Chemotherapies based on platinum have been the standard first-line treatment for patients with small-cell lung cancer(SCLC) in the past years. However, the progression of patients occurs mostly due to rapid development of resistance to chemotherapy. In addition, the mechanisms involved in development of cisplatin-resistance in SCLC remain undetermined. Here, we analyzed whole-exome sequencing(WES) datasets from Genomics of Drug Sensitivity in Cancer and WES data and overall survival(OS) from a published cohort(N=101) to search for cisplatin-resistant target genes and genes associated with poor prognosis. We use our cohort(NCT03162705) as the validation set. We applied single sample gene set enrichment analysis(ssGSEA) to explore the potential molecular mechanisms of cisplatin-resistance. DNAH10 mutations in SCLC was significantly associated with cisplatin-resistance(P=0.0350), poor OS and worse progression-free survival (PFS)(P=0.0142). ssGSEA showed that the negative regulation of FGFR, the SPRY regulation of FGF, and the positive regulation of noncanonical WNT and PI3K/AKT/IKK signaling pathways are differentially up- or downregulated in DNAH10-mutated cell lines. A higher TMB was observed in DNAH10-mutated cell lines. Taken together, DNAH10 mutations may have a potential value in prediction of cisplatin resistance and poor survival in SCLC. Moreover, DNAH10 mutations may have a positive correlation with high TMB in SCLC. Small-cell lung cancer (SCLC) is a highly invasive neuroendocrine tumor characterized by rapid growth and early metastasis and accounts for about 15%–20% of lung cancers . In the Next-generation sequencing allows understanding of SCLC from the perspective of biologic drivers and molecular pathogenesis . SCLC haGene mutations are one of the molecular mechanisms of cisplatin resistance . Sakai eWe analyzed the whole-exome sequencing (WES) datasets, the drug response data of Genomics of Drug Sensitivity in Cancer (GDSC) database, and the reported WES and corresponding clinical data of patients with SCLC  to screGDSC characterized about 1000 human cancer cell lines and screened them with 100s of compounds. Using this database, we obtained the IC50 distribution for cisplatin by tissue type . We alsoTo further validate our hypothesis, we assessed DNAH10 mutation in our cohort comprising 47 patients with ES-SCLC who had not undergone any treatment. Patients with DNAH10 mutation had poor progression-free survival (PFS)  are liThe mRNA expression matrix files of GDSC-SCLC cell lines were analyzed in R software by the limma package. The P-values < 0.05 and FC was ≥3/2 or ≤ 2/3. were chosen as cut-off criteria. A heatmap of all 157 DEGs is shown in To understand potential DNAH10-related pathways, we conducted ssGSEA among 55 GDSC-SCLC cell lines  followedTMB was higher in patients with DNAH10 mutation compared with patients without the mutation -related pathways and response or resistance to platinum compound. DNAH10-mutated cell lines had significantly higher mutation count in the mismatch repair (MMR) pathway compared with the wild-type DNAH10 cell lines  or irinotecan and cisplatin (IP)  showed tIn this study, we analyzed the WES and transcriptome data of 55 GDSC-SCLC cell lines as well as the cisplatin response, WES, and clinical data of 101 patients . We fouPlatinum drugs form a complex with DNA, leading to DNA damage, which is recognized by the MMR system and leads to apoptosis . HypotheThis study presents some limitations. First, the precise molecular mechanisms of DNAH10 mutation on chemoresistance in SCLC remain unclear. Second, we did not compare the conservation of different mutation across evolution mainly because we found that the DNAH10 mutation sites are randomly occurred in different samples. Third, no animal models are available for providing further evidence for our hypothesis. In this regard, relevant animal models should be established to verify our hypothesis.In conclusion, DNAH10 mutation might be a potential biomarker for cisplatin resistance and poor survival in patients with SCLC. Our results showed the good association of DNAH10 mutations with high TMB in SCLC. Moreover, the negative regulation of FGFR, the SPRY regulation of FGF, and the positive regulation of noncanonical WNT and PI3K/AKT/IKK signaling pathways may be the key pathways regulated by DNAH10 mutation for promoting cisplatin resistance in SCLC. If new lung cancer patients are diagnosed with SCLC, patients could be sequenced using WES or target sequencing (including DNAH10). With DNAH10 mutations were detected with next-generation sequencing assays or target sequencing, and its presence in the current analysis was strongly associated with cisplatin-resistance. clinicians can try not to use cisplatin, consider other combinations of chemotherapy; or consider using ICIs for first-line treatment of DNAH10 mutant SCLC. Our findings provide potential therapeutic targets for improving the efficacy of chemotherapy in patients with SCLC and DNAH10 mutation. Further experimental validation should be performed to prove the biological effect of DNAH10 mutation on SCLC.We analyzed WES data (Illumina HiSeq 2000) of SCLC cell lines from Genomics of Drug Sensitivity in Cancer  databases . Fifty-fHuman SCLC cell lines, namely, NCI-H69, NCI-H69AR, and NCI-H446, were acquired from the American Type Culture Collection . Chemo-resistant H446DDP cells were obtained by incubating H446 cells in progressively increasing cisplatin doses of up to 5 μg/mL for 6 months. RNA was extracted from human cell lines such as NCI-H69, NCI-H69AR, NCI-H446, and NCI-H446DDP by using Trizol method and quantified by Qubit3.0. RNA quality was assessed by 4200 TapeStation (Agilent Technologies). RNA libraries were prepared with VAHTSTMmRNA-seq V3 preparation kit (Illumine). The libraries were then quantified and sequenced with Illumina Novaseq6000 platform.https://www.clinicaltrials.gov/). The patients received care and follow-up in Zhujiang Hospital , the First Affiliated Hospital of Guangzhou Medical University , the Collaborative Innovation Center for Cancer Medicine , or the Nangfang Hospital . Informed consent was obtained from all patients before specimen collection. The experiments were approved by the Ethics Committee of the Southern Medical University .Forty-seven fresh–frozen SCLC samples were collected from patients between May 2017 and January 2019 , respectively. DNAs were quantified by Qubit 3.0 by using the dsDNA HS Assay Kit (ThermoFisher Scientific). Library preparations were performed with KAPA Hyper Prep Kit (KAPA Biosystems). The average coverage depth for tumors was set as 157×. Trimmomatic was used for FASTQ file quality control. Paired-end reads were aligned to the reference human genome (build hg19) by using Burrows–Wheeler Aligner. PCR deduplication was performed using Picard. Local realignment around indels and base quality score recalibration were performed using GATK3.Predictive genes for patients’ survival and sensitivity to cisplatin were detected by three steps. (1) The association of gene mutations (mutant frequency>10% in GDSC databases) with the IC50 values of cisplatin was assessed by Wilcoxon test (P <0.05). (2) The association of gene mutations (mutant frequency>10%) with OS of patients with SCLC in a reported cohort was determined by univariate Cox proportional hazard regression model (P <0.05). (3) Common genes in the first and second steps were identified. The overall process yielded two predictive genes, namely, DNAH10 and WDR87, for SCLC tumors.To investigate the genomic profile between DNAH10 mutation and wild-type groups, we performed differential expression gene (DEG) analysis in SCLC cell lines by the limma  package For clinical data, ssGSEA ES, DNAH10 expression, immune checkpoint expression, IC50, TMB, and DNA damage response and repair (DDR)-related pathway alternation were compared between the two groups. Wilcoxon test/Mann–Whitney U test was used for data with abnormal distribution, and independent-sample Student’s t-test was used for data with normal distribution. Survival curves were estimated with R software (version.3.6) by using survival  and survSupplementary FiguresSupplementary TablesSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7Supplementary Table 8


