid model. We suspect that this may be related to their low oxygen culture conditions (7%), which was specific for only these two cell lines. Hirst et al.40 showed that an increase in hypoxia-regulated genes and markers of stemness were present in the core of 3D spheroids but not in monolayered cells and that this induced chemoresistance and phenotypic changes. In addition, these cell lines formed spheroid aggregates that were not compact and had larger spheroid diameters, which has been shown to influence drug resistance41.Given that each model has unique features, their relative response to cytotoxic therapy may vary. Immortalized EOC monolayer cultures offer little cell–cell interaction and consist uniquely of a sub-clonal population of epithelial cancer cells. While spheroids also consist of mostly epithelial cancer cells, they offer a 3D structure with inherent cell layers, cell–cell interactions and chemical/nutrient gradients24, control of fluids and constant supply of nutrients42, long-term viability44 and the maintenance of MDTs and their TME24 without need of growth supplements46. This model further allows testing multiple cycles of cytotoxic therapies as well as studying the effects of cytostatic drugs that require longer incubation periods. This underscores the need to incorporate these models into the drug development pipeline to better evaluate the potential efficacy of new drugs or combinations prior to entering in expensive clinical trial settings.Importantly, our 3D ex vivo model provided a complete concordant correlation with in vivo responses. Ex vivo models are attractive for fundamental and translational research as they can predict patient response to drugs in a clinically relevant timeframe. Important advantages of this model include minimal waste of tissue and culture/drug reagentsAlthough this study has some limitations, such as use of a single drug, choice of flow cytometry for 3D spheroid analyses and limited utility of ex vivo tumors with low epithelial count, the reproducible comparison between model systems while using the same cell lines clearly shows the relevance of using various preclinical models to better characterize response to novel therapies. We are aware that cell lines are often devoid of many elements of the natural TME such as stromal and immune cells, which have been shown to influence response, and may not fully represent the primary tumor heterogeneity. However, the use of cell lines currently remains common practice in most preclinical models. Furthermore, a mouse model may not entirely reflect the human drug response. Hence, ex vivo models derived directly from patient samples would eliminate this bias in the drug development pipeline. Alternatively, similar analyses in comparing these preclinical models could be applied to other drugs in ovarian cancer treatment, such as paclitaxel, poly (ADP-ribose) polymerase (PARP) inhibitors and other drugs currently in preclinical studies such as HIF, WEE1 and TGFß inhibitors. Unfortunately, 3D analysis methods have traditionally relied on 2D culture methods or confocal microscopy analyses, which have their limitations. Thus, it would be interesting to include novel techniques such as light sheet microscopy and tissue clearing as well as IF staining of MDTMAs to allow optimal analysis of tissue without disturbing its natural environment.23, which are more difficult to accrue for clinical trials. In the era of personalized medicine, future applications would be to optimize treatment selection based on the individual tumor and patient characteristics rather than a ‘one treatment fits all’ approach. Thus, validation and feasibility studies of newer and more complex models are needed to enhance the current standards.Platinum resistance remains an important obstacle in EOC with dismal survival and limited options at advanced stages of disease progression. With the overall high attrition rate of oncologic treatments, more cost-effective predictive cancer models that accurately reflect patient response are needed. With this study, we clearly demonstrate a heterogeneity in therapeutic responses of EOC cell lines when cultured in different systems, which underscores the need to consider multiple factors when selecting a preclinical model for drug discovery and screening studies. This may avoid rejecting potentially effective drugs while eliminating ineffective drugs at the preclinical stage. This could also help reduce the rate of failed clinical trials in which patients experience drug toxicities with minimal efficacy, particularly for rare cancersSupplementary Information.


Tumor cells detached from the extracellular matrix (ECM) undergo anoikis resistance and metabolic reprogramming to facilitate cancer cell survival and promote metastasis. During ECM detachment, cancer cells utilize genomic methylation to regulate transcriptional events. One-carbon (1C) metabolism is a well-known contributor of SAM, a global substrate for methylation reactions, especially DNA methylation. DNA methylation-mediated repression of NK cell ligands MICA and MICB during ECM detachment has been overlooked. In the current work, we quantitated the impact of ECM detachment on one-carbon metabolites, expression of 1C regulatory pathway genes, and total methylation levels. Our results showed that ECM detachment promotes the accumulation of one-carbon metabolites and induces regulatory pathway genes and total DNA methylation. Furthermore, we measured the expression of well-known targets of DNA methylation in NK cell ligands in cancer cells, namely, MICA/B, during ECM detachment and observed low expression compared to ECM-attached cancer cells. Finally, we treated the ECM-detached cancer cells with vitamin C  and observed a reduction in the promoter methylation of NK cell ligands, resulting in MICA/B re-expression. Treatment with vitamin C was also found to reduce global DNA methylation levels in ECM-detached cancer cells. Extracellular matrix (ECM) attachment is required for cell survival , whereasNatural killer (NK) cells are innate immune cells and, in physiologically damaged conditions, such as tumor cell growth, they act as robust cytotoxic functioning cells. Although healthy cells express MHC-I, an inhibitory ligand for NK cells, cancer cell surfaces lose this molecule ,20. MoreHowever, the study also showed that MICA expression is reduced when a cell loses its adherence matrix . It has First, both HeLa and MCF-7 cell lines were grown in poly-HEMA gel-coated 6-well plates for six days to induce the ECM detachment model in cell lines. After that, both HeLa and MCF-7 cell lines, attached and 6 days ECM-detached cell aliquots, were taken for whole experiments a. The exIn contrast, the peak intensity results for the ECM-detached MCF-7 cell lines showed that they also accumulated higher levels of core one-carbon metabolites, such as SAM, methionine, cysteine, homocysteine, spermidine, pyruvic acid, adenine, etc. Compared with the ECM-detached HeLa cell line, the ECM-detached MCF-7 cell line had accumulated a higher level of core one-carbon metabolites to a lesser extent b,c. Howe2 flasks. Simultaneously, they were grown in poly-HEMA-coated 24-well plates for 6 days. After extracting RNA, cDNA conversion was performed for conducting qRT-PCR. Our experimental results showed that the methionine cycle-driving genes, such as methionine adenosyltransferase-1A/2A (MAT1A/2A), were significantly upregulated in the HeLa cell line. In addition, two genes (~33%) involved in the folate cycle were induced; the thymidylate synthetase (TYMS) gene was highly induced, while methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) was slightly induced. The cystathionine β-synthase (CBS) gene was marginally induced. The rest of the genes were downregulated (BHMT), and S-adenosyl homocysteine hydrolase (AHCY), were upregulated, with marginal induction of MAT1A/2A genes. Only the CBS gene was found to be upregulated among the folate cycles genes, with marginal induction of dihydrofolate reductase (DHFR), TYMS, and MTHFD1  is known as a potent DNMT inhibitor [In contrast, in MCF-7 cell lines, vitamin