TP53 mutations were detected in two cell lines with complex array profiles, indicating chromosomal instability (CIN). A quantitative assessment of tumor genomes at the chromosomal level was newly introduced to reveal total DNA sizes and Scales of Genomic Alterations (SGA) for each cell line. Kasumi-1 and 6 derived from relapsed phases demonstrated high levels of SGA, implying that the level of SGA would reflect on the tumor progression and could serve as an index of CIN. Our results extend the leukemia cellular resources with an additional five cell lines and provide reference genome data with ethnic identities for the ten Kasumi cell lines.Kasumi-1 has played an important role in an experimental model with t translocation, which is a representative example of leukemia cell lines. However, previous studies using Kasumi-1 show discrepancies in the genome profile. The wide use of leukemia cell lines is limited to lines that are well-characterized. The use of additional cell lines extends research to various types of leukemia, and to further explore leukemia pathogenesis, which can be achieved by uncovering the fundamental features of each cell line with accurate data. In this study, ten Kasumi cell lines established in Japan, including five that were previously unknown, have been characterized by SNP microarray and targeted sequencing. SNP genotyping suggested that the genetic ancestry in four of the ten Kasumi cell lines was not classified as Japanese but covered several different east-Asian ethnicities, suggesting that patients in Japan are genetically diverse. The online version of this article (10.1007/s13577-020-00347-5) contains supplementary material, which is available to authorized users. Leukemia genomes are primarily characterized by abnormal karyotypes, which often include the formation of a fusion gene . ChromosRUNX1-RUNX1T1, is well recognized as an important diagnostic and prognostic marker frequently observed in acute myeloid leukemia, according to the French–American–British classification M2 subtype (AML-M2), which was first identified in the Kasumi-1 cell line [Kasumi cell lines have been established at Hiroshima University from patients with leukemia in Japan since 1989. They were named after the location of the laboratory in the Kasumi area of Hiroshima city [ell line . Kasumi-Cell lines have attractive features, namely their ‘continuous’ or ‘immortalized’ abilities. This enables us to use the same cellular material across different laboratories and allows us to compare results using cell lines. However, three previous studies which analyzed Kasumi-1 by DNA microarray showed discordance in the genome profiles –17 .Kasumi-1–10 cell lines have been registered with the JCRB cell bank . The data analysis was performed based on the GRCh37 (hg19) reference using the Chromosome Analysis Suite software, ChAS 4.0 (Thermo Fisher Scientific). To measure genomic changes at chromosomal level, regions of gains, losses and uniparental disomy (UPD) larger than 1 Mb were counted. Compared with normal diploid male and female DNA sizes of 5977 Mb and 6073 Mb respectively, from the human genome reference hg19, nuclear DNA size of each cell line was estimated from the difference between gains and losses. The sum of sizes from gains, losses and UPD were calculated as the Scale of Genomic Alterations (SGA).Mutation analysis by target sequencing was conducted using a multiplex panel, Oncomine™ Myeloid Research Assay , consisting of 40 DNA genes analyzed by 526 amplicons and 29 RNA genes representing 700 fusion isoforms found in major myeloid disorders. An On-Demand panel, IAD178152, consisting of 6236 amplicons was designed and applied to AML cell lines and the two normal cell lines. This custom panel covered 286 leukemia-related genes and extended to 1.19 Mb in total (Table S2). Ethnic origin of each cell line was assessed by the Precision ID Ancestry Panel . Sequence libraries and templates were prepared using the Ion AmpliSeq Kit for Chef DL8  and the Ion PGM Hi-Q View Chef Kit , respectively. Sequencing was run on the Ion PGM using the Ion 318 Chip v2 BC . Reads were aligned to the hg19 reference and the analysis was carried out using the Ion Torrent Suite and the Ion Reporter (Thermo Fisher Scientific). To eliminate SNPs from variants, variants specific for each Kasumi cell line were extracted by comparisons with the two normal cell lines. Filter settings used for variant analysis of sequence data from the custom panel are given in Table S3. To obtain biogeographic ancestry information, sequence data from the ID panel were analyzed using the Ion Torrent HID SNP Genotyper Plugin (Thermo Fisher Scientific).Cells were washed and incubated for 20 min at 4 °C with antibodies and their isotype controls are listed in Table S4. The cells were washed with 5% FBS/PBS twice and resuspended in 4% PFA, and analyzed on a flow cytometer, FACSCanto using the FlowJo software (BD Biosciences).BCR-ABL1 fusion has been reported in Kasumi-4. Kasumi-10 has been established from a female infant with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), which does not show detectable copy number changes in autosomes, but has a balanced translocation, t. Due to a limitation of this technique [SNP microarray profiles of each cell line are shown in Fig. S1 and described in Table S5. Microarray data, CHP and CEL files, have been submitted to the NCBI repository databases under the BioProject ID PRJNA598005. Copy number alterations were identified in nine of the ten cell lines, with the exception of Kasumi-4. Although the array profile of Kasumi-4 represented an apparently normal female, a translocation, t9;11;22), which forms the echnique , array p, which fGains and losses were observed in 6 and 8 cell lines, respectively Table , S6. TriBecause microarray analysis is performed using genomic DNA samples extracted from an admixture of cells, a non-integer copy number appears in the profiles when the cell population is heterogeneous. This was observed at 9q and 18q in Kasumi-9 , which showed copy numbers of 1.3 and indicated mosaic losses, reflecting heterogeneous cell populations.CDKN2A/p16 locus (9p21.3) was observed in three ALL cell lines, which involved LOH across the boundary of the deletion . Additional losses were detected in other 9p regions, which were different between the three samples. Kasumi-9 had two additional homozygous deletions at 9p23 and 9p21.3 which extended to 5 Mb and 1 Mb, respectively, implying the occurrence of complex rearrangements on its chromosome 9.Cryptic homozygous deletion measured between 300 and 1200 kb at the TP53 mutations were detected in Kasumi-1 and 6, both of them exhibited 17p LOH in the array profiles , resulting in a 100% allele frequency (Table S8). FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) was detected in Kasumi-6 and -10, derived from elderly AML and infant ALL patients, respectively. Kasumi-1 has been used as a KIT mutant AML model [NSD1 was detected in all of the six cell lines at about a 1:3 frequency ratio, which would be explained by a nucleotide change after the gene duplication.Sequence variants detected by the Oncomine™ myeloid panel are listed in Table S7. Of them, hotspot mutations were identified in 7 cell lines Table , S8. TP5ML model , which wTCF3-PBX1 expression was observed in Kasumi-2, compared with other fusions. NUP98-RAP1GDS1 has been identified in Kasumi-5, but it was not covered as a target in the panel. Although the panel is limited to major fusion genes, these chimeras were undetected in Kasumi-3, 6, 7 and 9, which tested negative for those fusions.Fusion transcripts were detected in five cell lines Table , S11. A Analysis of SNP associated with population groups from which the cell lines originated revealed that six of them were classified into the Japanese population and two showed association between Korean and Japanese Table . The othNUP98-RAP1GDS1 [Flow cytometric histograms of 20 cell surface markers are shown in Figure S3. Positive cells were calculated from the histograms and listed as a percentage in Table S13. CD33 positive cells appeared in Kasumi-1, 3, 4 and 6, distinguishing them from the other 6 lymphoid cell lines. In contrast to the 4 myeloid cell lines, five BCP-ALL cell lines, Kasumi-2, 7, 8, 9 and 10, were positive for CD19. Although CD22 and CD38 are B-cell markers, these were detected in an AML cell line, Kasumi-3. CD13 expression was reported in the original leukemia cells of Kasumi-1 [RAP1GDS1 . Kasumi-Expression of HLA-DR was observed in Kasumi-1, 3 and 6 when established , 13, 15,A series of ten Kasumi cell lines have been evaluated by SNP microarray, each having a unique genome profile. Because gross DNA changes could affect gene expression , our quaTP53 mutations [TP53 mutations occur during the later development of leukemic cells and promote disease progression [TP53 alterations can be specified by DNA sequencing, CIN is described qualitatively. Among the 10 cell lines, a high level of SGA, more than 10% of total DNA, is marked in Kasumi-1 and 6 . Because all four cell lines with 9p changes have p16 alteration, instability of 9p regions would be associated with rearrangements at the p16 locus.Homozygous deletion of both se genes . Analysihylation . SNP micDiversity is found not only in leukemia genomes but also in human populations. It is reported that sensitivity to chemotherapy is sometimes different between ethnic groups . SNP anaBCR-ABL1 in CML [KMT2A-MLLT3 in ALL [In contrast to solid tumors, hematopoietic samples are readily available from bone marrow or peripheral blood, allowing diagnosis to be made by direct examination of leukemic cells. Karyotyping can be achieved by a short-term cell culture of these samples, which led to the discovery of disease-specific translocations such as 1 in CML  and KMT23 in ALL . ExperimCancer cells frequently undergo genomic changes through proliferation, known as clonal evolution, resulting in intra-tumor heterogeneity . As thisc-Kit mutation (Kasumi-1), MECOM mutation/activation , FLT3-ITD (Kasumi-6 and 10), GATA-2 mutation (Kasumi-4), C/EBPα mutation (Kasumi-6), NUP98-RAP1GDS1 fusion (Kasumi-5), and other well-known fusion genes . Our panel of Kasumi cell lines promises to serve as a unique resource, which helps to develop a novel molecular target therapy.Leukemia cell lines play a key role in drug development ; howeverSupplementary file1 (PDF 2225 kb)Supplementary file2 (XLSX 230 kb)Supplementary file3 (XLSX 1479 kb)Supplementary file4 (XLSX 504 kb)Below is the link to the electronic supplementary material.