C significantly repressed the global methylation level a. Not onDNMTs b. Moreoveriments c. Additipression d,e. Howenhibitor , we chosAnoikis-resistant ECM-detached cancer cells undergo various metabolic adaptations and reprogramming to survive. For example, upon loss of ECM attachment, AMPK is induced to promote the uptake of glutamine, which lowers excessive oxidative stress. This metabolic reprogramming helps ECM-detached cancer cells to survive ,41. MoreMTHFD1 gene, affects survival rates in acute leukemia patients [SHMT1 gene, there was evidence of apoptosis induction in lung cancer [Moreover, several previous studies have measured the expression levels of one-carbon metabolic genes among healthy cohorts, inflammatory macrophages, and liver cancer cells ,43,44. Apatients . Even wig cancer .Moreover, therapeutic aspects have previously been revealed by several studies. For example, a study showed that 5-fluorouracil (5-FU—an inhibitor of TYMS) and methotrexate target the DHFR in ALL as a chemotherapeutic drug. Combined treatment with cyclophosphamide, 5-FU, and methotrexate showed effectiveness in node-negative breast cancer patients . AnotherDNMT mRNA expression and repressed MICA/B mRNA expression. Even the expression of surface proteins was downregulated after ECM detachment.Similarly, the ECM-detached MCF-7 cell line also had high methionine, SAM, DHFR, and cystathionine synthesizing gene expression levels. The cell line also showed the slight induction of dTUMP, m-THF, and DHFR synthesizing gene expression. Moreover, both MCF-7 and HeLa cell lines showed that loss of ECM attachment induced 5mC levels, which indicated that DNMT activity could be induced . A previDNMTs expression and the global DNA methylation (5mC) level. However, vitamin C treatment-induction of NKG2D ligands did not significantly affect DNMT3b mRNA levels in ECM-detached HeLa cell line.However, currently, natural compounds-based oncological therapy is not so widely available for cancer patients. These therapies have been clinically neglected for a long time, though they have been found to have very negligible side effects. Considering this, we decided to check the epigenetic effect on cancer cells of one of the most promising natural compounds, vitamin C. We treated ECM-detached MCF-7 and HeLa cell lines with 5-aza-dc as a positive control and vitamin C for 6 Days. In the ECM-detached MCF-7 cell line, vitamin C treatment-induced NKG2D ligands and significantly downregulated DNMT1/3a mRNA expression in the HeLa cell line. A previous study may explain this controversy, for it was demonstrated there that ascorbate (vitamin C) could inhibit only DNMT enzyme activity in melanoma cells [Nevertheless, vitamin C treatment downregulated ma cells . Additioma cells .MICB gene showed that vitamin C reduced the methylation signal only in the MCF-7 but not in the HeLa cell line. However, in both cell lines, MICA gene methylation was not detected in MSPCR.Nevertheless, 5mC level assessment revealed that both cell lines had reduced global methylation levels after vitamin C treatment. Additionally, the MSPCR result for the DNMT1/3a expression while not affecting DNMT3b, DNMTi and vitamin C induced MICA/B expression, suggesting that the MICA/B genes in the ECM-detached HeLa cell line are probably regulated by other epigenetic mechanisms. A previous study also suggested this probability, having shown that MICA/B promoters are silenced by histone hypo-acetylation [Hence, our overall results demonstrate that the two cell lines showed almost similar outcomes. MICA/B gene and surface protein expression were induced in DNMTi- and vitamin C-treated MCF-7 cell lines, with significant downregulation of DNMT and global methylation (5mC) levels. This means that MICA/MICB genes may have been suppressed or downregulated by epigenetic mechanisms in ECM-detached MCF-7 cell lines. On the other hand, the DNMTi- and vitamin C-treated ECM-detached HeLa cell line had reduced global DNA methylation. Though treatment reduced tylation . Even DNtylation , which i2 incubator. Upon 85–90% confluence, cells were trypsinized and seeded in poly-HEMA-coated 96-well plates and incubated overnight to ensure that cells were healthy without any contamination. Then, cells were treated with 5-aza-dc  at 500 nM, 1.5 µM, and 3 µM for 48 h. For vitamin C  treatment, cells were treated at 5 µM, 10 µM, 15 µM, 20 µM, 25 µM, and 30 µM. After 48 h, WST-1 was added, and a cell viability assay was performed with a BioTek ELISA microplate reader at 450 nm. Based on the cell viability assay, we selected doses of 5-aza-dc of 60 µM and 240 µM for vitamin C treatment for 6 days.Poly-HEMA gel was purchased from Sigma, dissolved in 95% ethanol at 65 °C, and incubated overnight. Cells were then seeded in a poly-HEMA-  coated plate. MCF-7 and HeLa cell lines were collected and grown and subcultured in DMEM media  supplemented with 10% FBS and 1% penicillin and incubated in a COv/v). Then, 1 mL of ice-cold solvent was added to the cell supernatant, followed by quick vortexing for 30s and incubation for 1 h at −20 °C. The supernatant was then spun for 15 min at 13,000 rpm at 4 °C, after which it was removed and the sample was dried, reconstituted in 100 μL of acetonitrile: water , vortexed for 10 min, and spun for 15 min at 13,000 rpm at 4 °C to remove insoluble debris. Finally, supernatants were taken for LC–MS/MS [Metabolites were extracted from attached and detached cells with methanol: acetonitrile: water at a ratio of 2:2:1 . The full scan range was chosen from 100 to 1000 m/z, with spray voltage at −3.0 kV and capillary voltage at 4.0 V. The temperature was fixed at 270 °C. Helium was used as a buffer gas, nitrogen as a sheath gas. The flow rate was fixed at 40 arbitrary units. Next, the obtained data were processed with XCMS online server data processing software .AHCY, MICA, MICB, and DNMTs primers sequences were obtained from the UCSC genome browser. One-carbon metabolic gene primers were obtained from other studies [−(ΔΔCt) method [Total RNA was extracted using a PureLink™ RNA Mini Kit . Then, 100 ng/uL RNA was transcribed into cDNA using a high-capacity cDNA synthesis kit .  studies ,60,61,62) method .DNA was extracted using an easy DNA kit . The 5mC kit was purchased from Abcam . The extracted DNA was seeded for binding the DNA to the assay well. Then, the wells were washed and the capture antibody was added. After that, the wells were rewashed, and detection antibody, enhancer solution, and developing solution were added. Finally, absorbance was observed at 450 nm. The whole procedure and data calculation were performed as per the manufacturer’s protocol.TM, Waltham, MA, USA) was diluted as 1:100 in 2% FBS for staining. Stained samples were put in a shaker for 1 h, followed by 1X PBS wash. After that, the surface expression of MICA/B was analyzed by Guava easyCyte HT.Both HeLa and MCF-7 cell lines were seeded in poly-HEMA-coated 24-well plates and treated with 5-aza-dc at 60 µM and 240 µM for vitamin C treatment for 6 days. Then, the collected cells were washed with 1X PBS, followed by thorough mixing with 100 uL 2% FBS. Anti-Hu MICA/B Ab  for methylated and unmethylated primer design , bisulfite conversion of extracted DNA was performed according to the supplier’s protocol. The bisulfite-modified DNA template was used for further PCR reactions using the DNA Master Mix Kit (Molequle On). For PCR reactions, whole gene sequences were selected for both t-tests or two ANOVA mixed models using GraphPad Prism 9 software. The Holme–Sidak and Tukey test methods were chosen, and the alpha was set as ≤0.5 for considering statistical significance. The data were presented as mean ± SEM.The statistical analysis was carried out by multiple ECM-detached cancer cells possess high levels of one-carbon metabolites that mediate the induction of global methylation in these conditions. Higher global DNA methylation levels may suppress one of the major NK cell immune ligands-MICB. Hence, targeting the one-carbon metabolic pathway and/or global methylation levels constitutes a beneficial strategy for chemotherapy outcomes in patients.