Solanum tuberosum (potato) macro-chloroplast lines overexpressing the tubulin-like GTPase protein gene FtsZ1 from Arabidopsis thaliana. Macro-chloroplast lines exhibited delayed growth at anthesis; however, at the time of harvest there was no significant difference in height between macro-chloroplast and wild-type lines. Macro-chloroplasts were successfully transformed by biolistic DNA-delivery and efficiently regenerated into homoplasmic transplastomic lines. We also demonstrated that macro-chloroplasts accumulate the same amount of heterologous protein than wild-type organelles, confirming efficient usage in plastid engineering. Advantages and limitations of using enlarge compartments in chloroplast biotechnology are discussed.Chloroplast biotechnology is a route for novel crop metabolic engineering. The potential bio-confinement of transgenes, the high protein expression and the possibility to organize genes into operons represent considerable advantages that make chloroplasts valuable targets in agricultural biotechnology. In the last 3 decades, chloroplast genomes from a few economically important crops have been successfully transformed. The main bottlenecks that prevent efficient transformation in a greater number of crops include the dearth of proven selectable marker gene-selection combinations and tissue culture methods for efficient regeneration of transplastomic plants. The prospects of increasing organelle size are attractive from several perspectives, including an increase in the surface area of potential targets. As a proof-of-concept, we generated  While metabolic engineering in plastids has been used for bioproduction of carotenoids4, terpenoids5, vaccines7, pharmaceutical drugs9 and valuable enzymes of industrial interest11, no commercial transplastomic crops have been introduced in the market. Even fewer studies have focused on plastid engineering for crop improvement, such as increasing photosynthetic efficiency13 or mitigating biotic and abiotic stresses15. Given the inherent bioconfinement of transgenes in plastids17, the lack of transgene position effects18, absence of gene silencing mechanisms, extremely high heterologous protein production20, and ability to coordinate gene expression in operons22, plastid engineering appears to be an attractive strategy. Nonetheless, there are some technological barriers that are impediments in need of addressing in order to widely implement chloroplast transformation for crop agriculture.Chloroplast biotechnology has long been recognized as a relatively untapped resource for enabling metabolic engineering and crop improvement23, chloroplasts of few agronomically important dicotyledon crops including tomato, potato and lettuce26 have been successfully transformed and regenerated to homoplasmic transplastomic lines. All monocotyledon cereals have proven recalcitrant to current methods to generate stable transplastomic plants. Only few examples of transient chloroplast transformation in rice (Oryza sativa) have been published28. Successful chloroplast transformation in key crops, including cereals, will depend on the ability to find more efficient selection  and tissue culture methods to efficiently regenerate transplastomic plants.Together with the model species tobacco and more recently, Arabidopsis29, for alternative methods of plastid transformation (e.g. microinjection), an increase in plastid size would be beneficial if not necessary. For example, the installation of large DNA molecules (synthetic plastid genomes) will likely require alternative transformation strategies.The ability to significantly increase plastid size provides an intriguing avenue with regards to plastid biotechnology. While the small size of plastids has been shown to not be a limiting factor for biolistic transformation, assuming a strong selection system31. FtsZ1 is a tubulin-like GTPase that, in association with FtsZ2 and other division-related components, regulate chloroplast division by participating in the formation of a z-ring at the mid-organelle35. Considering that the stoichiometry between FtsZ proteins is important for their correct function in z-ring formation, the most common strategy to increase chloroplast size is through altering FtsZ1 gene expression34. Overexpression of FtsZ1 in Arabidopsis was altered chloroplast division resulting in a reduction of organelle number and drastic increase in size, leading to the term “macro-chloroplast”36. Surprisingly, despite this extreme chloroplast phenotype, macro-chloroplast lines were indistinguishable from wild-type lines with regards to plant growth and morphology30. However, in a follow-up study in Nicotiana tabacum (tobacco) a significant growth reduction was observed under high- and low-light irradiation in lines with a severe macro-chloroplast phenotype (defined as 1–3 large macro-chloroplasts per cell)37. In medium-light irradiation, the growth of all macro-chloroplast lines were indistinguishable from controls37. In potato, macro-chloroplast lines produced alterations in both chloroplasts and amyloplasts38. Macro-chloroplast potato demonstrated morphological changes in starch granule size as well as increased phosphorous content in isolated starch grana compared to wild-type lines38. The ability to alter the size and content of non-green plastids is particularly relevant to plastid biotechnology for food crop improvement, where non-green plastids represent a primary source of nutrition (e.g. chromoplasts of tomato)40.In higher plants, chloroplast division is regulated by two FtsZ protein homologous (FtsZ1 and 2)41. In addition, researchers demonstrated that homoplasmic macro-chloroplast plants could be regenerated in tissue culture where they accumulated a similar amount of foreign protein compared to traditional chloroplast engineering42. Based on this foundational work, we sought to develop macro-chloroplast potato lines specifically for the purposes of evaluating their potential for chloroplast biotechnology. We hypothesized that the use of macro-chloroplast containing leaves would increase the transformation efficiency of potato, providing a superior method for potato chloroplast engineering. Secondarily, we hypothesized that the use of macro-chloroplasts may increase the capacity for protein overproduction without negatively affecting chloroplast function. This would be particularly useful for production of plant bioreactors for high production of foreign proteins.While methods to generate macro-chloroplasts have been established for decades, there are few examples of chloroplast engineering using these altered plastids. The first successful transformation of macro-chloroplasts in tobacco, using biolistics, demonstrated a 40% increase in chloroplast transformation efficiency relative to wild-type controlsAtFtsZ1 overexpressing potato lines and selected for macro-chloroplast phenotypes for use in chloroplast biotechnology. The phenotypes of macro-chloroplast lines were characterized, as well as, the efficiency of chloroplast transformation, selection, and regeneration in selected lines.Specifically, in this work we engineered AtFtsZ1 under the control of the CaMV 35S promoter were generated by Agrobacterium-mediated transformation. In a first screen, six independent lines were chosen for in-depth analysis from > 200 putative transgenic lines using microscopic analysis of chloroplast morphology in leaf mesophyll-derived protoplasts  or SSC (ndhG/ndhI) regions of plastome were used  were conducted  in all wild-type and macro-chloroplast transplastomic lines  or photodamage of the photosynthetic machinery (at high-light irradiation), resulting in reduced plant growth in both cases44.The results from this work suggest that macro-chloroplast lines have a delay in growth and significant reduction in tuber biomass compared to wild-type controls Fig. . Despitenes Fig. g,h. AlteAtFtsZ1 transgenic lines can be transformed using biolistics and transplastomic lines can be regenerated using standard tissue culture. We anticipated that increasing the size of the target organelle may provide an advantage in transformation efficiency in potato as it has been previously demonstrated in tobacco41. Considering the transformation efficiencies of potato chloroplasts reported in the literature47, our macro-chloroplast lines can be efficiently transformed, yielding approximately 1 transplastomic plant per each plate bombarded  transformation53 and electroporation55 have been used to transform chloroplasts. Different kinds of commercial carbon-nanotubes have also been used to deliver DNA into plants cells57, and these delivery techniques could also represent an alternative to deliver vectors more efficiently into macro-chloroplasts. Thus, macro-chloroplasts could provide considerable advantages in increasing transformation efficiency using these methods.Nevertheless, this result does not exclude the potential for macro-chloroplasts to be desirable targets for other gene-delivery methods in which the transformable surface is an important limiting factor. For almost 3 decades, the gene gun has been the most efficient and reproducible method used for chloroplast transformationtrnI/trnA and ndhG/ndhI integration sites  in both macro- and wild-type chloroplast-engineered plants. While a difference in plastome copy number was previously observed in macro-chloroplast and wild-type tobacco58, our results suggest that potato AtFtsZ1 overexpressing lines have similar plastome copy number  var. ‘Desirée’ and AtFtsZ1 overexpressing lines were grown in Magenta boxes containing MS Reg media . Transplastomic lines of both genotypes (wild-type and macro) were grown in selective MS rooting media60.AtFtsZ1 lines, respectively) or full maturity (24-weeks-old for both genotypes). Pots of 3.8 or 11.4 l volume were used to grow plants at anthesis or full maturity, respectively. Plant height (cm), fresh and dry weight (g) of leaves and total biomass were measured. The total number, along with the dry and fresh weights (g) of tubers were also measured. For determination of dry weight, plant tissue was dried for 1 week at 50 °C. Both leaf and tuber areas were obtained by image analysis using ImageJ 1.41o software .For determination of growth characteristics, apical shoots (same length and age) from both transgenic and wild-type plants were in vitro propagated. After 2 weeks, small plantlets with roots were transferred to soil, Pro-Mix BK25 , and grown to anthesis (9- and 10-weeks-old for wild-type and 2 assimilation (A) values per unit of leaf area (µmol m−2 s−1) were obtained using a LI-6800 portable photosynthesis system  at atmospheric CO2 concentrations (400 µmol mol air−1), constant irradiance (1000 µmol photons m−2 s−1), ambient temperature of 23 °C, a vapour pressure deficit (VPD leaf) of 0.8–1.2 kPa and a flow rate of 200 µmol s−1). The leaf chlorophyll content index (CCI) was obtained using a portable CCM-200 plus chlorophyll content meter .COAtFtsZ1 independent lines (Macro 1 and 2) along with wild-type control) for the growth experiments at anthesis and full maturity, respectively. ANOVAs with post-hoc Tukey statistical analysis (IBM SPSS software) (p < 0.05) were performed to determine if differences were statistically significant among wild-type and AtFtsZ1 lines. Plants grown in vitro and in pots were subjected to congruent environmental conditions, 16/8 h of light and dark respectively, and the temperature was kept constant at 24 °C.Results were expressed as mean ± standard deviation (sd) of six and four biological replicates for each genotype  plastome IR  and SSC  regions, respectively, were synthesized by GeneArt . The two homologous regions were modified including several SNPs to facilitate cloning. Before introducing the selection cassette between homologous arms, the trnI/trnA and ndhG/ndhI regions were cloned into pMK vector . A chloroplast selection cassette (Prrn-SD::aadA::5′UTR::GFP::psbA3′UTR) was PCR amplified from the pLD-PTD-GFP plasmid61 using 1Fw/1Rv primers and cloned into the PmeI site of trnI/trnA and ndhG/ndhI sites generating pIR and pSSC plasmids, respectively. A list of primers used in this work is shown in Supplementary Table The vector pAP202 containing the full-length cDNA of AtFtsZ1 overexpressing lines (macro-chloroplast lines) were generated by Agrobacterium-mediated transformation. The vector pAP20236 was introduced into Agrobacterium tumefaciens LBA4404 by the freeze thaw method62. For each transformation, 20 internodes of ~ 1 cm-length from 1-month-old in vitro potato plants were used. Internodes were then transformed via Agrobacterium and transgenic plants were regenerated in selective media, as previously described63. Putative transgenic lines were screened by fluorescent microscopy for clearly-defined enlarged chloroplasts. Thereafter, selected lines were genetically characterized for construct integration and transgene expression.Potato 64. Macro-chloroplast line 1 was used for chloroplast transformation. Per each transformation ~ 6 cm2 of leaf tissue from 1-month-old in vitro potato plants were used. Per each shoot 0.3 mg of gold-particles (0.6 µm in diameter) binding 1 µg of plasmid were used following the manufacturer protocol . Transplastomic plants of both genotypes (wild-type and macro-chloroplast) were then regenerated from transformed leaf material incubated in selective media as described previously60. The second round of transplastomic plants were obtained by applying the same protocol of tissue culture/selection/regeneration60. All lines were genetically characterized for the presence of the selection cassette integrated and GFP expression.The PDS-1000/He biolistics device  was subsequently used to transform chloroplasts of both macro-chloroplast and wild-type potato plants64. PCR (25 cycles) was performed in 25 µl reaction-volume by using 5 ng of total genomic DNA and the DreamTaq Green PCR Master Mix . For molecular characterization of AtFtsZ1 overexpressing lines, the pairs of primers 2Fw/2Rv and 3Fw/3Rv were used to check for nptII/AtFtsZ1 cassette integration and the internal control actin (GeneID-102593904), respectively. For genotyping of transplastomic lines, the two pairs of primers 4Fw/4Rv and 5Fw/5Rv were used to verify cassette integration in the trnI/trnA and ndhG/ndhI sites of plastome, respectively. The two pair of primers 6Fw/6Rv and 7Fw/7Rv were used to amplify full-length GFP (NCBI ID: AEX93343.1) and aadA (NCBI ID: AAR14532.1) genes, respectively. The primers 8Fw/8Rv were used to amplify an internal portion of rbcL (NCBI ID: 4099985).Total genomic-DNA preparations from leaves were obtained using ~ 50 mg of tissue and the CTAB-based procedure of extractionAtFtsZ, whereas the two internal controls rbcL (plastome) and ef1α (nuclear) were used for normalization. The pair of primers 9Fw/9Rv, 10Fw/10Rv and 11Fw/11Rv were used to amplify and internal fragment (~ 100 bp) of AtFtsZ (Tair ID: AT5G55280), rbcL  and ef1α (NCBI ID: NM_001288491.1), respectively.Total RNA extraction was performed using Tri-Reagent  according to manufacturer’s protocol. Per each extraction ~ 50 mg healthy leaf tissue was used. Total RNA preparations were cleaned and subjected to DNase treatment using the RNA Clean & Concentrator Kit  according to manufacturer’s instruction. The cDNA synthesis was performed using the Super Script III Reverse Transcriptase  following the manufacturer’s instruction. cDNA was quantified using a NanoDrop , and several serial cDNA dilutions were tested by PCR (as described before) for the presence of the transgene KasI/HindIII and FspI/ScaI restriction enzymes, respectively. The DNA fragments were separated on 0.9% agarose gel, and after that, the gel was depurinated, denatured and transferred on a nylon membrane as described previously13. The membrane was then incubated with the DIG-labelled probe and detected using the anti- digoxigenin-AP Fab fragments detection kit  accordingly to the manufacturer’s protocol.DNA probes for detection of pIR and pSSC constructs integrated into potato plastome (GenBank: NC_008096.2) were designed on IR  and SSC  regions, respectively. The PCR DIG Probe Synthesis Kit  was used to synthesize digoxigenin(DIG)-sUTP-labelled IR and SSC DNA-probes using the pair of primers 12Fw/12Rv