NUDT15 variant genotype is reportedly highly associated with severe myelosuppression during maintenance therapy, particularly in Asian and Hispanic populations. It has also been demonstrated that acquired somatic mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism, are detectable in a portion of relapsed childhood ALL. To directly confirm the significance of the NUDT15 variant genotype and NT5C2 and PRPS1 mutations in thiopurine sensitivity of leukaemia cells in the intrinsic genes, we investigated 84 B‐cell precursor‐ALL (BCP‐ALL) cell lines. Three and 14 cell lines had homozygous and heterozygous variant diplotypes of the NUDT15 gene, respectively, while 4 and 2 cell lines that were exclusively established from the samples at relapse had the NT5C2 and PRPS1 mutations, respectively. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with DNA‐incorporated thioguanine levels after exposure to thioguanine at therapeutic concentration. Considering the continuous exposure during the maintenance therapy, we evaluated in vitro mercaptopurine sensitivity after 7‐day exposure. Mercaptopurine concentrations lethal to 50% of the leukaemia cells were comparable to therapeutic serum concentration of mercaptopurine. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with mercaptopurine sensitivity in 83 BCP‐ALL and 23 T‐ALL cell lines. The present study provides direct evidence to support the general principle showing that both inherited genotype and somatically acquired mutation are crucially implicated in the drug sensitivity of leukaemia cells.In chemotherapy for childhood acute lymphoblastic leukaemia (ALL), maintenance therapy consisting of oral daily mercaptopurine and weekly methotrexate is important.  In contemporary regimens for childhood acute lymphoblastic leukaemia (ALL), maintenance therapy consisting of oral daily 6MP and weekly methotrexate is an important component.NUDT15 may influence the thiopurine sensitivity of leukaemia cells.NUDT15 in thiopurine sensitivity of ALL cells is not fully elucidated in vitro in the human model.NUDT15 is a nucleotide diphosphatase; it converts active thioguanine triphosphate (TGTP) to inactive thioguanine monophosphate (TGMP) in thiopurine metabolism.NT5C2PRPS1.NT5C2 mutations reduce the cytotoxic effect of thiopurine due to a degradation of critical intermediate metabolites of thiopurine. PRPS1 is an enzyme essential for purine biosynthesis.PRPS1 mutations impair thiopurine metabolism due to a defect in the purine salvage pathway. In previous reports, the significances of NT5C2 and PRPS1 mutations in thiopurine sensitivity were confirmed using ALL cell lines that were transduced with wild‐type or mutated cDNA by lentivirus vector.NT5C2 and PRPS1 mutations in thiopurine sensitivity of ALL cells remain to be directly confirmed in the intrinsic genes.In addition to the inherited genetic variants, acquired somatic mutations may also influence the thiopurine sensitivity of leukaemia cells. Recent studies in relapsed childhood ALL revealed that relapse‐specific somatic mutations were detectable in the genes involved in thiopurine metabolism such as NUDT15 genotype and NT5C2 and PRPS1 mutations in thiopurine activation and sensitivity of leukaemia cells using a large series of B‐cell precursor‐ALL (BCP‐ALL) and T‐cell ALL (T‐ALL) cell lines. We found that both variant NUDT15 genotype and NT5C2 and PRPS1 mutations were significantly associated with the activation of thiopurine. However, neither variant NUDT15 genotype nor NT5C2 and PRPS1 mutations were significantly associated with the sensitivities to thiopurine in the standard 3‐day incubation assay. As 6MP is administrated daily in maintenance therapy, we evaluated in vitro 6MP sensitivity after contentious exposure for 7 days, and found that both variant NUDT15 genotype and NT5C2 and PRPS1 mutations were significantly associated with 6MP sensitivity.Previous studies demonstrated that in vitro drug sensitivity profiles of primary leukaemia cells were significantly associated with clinical outcome in childhood ALL.22.12 at 37°C.We used 84 BCP‐ALL cell lines Table . Seventy2.2NUDT15 genotype was analysed in the 84 BCP‐ALL cell lines and 23 T‐ALL cell lines. Coding regions (exons 1 and 3) of the NUDT15 gene were first amplified by polymerase chain reaction (PCR) from germline DNA using the identical primers described in a previous report  and reverse (5′‐TAGTGCAGTTCCTCCACTCAG‐3′) primers. Exons 4–7 were amplified using forward (5′‐CTGAGTGGAGGAACTGCACTAT‐3′) and reverse (5′‐TACCAAGGGGAAACAAGGGTG‐3′) primers. We also performed the target‐exon sequence analysis of the NT5C2 and PRPS1 genes . The library was constructed using an Ion AmpliSeq Library Kit v2.0 and Ion Xpress Barcode Adaptors Kit (Thermo Fisher Scientific). After Agencourt AMPure XP purification (Beckman Coulter), individual libraries were amplified. The libraries were then processed with an Ion Chef System using an Ion PG Hi‐Q Chef Kit (Thermo Fisher Scientific). Next‐generation sequencing (NGS) was performed by using an Ion PGM Hi‐Q Sequencing Kit (Thermo Fisher Scientific) and 850 flows on an Ion 318 Chip Kit v2 (Thermo Fisher Scientific). After sequencing, single processing and base calling were performed using Torrent Suite 5.0.2 (Thermo Fisher Scientific). Output data were analysed using the Ion Reporter™ 5.16.0.2. Variant Call Format (VCF) files, then, were converted to Mutation Annotation Format (MAF) files using vcf2maf utility (https://github.com/mskcc/vcf2maf).Three regions of the 2.42 pH 5.3) at 50°C for 60 min. The digested DNA was treated with 3 U of calf intestinal alkaline phosphatase in the presence of 10× reaction buffer (Toyobo) at 37°C for 30 min and measured by LC‐MS/MS . TG concentration was adjusted according to the amount of DNA.To determine thiopurine metabolism in vitro, cells were treated with 0.1 µM of 6TG for 48 h. DNA‐incorporated thioguanine (DNA‐TG) levels were analysed with liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) as described previously2.55 cells were plated into each well of a 96‐well flat‐bottom plate in triplicate in the absence or presence of seven different concentrations of 6MP  and 6TG (0.25–180 µM). Cells were cultured for 68 h without washing the cells, and then, alamarBlue was added. In the 7‐day incubation assay, after initial culture for 3 days in the absence or presence of seven different concentrations of 6MP (0.012–3 µM), equal volumes of fresh medium containing the same concentrations of 6MP were added into each corresponding well. Each cell line was cultured for a total of 7 days, and then, alamarBlue was added. To determine the IC50 values of representative agents, each cell line was incubated for 2 days  or 3 days .To determine the IC50 of 6MP and 6TG, an alamarBlue cell viability assay (Bio‐Rad Laboratory) was performed,2.6Correlation coefficient was evaluated using Excel software (version 16.16.27). In each evaluation, median value was compared. Mann‐Whitney test, Fisher's exact test and Pearson's correlation analysis were performed using R (version 3.5.2) software.33.1max value of 6MP  during daily oral administration of standard dose of 6MP in childhood ALL patients.max values of 6TG  during daily oral administration of standard dose of 6TG in childhood ALL patients.We evaluated thiopurine sensitivities of BCP‐ALL cell lines by alamarBlue assay after a 3‐day incubation period Figure . In the 3.2max level of 6MP during maintenance therapy.MLL fusion was significantly higher than that in 16 cell lines with BCR/ABL1 or BCR/ABL1‐like fusions (p = 0.048 in Mann‐Whitney test) and in 4 cell lines with TCF3/HLF fusion (p = 0.044)   Figure . Moreove) Figure .max level) of 6MP, cell viability was decreased to less than 70% in three cell lines. In contrast, after 7‐day exposure to 0.2 µM  of 6MP, cell viability was decreased to less than 70% and 40% in four and two cell lines, respectively. These observations indicated that the prolonged continuous exposure to 6MP for 7 days is a reliable in vitro model system that may mimic the anti‐leukemic activity of 6MP during maintenance therapy.Next, we evaluated induction of apoptosis in five representative cell lines Figure . After 33.32 = 0.55, p < 0.0001; Figure We compared the anti‐leukemic properties of 6MP in BCP‐ALL cell lines during 3‐day exposure and 7‐day exposure. In the 3‐day incubation assay, a significant positive correlation was observed between the IC50 (day3) value of 6MP and that of 6TG  of 6TG  and those in 67 wild‐type cell lines  . These observations demonstrated that NUDT15 genotype is strongly associated with thiopurine metabolism in BCP‐ALL cell lines.Then, we evaluated the level of thioguanine incorporated into cellular DNA (DNA‐TG) in each cell line 48 h after exposure to 0.1 µM  Figure . Moreove3.5NT5C2PRPS1NT5C2 (located on 10q24.32‐q24.33) and PRPS1 (Xq22.3) mutations in 84 BCP‐ALL cell lines   were significantly lower than 78 cell lines without mutations   were significantly lower than the 61 cell lines without mutations  , while no mutations were observed in the cell lines established from the samples at diagnosis (0/29 cell lines)  Figure . We next) Figure . We focu) Figure . These o3.6NUDT15 genotype and NT5C2 and PRPS1 mutations in thiopurine sensitivities. Prior to the evaluation, we verified whether DNA‐TG levels in BCP‐ALL cell lines were associated with thiopurine sensitivities  were significantly more resistant than the 14 cell lines with heterozygous variant genotype  and tended to be more resistant than the 3 cell lines with homozygous variant genotype . Moreover, among 46 cell lines established at relapse, variant NUDT15 genotype tended to be associated with better 6MP sensitivity  were significantly more resistant to 6MP than 77 cell lines without mutations   value in six cell lines with NT5C2 and PRPS1 mutations was significantly higher than 61 cell lines without mutations   Figure . In 46 Be Figure . In 67 B) Figure . These o3.7NUDT15 and TPMT genes, and confirmed heterozygous variant genotypes of the NUDT15 and TPMT genes in two and one cell lines, respectively. We also performed the exon sequencing analysis of the NT5C2 and PRPS1 genes, and confirmed heterozygous mutation of the NT5C2 gene  in one cell line (BE‐13) (Table p = 0.014 in Mann‐Whitney test)  tended to be more sensitive to 6MP than 20 cell lines with wild‐type genotypes   was highly resistant to 6MP .In 23 T‐ALL cell lines Table , we eval3) Table . Then, w) Figure . Althoug) Figure . In contp = 0.0076 in Mann‐Whitney test)  values in 106 ALL cell lines (83 BCP‐ALL cell lines and 23 T‐ALL cell lines). Sixty‐three cell lines established at relapse  were significantly more resistant to 6MP than 35 cell lines established at diagnosis  Figure . The sigs Figure  and in 6s Figure . Moreoves Figure , in 63 cs Figure  and in 8e Figure . No cleas Figure . These o4NUDT15 gene. Among 77 Japanese BCP‐ALL cell lines, 14 (18.2%) and 3 (3.9%) cell lines had the heterozygous and homozygous variant diplotypes of the NUDT15 gene, respectively. Regarding the rs116855232 genotype—the most common variant of the NUDT15 geneNUDT15 genotypes in our Japanese BCP‐ALL cell lines was similar to that in Japanese childhood ALL patients. Using 84 BCP‐ALL cell lines that included 7 non‐Japanese cell lines, we confirmed the significant association of variant NUDT15 genotype with higher intra‐cellular DNA‐TG levels after exposure to therapeutic concentration of 6TG. Our observation in BCP‐ALL cell lines was consistent with the previous clinical notion that variant genotype of the NUDT15 gene was associated with higher DNA‐TG level in white blood cells from children with ALL receiving daily 6MP treatment.In the present study, using a large panel of BCP‐ALL cell lines, we investigated the significance of both inherited variant genotype and somatically acquired mutation on thiopurine activation and sensitivity. First, we focused on the variant genotypes of the NT5C2 and PRPS1 genes in BCP‐ALL cell lines. In the previous reports of childhood ALL, NT5C2 and PRPS1 mutations are exclusively observed in the samples at disease relapse.NT5C2 and PRPS1 mutations were exclusively observed in the cell lines established from the samples at relapse. Using these BCP‐ALL cell lines, we confirmed that intra‐cellular DNA‐TG levels (after exposure to a therapeutic concentration of 6TG for 2 days) were significantly lower in the cell lines with NT5C2 and PRPS1 mutations than in the cell lines without mutations. Thus, our observation in BCP‐ALL cell lines was consistent with the previous notion that these gain‐of‐function mutations of NT5C2PRPS1Next, we investigated somatic mutations of the NUDT15 and mutations of NT5C2 and PRPS1 must be associated with in vitro thiopurine sensitivity of BCP‐ALL cell lines. To evaluate in vitro drug sensitivity of leukaemia cells, IC50 values determined by MTT or its derivative assays after 2–4 days exposures to the drug are widely used as a standard index.NUDT15 genotype or NT5C2 and PRPS1 mutations. In this context, we noticed that the IC50 (day 3) values of 6MP and 6TG in our BCP‐ALL cell lines were approximately 800 and 30 times higher than previously reported serum Cmax levels of 6MPUnder these circumstances, both variant genotype of max level of 6MP during maintenance therapy.NUDT15 genotype and NT5C2 and PRPS1 mutations were significantly associated with 6MP sensitivity of BCP‐ALL and T‐ALL cell lines in the prolonged 7‐day assay. When focused on the cell lines with the wild‐type NUDT15 genotype, NT5C2 and PRPS1 mutations were significantly associated with 6MP resistance. These observations indicated that both variant genotype of NUDT15 and mutations of NT5C2 and PRPS1 are independently associated with thiopurine sensitivity of BCP‐ALL and T‐ALL  value and IC50 (day 3) value of 6MP. Of clinical importance, the range of IC50 values of 6MP in the prolonged 