and 13Fw/13Rv, respectively. Total genomic DNA from leaf tissue was extracted using CTAB, as described above. After quantification, 1 µg of DNA for each pIR and pSSC sample was digested using 65. The primers 10Fw/10Rv and 14Fw/14Rv were use to detect the plastome gene rbcL (NCBI ID: 4099985) and the nuclear gene actin (GeneID-102593904), respectively (Supplementary Table 2(2−ΔCT) of rbcL vs actin. Results were expressed as mean ± standard deviation (sd) of 3 biological replicates and 8 technical replicates per biological replicate per each genotype. An ANOVA post-hoc Tukey statistical analysis (p < 0.05) was performed to determine statistically significant difference among means (IBM SPSS software).PCRs were performed in 96-well plates , in a total volume of 15 µl per reaction using 1X PowerUp SYBR Green Master Mix , 5 ng of total genomic DNA from leaves and 0.5 µM of each primer. Primers 20 bp-long with an annealing temperature of ~ 57 °C and able to amplify a ~ 100 bp-fragment were design using the online software Primer3 input v. 0.4.0 66. The results were expressed as mean ± standard deviation (ng GFP/mg fresh weight) of two indipendent experiments, resulting in a total of 2 biological replicates and 2 technical replicates per biological replicate for each independent transplastomic line. We analyzed 15 pIR and 8 pSSC lines for each genotype, normal and macro-chloroplast.The amount of GFP accumulated in leaf tissue of transplastomic lines was quantified by using the Fluorometric GFP Quantification Kit  according to the manufacturer’s protocolAtFtsZ1 overexpressing lines and wild-type controls. The results were expressed as mean ± standard deviation (sd) and the statistical analysis was performed using SPSS statistics 25 software (IBM).Leaf tissue from 3-week-old in vitro plants was imaged using an Olympus Fv1200 confocal microscope . GFP was excited at 488 nm and detected at an emission wavelength of 509 nm. Chlorophyll autofluorescence was visualized using an excitation wavelength of 543 nm and an emission wavelength of 667 nm. Confocal images were acquired using the manufacturer’s Olympus FV10-ASW Viewer software Ver.4.2a . Confocal images were processed using the online software ImageJ 1.41o . The same ImageJ software was also used to estimate chloroplast size in both 67. Ultrathin sections (60–100 nm) of embedded tissue were cut using a diamond knife and a Leica EM UC7 ultramicrotome . Ultrathin sections were post strained using aqueous solutions of uranyl acetate and lead citrate as described previously67. Images were obtained using a JEOL 1400 transmission electron microscope operating at 80 kV  equipped with a Gatan OneView camera . TEM micrographs were processed using the software ImageJ 1.41o .Leaf tissue from 3-week-old in vitro plants were chemically fixed in glutaraldehyde/ paraformaldehyde along with an osmium tetroxide solution and embedded in EMBed-821 resin  as described previously43. GFP fluorescence in plants was acquired at 150 ms exposure time using 465 nm excitation and 525 nm emission notch (50 nm) filter. Standoff detection was performed at 3 m from the laser source. FILP images were processed using ImageJ 1.41o software .Plant fluorescence patterns were characterized using the Fluorescence-Inducing Laser Projector (FILP), a custom-built instrument for imaging fluorescence in plants. FILP imaging was performed on 3-week-old transplastomic lines in potsSupplementary Information 1.Supplementary Video 1.


The WW domain-containing oxidoreductase (WWOX) tumor suppressor gene is frequently lost in a variety of solid and hematopoietic malignancies in humans. Dysregulation of WWOX has been implicated as playing a key role in tumor cell survival, DNA damage repair, and genomic stability. The purpose of this study was to characterize WWOX expression in spontaneous canine mast cell tumors (MCTs) and malignant cell lines and investigate the potential contribution of WWOX loss on malignant mast cell behavior.WWOX expression is decreased in primary canine MCTs and malignant mast cell lines compared to normal canine bone marrow-cultured mast cells. In transformed canine mastocytoma cell lines, overexpression of WWOX or WWOX knockdown had no effect on mast cell viability. Inhibition of WWOX enhanced clonogenic survival following treatment with ionizing radiation in the C2 mast cell line. Lastly, immunohistochemistry for WWOX was performed using a canine MCT tissue microarray, demonstrating that WWOX staining intensity and percent of cells staining for WWOX is decreased in high-grade MCTs compared to low-grade MCTs.These data suggest that WWOX expression is attenuated or lost in primary canine MCTs and malignant mast cell lines. Given the observed increase in clonogenic survival in WWOX-deficient C2 mast cells treated with ionizing radiation, further investigation of WWOX and its role in mediating the DNA damage response in malignant mast cells is warranted.Supplementary information accompanies this paper at 10.1186/s12917-020-02638-3. Mast cell tumors (MCTs) are the most common malignant skin tumor in dogs, accounting for approximately 7–20% of all cutaneous tumors –5. The bc-KIT in approximately 30% of dogs with aggressive MCTs has provided insight into the genetic changes that mediate the biological behavior of MCTs [The etiology of canine MCTs is largely unknown; however, the identification of activating mutations in the proto-oncogene  of MCTs –10. It h of MCTs . While d of MCTs , 8. Whil of MCTs , 13. The of MCTs .WWOX gene resides in the genomic region FRA16D at 16q23, the second most common human chromosomal fragile site that is prone to frequent chromosome breakage [WWOX and study its relevance in human health and disease.The WW domain-containing oxidoreductase (WWOX) is a highly conserved, 46 kDa protein consisting of two N-terminal WW domains and a C-terminal short-chain dehydrogenase/reductase domain . The firbreakage , 23. Thebreakage . The intWWOX makes the locus susceptible to loss of heterozygosity (LOH) or homozygous deletion of the gene, resulting in reduced gene expression [WWOX promoter has been shown to transcriptionally inactivate WWOX in ovarian, pancreatic, lung, and breast cancer cell lines [WWOX gene promoter and aberrant splicing events result in atypical nuclear localization and attenuated WWOX function [There is now substantial data supporting the role of WWOX in cancer biology, including the observation that loss or attenuation of WWOX occurs in a number of human malignancies including bone, breast, lung, liver, ovarian, pancreatic, and many hematopoietic cancers , 25–28. pression . Alternall lines . In contfunction .Wwox deletion found an increased incidence of spontaneous tumor formation in mice, suggesting a critical role for WWOX in tumorigenesis [Wwox (+/−) mice developed significantly more ethyl nitrosourea-induced lung tumors and lymphoma compared to wild-type littermate mice, suggesting that Wwox haploinsufficiency is cancer predisposing. The versatile nature of WWOX is attributed, in part, to its ability to directly interact with proteins in a variety of cellular processes, including cell differentiation and growth. One of the first binding partners identified to interact with WWOX was p73, a p53 family homolog that mediates cellular response to stress, including regulating cell cycle checkpoint activation and apoptosis [Importantly, in vivo studies investigating the phenotypic consequences of global igenesis . Additiopoptosis . Loss ofpoptosis –36. Givepoptosis , 37–41.It is well established that WWOX expression is altered in many human malignancies and that WWOX functions as a tumor suppressor gene through dysregulation of target binding partners , 20. CurThe presence of genetic alterations in WWOX, largely due to loss of heterozygosity, and its reduced expression has been observed in a number of human cancers , 43. MorCanine BMCMCs possess phenotypic and functional properties similar to those described of mast cells directly isolated from canine skin; however, BMCMCs are dependent on recombinant canine stem cell factor (rcSCF) for survival in vitro . In