7‐day assay was comparable to the serum Ceg less than 1 µM) may be crucial for anti‐leukemic properties of 6MP during the maintenance therapy. To verify the unique anti‐leukemic properties of 6MP during the maintenance therapy, we evaluated the correlation between 6MP sensitivity and sensitivities to other chemotherapeutic agents in BCP‐ALL cell lines. However, no significant correlation was observed between 6MP sensitivity in the continuous 7‐day exposure and sensitivities to representative agents including glucocorticoids, vincristine, l‐asparaginase and daunorubicin. These observations suggested that the anti‐leukemic properties of 6MP in the maintenance therapy may not overlap with anti‐leukemic properties of other agents. In some regimens for childhood ALL, maintenance therapy is intensified with vincristine plus prednisolone or dexamethasone pulses.Even in recent intensified multi‐agent chemotherapeutic regimens for childhood ALL, maintenance therapy is an indispensable component.NUDT15 genotype and NT5C2 and PRPS1 mutations with a resistance to 6MP at concentrations pharmacologically relevant to the clinical setting. Although these notions were obtained in the study of cell lines, our observations validate the clinical benefit of 6MP dose adjustment for BCP‐ALL and T‐ALL patients who carry the NUDT15 variant allele by reducing myelosuppression without compromising anti‐leukemic efficacy. More generally, the present study provides direct evidence supporting the principle that both inherited genotypes and somatically acquired mutations are crucially implicated in the drug sensitivity of cancer cells.In conclusion, 7‐day incubation assay of 6MP sensitivity is a reasonable in vitro system to evaluate the anti‐leukemic properties of 6MP in the maintenance therapy for ALL. Using this system (in a large series of BCP‐ALL and T‐ALL cell lines), we verified the association of both wild‐type The authors declare no competing financial interests.Shinpei Somazu: Data curation ; Formal analysis ; Investigation ; Project administration ; Visualization ; Writing‐original draft ; Writing‐review & editing . Yoichi Tanaka: Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Visualization ; Writing‐original draft ; Writing‐review & editing . Minori Tamai: Formal analysis ; Writing‐review & editing . Atsushi Watanabe: Formal analysis ; Visualization ; Writing‐review & editing . Keiko Kagami: Data curation ; Formal analysis ; Writing‐review & editing . Masako Abe: Data curation ; Formal analysis ; Writing‐review & editing . Daisuke Harama: Formal analysis ; Writing‐review & editing . Tamao Shinohara: Formal analysis ; Writing‐review & editing . Koshi Akahane: Formal analysis ; Writing‐review & editing . Kumiko Goi: Formal analysis ; Writing‐review & editing . Kanji Sugita: Resources ; Supervision ; Writing‐review & editing . Takaya Moriyama: Conceptualization ; Methodology ; Writing‐review & editing . Jun Yang: Conceptualization ; Methodology ; Writing‐review & editing . Hiroaki Goto: Resources ; Writing‐review & editing . Masayoshi Minegishi: Resources ; Writing‐review & editing . Shotaro Iwamoto: Resources ; Writing‐review & editing . Junko Takita: Resources ; Writing‐review & editing . Takeshi Inukai: Conceptualization ; Data curation ; Funding acquisition ; Project administration ; Supervision ; Visualization ; Writing‐original draft ; Writing‐review & editing .Supplementary MaterialClick here for additional data file.


The median-effect principle proposed by Chou and Talalay is the most effective approach to parameterize interactions between several agents in combination. However, this method cannot be used to evaluate the effectiveness of equimolar drug combinations, which are comparative references for dual-targeting molecular design. Here, using data acquired through the development of “combi-molecules” blocking two kinases , we established potency indices for equimolar and dual-targeted inhibitors. If the fold difference (κ) between the IC50 of the two individual kinase inhibitors was >6, the IC50 of their equimolar combination resembled that of the more potent inhibitor. Hence, the “combi-targeting” of the two kinases was considered “imbalanced” and the combination ineffective. However, if κ ≤ 6, the IC50 of the combination fell below that of each individual drug and the combi-targeting was considered “balanced” and the combination effective. We also showed that combi-molecules should be compared with equimolar combinations only under balanced conditions and propose a new parameter Ω for validating their effectiveness. A multi-targeted drug is effective if Ω < 1, where Ω is defined as the IC50 of the drug divided by that of the corresponding equimolar combination. Our study provides a methodology to determine the in vitro potency of equimolar two-drug combinations as well as combi-/hybrid molecules inhibiting two different kinase targets. The implication of several signaling proteins in a complex network of signal transduction pathways is a commonly occurring event in advanced cancers. These signaling interactions between growth factor receptors , hepatocyte growth factor receptor (c-Met), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), etc.) and cytoplasmic non-receptor tyrosine kinases and transcription factors  not only synergize to promote tumor growth, survival, and metastasis, but also mediate resistance to targeted therapies through the activation of compensatory signaling pathways ,2,3. ThuRecent efforts toward inducing a tandem blockade of multiple signaling pathways have increased interest in the use of equimolar combinations. We and others have frequently used them as a reference for studying the biological effects of newly designed hybrid molecules ,29,30,31In the past, Chou and Talalay  demonstrThe approach we chose to study was to target tyrosine kinases such as c-Src, c-Met, and EGFR, which are known oncogenes driving various tumors through complex signaling interplay. Here, we first determined the IC50 values for growth inhibition induced by their clinical inhibitors both as single agents and equimolar combinations against human cancer cell lines of various histological origins. Subsequently, we analyzed the trends of the IC50 values of these combinations in comparison with individual drugs as well as combi-molecules synthesized in our lab, targeting EGFR, c-Src, and c-Met . “ImbalaIn order to profile the responses, we primarily screened a panel of cancer cell lines using single and equimolar combinations of clinically approved tyrosine kinase inhibitors (TKIs) targeting kinases engaged in synergistic crosstalk, and further extended the screening to kinase–DNA targeting drug combinations. The cell lines used in this study included breast, lung, prostate, ovarian, head and neck, and brain cancer cells along with the NIH3T3 panel of wild type, EGFR, and Her2 transfected cell lines. Clinical TKIs including gefitinib (EGFR inhibitor), crizotinib (c-Met inhibitor), dasatinib (c-Src inhibitor), and the DNA alkylating agent temozolomide were used a. In allFurther studies targeting c-Met and EGFR with crizotinib and gefitinib, respectively, showed a similar trend as described above, with IC50 values for crizotinib ranging from 0.5 ± 0.02 μM to 6.5 ± 1.0 μM and generally being lower than that for gefitinib (to 0.22 ± 0.01 μM to 73.7 ± 7.7 μM) a. The ICThe analysis was extended to c-Met and c-Src targeting using crizotinib and dasatinib , respectively. The results showed that the IC50 of dasatinib ranged from 0.01 ± 0.0003 μM to 15.1 ± 1.8 μM and that of crizotinib ranged from 0.5 ± 0.02 μM to 6.5 ± 1.0 μM a. As preThe analysis was also carried out using a more divergent type of targeting involving one kinase inhibitor  and a DNA damaging agent. We determined the growth inhibitory potency of gefitinib (EGFR TKI) or crizotinib (c-Met inhibitor) alone or in equimolar combination with temozolomide  on a panel of cell lines c. The rep < 0.001). Consequently, the average IC50 for the equimolar drug combination (γ3) was in the same range as that of γ2 (IC50 of kinase inhibitor-2). In contrast to balanced targeting, there was no significant difference between γ1 and γ2 (p > 0.05) for data with κ ≤ 6  and the more potent drug (γ2) is higher than 6-fold, the IC50 value for the equimolar combination referred to as γ3 is in the same range as γ2 (the more potent drug). However, if the difference in IC50 between γ1 and γ2 is less than or equal to 6-fold, which we consider to be a similar IC50 range, the equimolar combination exhibits superior potency when compared with each drug alone. We categorized the data into two groups based on the fold difference calculated as γ1/γ2; this term is referred to as κ. For imbalanced targeting (κ > 6), the average distribution of all the IC50 values of kinase inhibitor-1 (γ1) and kinase inhibitor-2 (γ2) across various cell lines showed a significant difference  gave Ω values far greater than 1 in cells expressing their target oncogenes, indicating that EGFR and c-Src are not adequately targeted by these combi-molecules b,c. OptiGiven the potency of LP121 in blocking both c-Met and EGFR tyrosine kinase activity, we evaluated its growth inhibitory properties in cell lines exhibiting less than a 6-fold difference in IC50 values between gefitinib and crizotinib, as previously determined. The cell lines chosen included DU145, PC3 (prostate cancer), 4T1 (mouse mammary tumor cells), IGROV-1, SKOV-3, and EFO-21 (ovarian cancer), which exhibited sensitivity to the equimolar combination of gefitinib + crizotinib when compared with an individual drug alone. The results showed that among all the cell lines analyzed, LP121 showed superior activity compared with the equimolar combination in SKOV-3 cells (IC50 = 0.29 ± 0.04 μM and Ω = 0.7) and similar potency to the equimolar combination in EFO-21 cells (IC50 = 0.01 ± 0.001 μM and Ω = 1.0), indicating that LP121 is an effective combi-molecule against these cells.The complexity of signaling networks driving advanced cancers has given rise to several multi-targeted strategies, including the use of complex drug cocktails, hybrid or chimeric molecules  which are in preclinical/clinical stages of evaluation or currently being used in the clinic ,40,41,42The general trend observed was that when the IC50 value of one drug was 6-fold higher than that of the other (fold difference of κ > 6), the IC50 of the equimolar combination of the two drugs resembled that of the more potent inhibitor. However, when the difference in IC50 values of the two drugs was equal to or less than 6-fold, the equimolar combination of the two drugs showed superior potency compared with a single drug alone, thereby exhibiting what we designate as “balanced targeting”. Our choice to define the κ threshold at 6-fold is based on the small sample size we have evaluated (10 drugs/combinations across 27 cell lines) and could be determined differently by other teams depending on their cell models and the nature of the drugs or combinations being tested.The biological significance of these observations can be analyzed considering the dominant pathways that control cell growth and the ability to undergo apoptosis in human tumor cells. Being at the crossroad of multiple signaling pathways, blockade of c-Src with the potent inhibitor dasatinib led to IC50 values in the nanomolar range, which renders the blockade of other receptors (such as EGFR or c-Met) a minor contributor to the overall IC50 in combination. It is also important to also note that dasatinib exerts its action through inhibiting multiple other targets (polypharmacology), which could also be contributing towards its overall superior potency in certain cell lines . As anotIn cells where balanced targeting is observed, perhaps the two targets synergize to promote growth and anti-apoptotic effects. Therefore, their tandem modulation leads to superior growth inhibitory potency compared with either drug alone. Importantly, these cell lines responding better to equimolar drug combinations are likely to benefit from hybrid/combi-molecules, as demonstrated in this study. Finally, if a combi-molecule achieves Ω < 1  in these cells, it can be considered worthy of further in vitro analysis and in vivo studies in xenograft models carrying the latter cell lines.If two drugs are combined at an equimolar ratio, and one drug shows a 6-fold greater IC50 than the other , the overall effect will resemble the IC50 of the combination.If two drugs are combined at an equimolar ratio, and the IC50 of one drug is 6-fold or less than that of the other , then the overall effect is superior to that of each individual drug, leading to balanced targeting.Under conditions of balanced targeting, a unimolecular combination  is said to be effective if the IC50 of the combi-molecule is equal to or a fraction of the IC50 of the equimolar 2-drug combination .Steps toward defining potency criteria for combi-molecules are schematized in The EGFR-c-Src combi-molecules AL660, AL690, AL692, AL739, and the EGFR-c-Met-targeting combi-molecule LP121 were synthesized according to the methods described in the 2 at 37 °C.The human cancer cell lines used in the present study include breast , prostate , lung , Chinese hamster lung cancer cells (VC8 and VC8-MGMT), ovarian , head and neck (UM22A), NIH3T3 wild type (Wt), EGFR (Her14) and Her2 (Neu) transfected cells, and mouse mammary tumor 4T1 cells. MGMT cells do not express the O6-methylguanine DNA methyltransferase (MGMT), which is a DNA repair enzyme that removes the cytotoxic O6-methyl group of guanine by transferring it to its internal cysteine residues. The prostate cancer cells were a generous gift from Dr. Amina Zoubeidi . The 4T1, U87, and U87-MGMT cells were a generous gift from Dr. Thierry Muanza . The 4T1 cells were originally isolated by Dr. Fred Miller  . The NIHv/v).Crizotinib was purchased