contAdditionally, Western blotting of protein lysates derived from fresh frozen canine MCT tissue samples revealed low or non-detectable levels of WWOX protein in 3 of the 5 tumors Fig. d. AlthouTo investigate the functional consequences of WWOX dysregulation in malignant canine mast cell lines, the canine BR mast cell line that exhibits low basal levels of WWOX was transduced with a full-length canine WWOX cDNA lentiviral expression vector. Stably transduced GFP + cells were sorted and WWOX overexpression was validated using qRT-PCR and Western blotting Fig. a.Fig. 2Reciprocal experiments were performed in the canine C2 mast cell line which expresses relatively high levels of WWOX. We designed several lentiviral-shRNAs targeting canine WWOX (shWWOX-554 and shWWOX-1304) to determine the impact of WWOX knockdown on the behavior of C2 mast cells in vitro. As shown in Fig. Prior studies have shown that ectopic expression of WWOX through various methods such as adenovirus-delivered WWOX cDNA (Ad-WWOX) or treatment with the DNA methyltransferase inhibitor,, 5-aza-2′-deoxycytidine to activate the endogenous WWOX gene results in decreased cell growth and viability in human breast and lung cancer-derived cells in vitro and in vivo , 47. To WWOX allele , highlighting the possibility that disruption of WWOX may be a key molecular alteration in aggressive MCTs. Furthermore, recent evidence supports a critical role for the tumor suppressor WWOX in DNA damage response and maintenance of genomic stability, in part, through its association with ataxia telangiectasia-mutated (ATM) following double strand DNA (dsDNA) breaks [p = 0.0416, Fig. p = 0.1200). In WWOX-deficient C2 mast cells, the shape of the survival curve had broader shoulder compared to the scramble control cells, providing support for the notion that WWOX attenuation may diminish sensitivity to ionizing radiation in this cell line.Genomic instability is a major risk factor for cancer development and progression. With respect to canine MCTs, genomic copy number profiles of tumors of different histological grades demonstrate a stepwise accumulation of numerical DNA copy number aberrations (CNAs) as histological grade increases, suggesting that these genomic alterations contribute to genomic instability and progression of canine MCTs. Interestingly, four CNA markers  were found to discriminate low-risk from high-risk MCTs with a sensitivity of 78–94% and specificity of 88–93% . Of note) breaks , 48. GivSimilar colony formation assay experiments were performed with the BR mast cell line; however, no colony growth was observed in the presence of methylcellulose-based media (data not shown), and thus the effect of WWOX overexpression on clonogenic survival of these cells could not be determined using this assay. Alternatively, we evaluated BR cell viability at multiple time points using an MTT assay as a means to assess cell proliferation following ionizing radiation treatment. While treated BR cells a demonstrated dose-dependent decrease in cell viability, no significant difference in cell viability and proliferation was found in BR cells expressing control vector or overexpressing WWOX (data not shown). The lack of observed differences in cell viability indicates that restoring WWOX does not significantly impact sensitivity to ionizing radiation in this cell line.N = 26) and paired normal skin samples (N = 19) and immunohistochemical (IHC) staining for WWOX was performed. All tumors were graded using the 2-tier grading system and IHC scoring was performed by two blinded board-certified veterinary pathologists using the specifications outlined in Table To more globally assess the prevalence of mast cell-specific WWOX expression in primary canine MCT samples, a tissue microarray was constructed from 45 archived canine MCT surgical biopsies  showed significantly lower staining intensity compared to low-grade tumors (N = 14)  Fig. a. We fou14) Fig. b. There WWOX allele. Concordantly, we found that primary canine MCTs and malignant mast cell lines express low basal levels of WWOX transcript compared to that found in normal canine BMCMCs. Low levels of WWOX were detected by Western blotting in malignant canine and mouse mast cell lines evaluated and this was independent of culture conditions and rcSCF stimulation. Similar to our findings in mast cell lines, WWOX protein expression was low to absent in a subset of fresh frozen MCTs derived from canine patients. These findings support previous studies demonstrating that WWOX is frequently attenuated or lost in human malignancies and suggest its loss may be involved the neoplastic transformation of canine mast cells [The molecular characteristics and chromosomal location of canine fragile sites have not been intensively studied; however, recent genomic profiling data demonstrate that canine MCTs exhibit a broad range of genome-wide copy number aberrations (CNAs) and that the frequency of CNAs increases with histological grade . These dst cells , 25, 49.To assess the prevalence of basal WWOX expression in primary canine MCT tissue samples, we constructed a TMA from archival formalin-fixed paraffin embedded tissue samples and performed IHC for WWOX. We analyzed WWOX expression levels in primary canine MCTs and found that the intensity of WWOX staining and percentage of stained neoplastic cells was significantly lower in high-grade MCTs. The limited number of primary MCTs evaluated in the current study precludes us from drawing conclusions about the expression of WWOX in high-risk MCTs. However, the observation that WWOX expression is attenuated in high-grade MCT provides support for the notion that loss of WWOX may correlate with the degree of mast cell differentiation and the acquisition of more a more aggressive biological behavior. Given the lack of complete patient information and follow-up accompanying the archival canine tumor samples, it was not possible to determine the prognostic significance of WWOX immunoreactivity in canine MCTs. Nevertheless, the finding that WWOX expression is frequently decreased in histologically high-grade tumors merits further investigation as to the role of WWOX in promoting an aggressive tumor phenotype.To define the functional consequences of altered WWOX expression on mast cell behavior, we generated canine BR mast cells stably overexpressing WWOX or inhibited WWOX function in the canine C2 cell line using shRNA techniques. Interestingly, enforced WWOX expression or WWOX knockdown had no effect on cell proliferation in the BR or C2 mast cell lines. Additionally, overexpression of WWOX in the canine BR mast cell lines did not significantly alter cell viability following treatment with ionizing radiation. In contrast to our study, numerous studies have shown that WWOX directly influences cell viability and survival in normal and malignant epithelial cells in vitro and in vivo in mouse xenograft tumor models . These oIn the current study, the canine BR mast cell line exhibited poor overall survival and colony formation using methycellulose-based media; as such, the effect of WWOX restoration on clonogenic survival following IR could not be fully assessed using a standard colony formation assay in this cell line. This cell line-intrinsic finding may reflect potential differences in cell surface protein expression that impacts the formation of clonogenic colonies in the BR cell line, further demonstrating the complicated genetic heterogeneity known in these tumors , 51. AltOne of the more intriguing findings in this study was that inhibition of WWOX enhanced clonogenic survival in the C2 mast cell line following radiation-induced double strand DNA (dsDNA) breaks. This finding was independent of cell viability and supports the idea that WWOX may play a role in mediating the DNA damage repair in response to dsDNA breaks. Prior studies have demonstrated that WWOX expression is increased following single- and double-stranded DNA breaks and it is recruited to sites of DNA damage. The recruitment and activation of WWOX facilitates DNA damage repair mediated in part, through its direct interaction with ATM and indirectly, through