from PharmaBlock USA, Inc.  and gefitinib from the Royal Victoria Hospital  pharmacy and extracted from pills in our laboratory. Dasatinib was purchased from Ark Pharm Inc., Arlington Heights, IL, USA. Temozolomide was extracted form Temodal pills purchased form Merck/Schering Plough . All drugs were dissolved in DMSO to obtain a concentration of 40 mM or lower. Drug dilutions were carried out under sterile conditions using RPMI or DMEM (10% FBS) medium and the final concentration of DMSO never exceeded 1% (Sulforhodamine B assay was used to measure growth inhibition in cells . Cells wEGFR and c-Met in vitro kinase assays were carried out in 96-well plates (Nunc Maxisorp) coated with PGT  and incubated at 37 °C for 48 h. PGT was the substrate to be phosphorylated by EGFR  or c-Met  in the presence of ATP (50 μm). Drugs  were added, followed by 13.3 ng/well of isolated EGFR (0.1 μg/μL) or 32 ng/well of c-Met (0.75 μg/μL). The HRP-conjugated anti-phosphotyrosine antibody  was used for phosphorylated substrate detection. The signal was developed using 3,3′,5,5′-tetramethylbenzidine peroxidase substrate  and assessed using a microplate reader ELx808 at 450 nm . GraphPad Prism 6.0  was used for IC50 determination and each experiment was repeated at least twice, in duplicate.NIH3T3-Her14 and 4T1 cells were plated and 24 h later rinsed twice with PBS and starved overnight using serum-free media. Cells were next treated with various concentrations of inhibitors, for 2 h, washed with PBS twice, and stimulated with 50 ng/mL EGF (NIH3T3-Her14) or EGF + HGF (4T1), each for 30 min at 37 °C. Western blot analysis was carried out according to methods previously described by Rao et al. . Phospho


The bottleneck arising from castration-resistant prostate cancer (CRPC) treatment is its high metastasis potential and antiandrogen drug resistance, which severely affects survival time of prostate cancer (PCa) patients. Secreted phosphoprotein 1 (SPP1) is a cardinal mediator of tumor-associated inflammation and facilitates metastasis. In our previous study, we firstly revealed SPP1 was a potential hub signature for predicting metastatic CRPC (mCRPC) development. Herein, we integrated multiple databases to explore the association of SPP1 expression with prognosis, survival, and metastatic levels in CRPC progression and investigated SPP1 expression in PCa tissues and cell lines. Next, PCa cell lines with overexpression or depletion of SPP1 were established to study the effect of SPP1 on enzalutamide sensitivity and adhesion and migration of prostate cancer cell lines and further explore the underlying regulatory mechanisms. Bioinformatics analysis, polymerase chain reaction (PCR), immunohistochemical staining, and western blot results suggested SPP1 upregulation had strong relationship with the malignant progression of CRPC and enzalutamide resistance. SPP1 knockdown enhanced enzalutamide sensitivity and repressed invasion and migration of prostate cancer cells. Importantly, upregulating SPP1 promoted, while silencing SPP1 attenuated epithelial-mesenchymal-transition (EMT). Our results further demonstrated that SPP1 overexpression maintains the activation of PI3K/AKT and ERK1/2 signaling pathways. Overall, our findings unraveled the functional role and clinical significance of SPP1 in PCa progression and help to discover new potential targets against mCRPC. Prostate cancer (PCa) is the most common malignancy, which causes the fifth rank of cancer-related mortality and is approximated 1,410,000 deaths and accounts for almost 14% of total cancer diagnosed in men . A signiSecreted phosphoprotein 1 (SPP1), also recognized as osteopontin (OPN), a secreted chemokine-like glycophosphoprotein, is a cardinal mediator of tumor-associated inflammation and facilitates metastasis . SPP1 isUnder certain physiological and pathological conditions, epithelial cells differentiate into mesenchyme, accompanied by changes in cell morphology and related genes , which iIn this study, we firstly integrated multiple databases, including TCGA, GEO, UALCAN, and HCMDB, to demonstrate the relationship of SPP1 expression with PCa prognosis, survival, metastatic levels, and CRPC progression. Next, we examined the effect of the regulation of SPP1 on enzalutamide sensitivity, migration, and adhesion of PCa cell lines, which had been genetically modified to overexpress or deplete SPP1. Moreover, we further explored the functional role of SPP1 in EMT activation and the underlying regulatory mechanisms. Overall, this study is aimed at identifying the clinical significance and biological functions of SPP1 in CRPC progression and helps in identifying novel potential biomarker and therapeutic strategy.P value as 1.4e − 06  (P = 0.305)  , whereasTo reveal the differential expression of SPP1 in PCa cells, we detected the RNA and protein expression level in four groups of cell lines  with SPP1 negative and positive expression cells as control groups. As shown in in vivo, we used the bone metastatic CRPC model developed by Prof. Qian lab, Queen's Medical Research Institute, University of Edinburgh, following intracardiac inoculation of an androgen-dependent murine prostate cancer cell line cells in FVB/N male mice. Through daily treatment with enzalutamide, bone metastatic CRPC model suffered from enzalutamide resistance. As the result of immunofluorescence staining ; more importantly, SPP1 knockdown remarkably enhanced apoptosis-promoting effect of enzalutamide (P < 0.001). Therefore, knockdown of SPP1 could induce the resensitization of enzalutamide-resistant CRPC cell line to enzalutamide.To clarify the role of SPP1 in the development of enzalutamide resistance in CRPC, 22Rv1 cell line with SPP1 knockdown was used to the effect of SPP1 on enzalutamide sensitivity. The results showed that SPP1 knockdown significantly inhibited 22Rv1 cell proliferation after enzalutamide treatment . In addi Figures  also demIn our previous study, based on single-gene Gene Set Enrichment Analysis (GSEA) results of GEO database mining, we found SPP1 could regulate AR signaling pathway in the SPP1 high-expression CRPC group , which cTumor cell adhesion to extracellular matrix proteins, for example, fibronectin, is important to provide a support for migration and metastasis. We next performed Matrigel transwell assay in SPP1 depleted and control groups  for 48 hP value of 0.014 (P < 0.01), compared with that in siRNA-NC ones. In contrast, vimentin and N-cadherin were significantly upregulated in lentivirus-transfected PC-3, compared with that in PC3-NC ones (P < 0.01). Specially, N-cadherin is remarkably downregulated in siRNA-SPP1 PC-3 (P < 0.01) and significantly upregulated in lentivirus-transfected LNCaP. Moreover, E-cadherin expression was both obviously upregulated in siRNA-SPP1 22Rv1 (P < 0.01) and PC-3 cell (P < 0.01), compared with that in siRNA-NC ones. Adversely, E-cadherin expression was both remarkably downregulated in