ATR , 48. LosThe present study investigated the impact of WWOX expression on certain aspects of cell behavior; however, the molecular factors by which WWOX influences clonogenic survival in mast cells requires further investigation. Furthermore, inhibition of WWOX binding partners that mediate this phenotype in canine mast cell lines would provide further convincing evidence of the importance of WWOX in the DNA damage repair response. As such, identifying proteins altered by WWOX that enhance clonogenic survival and validating these targets in canine mast cell lines/tumors represents an area of ongoing investigation.Our data demonstrate that WWOX expression is significantly decreased in primary canine MCT tissues and malignant canine mast cell lines compared to normal canine BMCMCs. In addition, the intensity and percentage of neoplastic cells staining for WWOX via IHC was negatively associated with histologic grade in canine MCTs, further supporting the notion that loss of WWOX contributes to the aggressive biological behavior of some canine MCTs. In the C2 canine mast cell line, shRNA-mediated WWOX knockdown enhanced clonogenic survival in response to ionizing radiation-induced dsDNA breaks, implying that WWOX may play a direct role in mediating the DNA damage repair response in malignant mast cells. This work serves as the foundation for future work to dissect the molecular mechanisms by which WWOX dysregulation contributes to the aggressive biological behavior of malignant canine mast cells, with the ultimate goal of identifying new targets for therapeutic intervention in this disease.Primary cutaneous mast cell tumor (MCT) tissue samples were collected from clinical cases that presented to The Ohio State University Veterinary Medical Center (OSU-VMC). Consent for tissue collection was obtained from all owners in accordance with an approved IACUC protocol (2010A0015) and collected by the OSU-VMC Blue Buffalo Clinical Trials Office and Veterinary Clinical Research Shared Resource. A total of 26 primary MCTs with 19 paired normal skin biopsies were identified. Surgical and post-mortem collected tumor samples were placed in formalin and processed for routine paraffin embedding for histopathology. This was a pilot study with the intent to characterize the expression of WWOX in primary canine MCTs and malignant cell lines, so no power calculation was made.Representative areas of tumor tissue and skin containing vasculature and normal mast cells were identified on hematoxylin and eosin (HE) stained sections by a single, board-certified veterinary pathologist (RJ) and graded using the Patnaik and Kiupel grading schemes. 2.0 mm core samples were extracted from the corresponding areas on 45 paraffin embedded tissue blocks and inserted into predetermined sites on the tissue microarray (TMA) recipient block. Immunohistochemical staining was performed on all MCT and normal skin samples for WWOX using rabbit anti-WWOX antiserum . This antiserum detects endogenous and recombinant human and murine WWOX amino acid residues 12–94 containing both of the WW domains. Human and canine WWOX exhibit 94.4% similarity at the protein level and the region corresponding to WWOX amino acids 12–94 shares 96% sequence homology , 56. NorSlides were evaluated by light microscopy to assess WWOX immunoreactivity. Overall WWOX signal intensity of normal and malignant mast cells was subjectively scored from 0 to 3  by two boarded veterinary pathologists  using the specifications detailed in Table KIT) and C2 (KIT ITD mutation in the JM domain) were generously provided by Dr. Warren Gold  and murine cell lines P815 (D814V KIT mutation) and C57 (wild-type KIT) were generously provided by Dr. Stephen Galli . The cells were maintained in Roswell Park Memorial Institute medium (RPMI) 1640 , supplemented with 10% fetal bovine serum (FBS) , Antibiotic-Antimycotic , GlutaMAX™ , non-essential amino acids, sodium pyruvate, and HEPES (4-(2-dydroxethyl)-1-piperazineethanesulfonic acid) at 37 °C, supplemented with 5% CO2 (media supplements from Gibco®). Canine bone marrow cultured mast cells (cBMCMCs) were generated from 2 dogs and maintained in Stemline  medium supplemented with recombinant canine stem cell factor  as previously described [The canine mastocytoma cell line BR  according to the manufacturer’s instructions. cDNA was made from 500 ng total RNA using Superscript III . Primers designed and utilized for canine WWOX and 18S endogenous control are listed in Table Real-time quantitative PCR (RT-qPCR) was performed to measure WWOX transcript expression using the methods described above. qRT-PCR was performed using Applied Biosystem’s StepOne Plus Real-Time PCR system (Applied Biosystems ThermoFisher Scientific). Canine WWOX and 18S were detected using Fast SYBR Green PCR master mix according to the manufacturer’s instructions. Data normalization was performed relative to 18S internal control. Experiments were repeated three times using samples in triplicate and included non-template controls for each gene. Relative gene expression for all RT-qPCR data was calculated using the comparative threshold cycle method .To assess the relative expression of WWOX, protein lysates were generated from primary MCT tissues or mast cell lines. Briefly, frozen tumor samples were pulverized using a frozen mortar and pestle. The resulting powder was resuspended in liquid nitrogen and transferred to 1.5 mL microcentrifuge tube. Tumor samples and mastocytoma cell lines were washed twice with 1X Dulbecco’s phosphate-buffered saline  and resuspended in complete lysis buffer consisting of 20 mM Tris-HCl pH 8.0, 137 nM NaCl, 10% glycerol, 1% IPEGAL CA-630, 10 mM ethylenediaminetetraacetic acid (EDTA), 1 mg/mL aprotinin, 1 mg/mL pepstatin A, 1 mM phenylmethylsulphonyl fluoride, 1 mM sodium orthovanadate, and 10 mM sodium fluoride. Samples were rocked for 1 h at 4 °C, centrifuged for 15 min at 14,000 RPM at 4 °C, and supernatants collected. Bradford protein quantification assay was performed on the extracts using BioRad Reagent  according to manufacturer’s instructions. Equal amounts of total protein were separated on 12% SDS-PAGE gels  and transferred onto PVDF membranes. Membranes were blocked in TBS-T containing 3% non-fat dry milk for 1 h and incubated overnight with polyclonal rabbit anti-WWOX antibody . The membranes were incubated with appropriate horseradish peroxidase linked secondary antibody , washed, and exposed to luminol enhancer Supersignal® West Dura Extended Duration Substrate developer . Blots were stripped using Restore™ Western Blot Stripping Buffer , washed, and reprobed for β-actin . Band intensities were calculated using ImageJ software  and relative intensity of WWOX was determined by dividing by β-actin.7) were cultured in complete media supplemented with 100 ng/mL canine recombinant SCF  or left untreated for 24 h at 37 °C supplemented with 5% CO2. Cells were collected, washed twice with 1X DPBS, and immunoblotted to detect WWOX expression was performed as previously described.To determine the potential effect of stem cell factor (SCF) stimulation on WWOX expression in canine mast cell lines, BR and C2 cells  and cells were infected with empty control (EV) lentivirus or WWOX lentivirus or TransDux™ alone for 24 h. FACS-mediated sorting based on GFP expression was performed 72 h post-transduction and WWOX overexpression was validated by qRT-PCR and Western blotting analysis.Full-length canine WWOX cDNA was amplified by RT-PCR from normal canine testes and the resultant product was gel purified and sequenced. Canine WWOX cDNA was ligated into the pGEMT plasmid vector , and subcloned into the pCDH-CMV-MSC-copGFP lentiviral expression plasmid . Packaging of the lentiviral constructs was performed using the pPACKH1 Lentivector Packaging Kit  according to manufacturer’s instructions. Briefly, 1 × 104 C2 cells were plated and left overnight in complete medium. The following day, medium was replaced with serum-free medium and target cells were infected with transfection agent TransDux™ (Systems Biosciences) and either pGreenPuro-Scramble or pGreenPuro-shWWOX