lentivirus-transfected LNCaP (P < 0.01) and PC-3 (P < 0.01). These results indicated that SPP1 could activate EMT pathway in PCa cells.EMT process plays a crucial role in tumor cell invasion and migration. In addition, after long-term treatment with enzalutamide, PCa cells undergo EMT changes, the epithelial markers in the tissue are significantly reduced, and the mesenchymal markers are significantly upregulated , 17. As of 0.014 . To clarP value of 0.004, and expression of MAPK1 and MAPK14 was positively associated with that of SPP1 significantly  [P < 0.05 was considered significance for survival analysis. The relationship of SPP1 expression level with individual cancer stages and lymph node metastasis status was analyzed by UALCAN database  [http://hcmdb.i-sanger.com/) [FPKM gene expression data, somatic mutation, and survival data of Pan-cancer analyzed in The Cancer Genome Atlas (TCGA) project were downloaded from University of California Santa Cruz (UCSC) Xena website (apages/) . Tumor mapages/) . The R pab.edu/) , which ier.com/) , which i2-humidified atmosphere. RNA interference was applied to knockdown SPP1 in cell lines. In our study, three different SPP1siRNAs and lentiviral vector were synthesized by JTS scientific . All these siRNAs were transfected into PCa cell lines by Lipofectamine 8000 based on the product manual (Invitrogen). The following siRNA sequences were finally used: SPP1(H)-508 . SPP1  was cloned into the lentiviral vector JLVO-CAG-GFP-Apuro. Lentiviral vector was transfected into PC-3 and LNCaP cell lines with polybrene, and cell lines with stable SPP1 expression were selected and amplified.PC-3, DU-145, LNCaP, and 22Rv1 were obtained from Cell Bank of Peking Union Medical College Culture Collection . LNCaP and 22Rv1 cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum, and 100 mg/mL penicillin and 100 mg/mL streptomycin were used to culture the cells. PC-3 and DU-145 cell line was cultured in F-12K Medium and Eagle's Minimum Essential Medium, respectively. Cell culture was at 37°C with 5% COEasy pure RNA Kits  were applied for total RNA extraction. TransScript First-Strand cDNA Synthesis SuperMix  was used for reverse transcription according to the manufacturer's protocol. The mRNA expression level was acquired through cyclic threshold method. SPP1 primers were used: SPP1 (5′-GTTAAACAGGCTGATTCTGG-3′ (forward), 5′-CATGGTCATCATCATCTTCA-3′ (reverse)). PCR reactions were performed in triplicates with following context: after an initial hot start at 95°C for 5 min, PCR amplification was conducted for 40 cycles of 95°C/10 s and 60°C/30 s, denaturation at 95°C/15 s, and annealing and extension at 60°C for 60 s. The relative level of gene expression was detected via the cyclic threshold method.α, PI3K p85, AKT, pAKT, AR, and β-actin were evaluated by Western blot. 2.5 × 105 cells were plated in 6-well plates at 37°C with 5% CO2 for 24 h and were washed thrice in ice-cold PBS and harvested, and cell pellets were resuspended in NP-40 lysis buffer  containing protease inhibitors and subjected to sonication. Protein concentrations were assessed using a Pierce bicinchoninic acid protein assay kit . Equal amounts of proteins (20 μg per sample) were separated by SDS-PAGE and transferred onto PVDF membranes. After being blocked with 5% milk in TBST, the membrane was incubated at 4°C overnight with the primary antibody . The following primary antibodies were used: anti-SPP1 , anti-vimentin , anti E-cadherin , anti-N-cadherin , anti-MAPK p38 , anti-ERK1/2 , anti-PI3K p110α , anti-PI3K p85 , anti-AKT , phopho-AKT (Ser 473) , anti-AR , and anti-β-actin . Subsequently, the membranes were washed thrice with PBS and incubated with horseradish peroxidase-conjugated secondary antibodies . Bands were visualized with BeyoECL Plus . BEZ235 was obtained from Med Chem Express .The protein expression levels of SPP1, vimentin, E-cadherin, N-cadherin, MAPK p38, ERK1/2, PI3K p110Immunohistochemical (IHC) staining was performed to determine the expression of SPP1 in primary PCa and bone metastasis CRPC. The details of IHC staining procedure are similar to those described in our previous study . The sliThrough the manufacturer's protocol, the cell proliferation test was performed by using the Celltiter-Glo assay (Promega P/N G7570). In this assay, the luminescence value is directly proportional to the amount of adenosine triphosphate (ATP), and ATP is positively related to the number of living cells, so cell viability can be obtained by detecting the ATP content. The cell lines were transfected with siRNA SPP1 or siRNA negative control (NC) and disposed by enzalutamide. Every 24 h, viable cell counts were detected, and both the SPP1 siRNA and siRNA-NC groups were conducted independently in triplicate.5/well. Four groups were involved in this experiment: the control group disposed with DMSO, enzalutamide treatment group, siRNA SPP1-transfected group, and SPP1 knockdown with enzalutamide treatment group. All the groups were stained with Annexin V and PI . Afterwards, the stained cells were examined by a flow cytometer (BD Biosciences) and data were processed by Cell Quest software. All the experiments were carried out in triplicate.22Rv1 and its siRNA SPP1-transfected cell lines were plated in 6-well plates at 3 × 10μL pipette's tip and washed several times using medium to wash dislodged cells. DU-145, 22Rv1, and their transfected cell lines migrated into wound area were photographed at the 48 h and 72 h, respectively.Migration ability of cells with or without SPP1 transfected was evaluated by wound healing assays. Monolayers of PCa cell lines and their transfected cells were cultured in 24-well plates. Cell layer was scratched with a 200 4 cells were added into the top chamber of a polycarbonate transwell filter chamber covered with Matrigel. After 48 h, the cells in the upper of membrane were wiped off and fixed with 4% paraformaldehyde and stained with 0.5% crystal violet for counting the number of cell. All experiments were carried out in triplicate.After being transfected with SPP1 siRNA or siRNA NC, the cell lines were cultured with 1% fetal bovine serum and plated into the top chamber; cell invasion abilities were evaluated by Matrigel transwell assays. 5 × 10t-test. P value < 0.05 was defined as significant.GraphPad Prism 8.0  was applied for statistical analysis. Data are showed as the mean ± S.E.M. Two groups were compared using Student's in vitro and in vivo. SPP1 could promote PCa cell metastasis by activating EMT, and we firstly reported that SPP1 could be a possible target against enzalutamide resistance and EMT in CRPC treatment, which could be regulated via the MAPK/ERK1/2 and PI3K/AKT pathways. This indicated that specific targeting of SPP1 can be a valid approach to treat antiandrogen resistance of metastatic CRPC.Our current data illustrated for the first time that overexpression of SPP1 in PCa bone lesions and enzalutamide resistance CRPC