virus or TransDux™ alone for 24 h. FACS-mediated sorting based on GFP expression was performed 72 h post-transduction. Cells were collected and processed for qRT-PCR and Western blotting as described below to detect levels of WWOX and efficiency of knock down. Sequences of template canine DNA were as follows: pGreenPuro-shWWOX-554 (5′-CCCTATGGATGGGAACAAGAA-3′) and pGreenPuro-shWWOX-1304 (5′-ACATGATGTACTCCTCCATTC-3′).Stable knock down of WWOX was performed using short hairpin RNA (shRNA) constructs cloned into the pGreenPuro shRNA cloning lentivector  and high-titer lentiviral stocks were generated as described above. Briefly, 1 × 103) transduced with EV control or WWOX lentiviral constructs and C2 cells (5 × 103) expressing scramble, shWWOX-554 or shWWOX-1304 constructs were seeded in complete medium in quadruplicate in 96-well plates and incubated for 24, 48, 72 or 96 h. To determine the influence of WWOX expression on mast cell viability following treatment with DNA damaging agents, BR cells (5 × 103) transduced with EV control or WWOX lentiviral constructs were treated with increasing doses  of ionizing radiation (IR) using a Siemen’s ONCOR linear accelerator and incubated for 24, 48, 72, or 96 h. 10 μL of MTT reagent  was added to each well and cells were incubated for an additional 4 h under normal culture conditions. Following incubation, 100 uL lysis buffer was added and the fluorescence was measured using a SpectraMax M2 microplate reader  with excitation at 570 nm and emission 670 nm filters. Cell viability and proliferation was calculated as the fold change of absorbance from 0 h control cells. Each experiment was repeated three times using samples in quadruplicate.To determine the effects of WWOX dysregulation on mast cell viability, BR cells  were placed above a solid water-equivalent plate (12 mm) and irradiated from below with the gantry at 180°. The dose distribution for this set up was verified by a medical physicist. Control (0 Gy treatment) cell culture plates were transported to the radiation therapy area but kept outside the radiation vault during treatments. After individual flasks were treated with doses of 0, 2, 4, and 6 Gy of radiation, cells were counted and plated to appropriate density based on dose received  of ionizing radiation at ambient temperature and pressure utilizing a linear accelerator . Cell culture flasks  and the ∆∆ Ct method was used to compare mRNA expression using Student’s t-test or one-way analysis of variance (ANOVA) . TMA datAdditional file 1: Figure S1. Uncropped Western blots.  Canine BR mast cell lines were left untreated (Lane 1) or incubated with 100 ng/mL rcSCF (Lane 2) and canine C2 mast cell lines were left untreated (Lane 3) or incubated with 100 ng/mL rcSCF. Western blotting for WWOX  and β-actin  was performed.  Canine BR (Lane 5) and C2 (Lane 6) cell lines and mouse C57 (Lane 7) and P815 (Lane 8) mast cell lines were processed for protein lysates. Western blotting for WWOX (upper panel) and β-actin (lower panel) was performed.  Canine primary mast cell tumors (Lanes 1–5) were probed for WWOX (upper panel) and β-actin (lower panel).  Canine BR-empty vector (Lane 1) and BR-WWOX (Lane 2) cell lines were probed for WWOX (upper panel) and β-actin (lower panel).  Canine C2-scramble (Lane 1), C2-shWWOX-554 (Lane 2) and C2-shWWOX-1304 (Lane 3) cell lines were probed for WWOX (upper panel) and β-actin (lower panel). Red arrows indicate ~ 47 kDa band or 43 kDa band corresponding to WWOX or β-actin, respectively. L = Protein Ladder. Dashed lines indicated cropped areas presented in main manuscript text.


The Triple-Negative Breast Cancer subtype (TNBC) is particularly aggressive and heterogeneous. Thus, Poly-ADP-Ribose Polymerase inhibitors were developed to improve the prognosis of patients and treatment protocols are still being evaluated. In this context, we modelized the efficacy of Olaparib , combined with fractioned irradiation  on two aggressive TNBC cell lines MDA-MB-231 (BRCAness) and SUM1315 (BRCA1-mutated). In 2D cell culture and for both models, the clonogenicity drop was 95-fold higher after 5 µM Olaparib and 10 Gy irradiation than Olaparib treatment alone and was only 2-fold higher after 50 µM and 10 Gy. Similar responses were obtained on TNBC tumor-like spheroid models after 10 days of co-treatment. Indeed, the ratio of metabolic activity decrease was of 1.2 for SUM1315 and 3.3 for MDA-MB-231 after 5 µM and 10 Gy and of only 0.9 (both models) after 50 µM and 10 Gy. MDA-MB-231, exhibiting a strong proliferation profile and an overexpression of AURKA, was more sensitive to the co-treatment than SUM1315 cell line, with a stem-cell like phenotype. These results suggest that, with the studied models, the potentiation of Olaparib treatment could be reached with low-dose and long-term exposure combined with fractioned irradiation. Among taZeneca) ,7.® (Olaparib) was adopted by the Committee for Medicinal Products for Human Use (CHMP from European Medicines Agency) for the treatment of adult patients with germline BRCA1/2-mutations, who have HER2-negative locally advanced or metastatic breast cancer. In addition to being used as monotherapy treatment agents, PARPi may also be used in combination with chemotherapy or radiotherapy [PARPi constitute a class of targeted therapeutics acting on DNA repair systems using the synthetic lethality concept. These open up new prospects for improving the treatment of TNBC aggressive subtype . Since Fotherapy . These cotherapy ,10.The pipeline of drug development is composed of several preclinical and clinical phases. The preclinical setting is divided into in vitro and in vivo experiments which goals are the determination of drug efficacy and toxicity. Recently, the three-dimensional cell culture (3D) has been integrated into these preclinical processes after the monolayer in vitro cell culture experiments. The 3D cell culture (spheroid) appears to be an adapted tool for the evaluation of drug efficacy for drug development and optimization in Oncology. Indeed, these 3D models mimic more precisely the tumour microenvironment by the formation of genetical, biochemical and phenotypical gradients, resembling intratumoral gradients and microenvironment ,12,13. In recent years, in order to optimize the treatment of these aggressive breast cancers, our research group has focused its works on the modeling of the balance between cytotoxicity/resistance of TNBC cell lines, in response to PARPi ,15. In t®, MO2) was cultured in Ham’s F12 medium supplemented with 5% decomplemented fetal calf serum, 10 mM HEPES buffer, 20 μg/mL gentamycin, 10 ng/mL EGF and 4 μg/mL insulin. MDA-MB-231 cell line  was cultured in RPMI medium 1640 supplemented with 10% decomplemented fetal calf serum and 20 μg/mL of gentamycin. Both cell lines were maintained at 37 °C under 5% CO2 in a humidified incubator. The absence of mycoplasma contamination was checked before every experiment by the mycoplasmacheck test . SUM1315 cell line , six-well flat bottom microplates  or 96-well flat-bottom microplates  at a concentration of 5000 cells/cmFor spheroid formation and treatments, the liquid overlay technique (LOT) was performed in Ultra-Low-Attachment round-bottom microplates according to our previous works .®) was solubilized in Dimethylsulfoxide (DMSO) at a concentration of 100 mM. For all experiments, the solubilized Olaparib was diluted in the culture medium at increasing concentrations with a final and constant DMSO concentration of 0.1%. Parallely, DMSO 0.1% controls without Olaparib were also performed. For Olaparib treatments and co-treatment (Olaparib/irradiation), Olaparib was added once, three hours before the first radiotherapy session. The treatment lasted for 24 h, 72 h or 120 h in 2D experiments, and for 6, 8 or 10 days in 3D experiments. The absence of DMSO 0.1% toxicity was systematically checked prior to results analysis.Olaparib . The dose rate applied to the accelerator was 400 Monitor Units per minute (MU/min), which corresponds to the dose rate conventionally used in radiotherapy. For this, the cells were placed 3 cm deep inside a 13 cm thick PolyMethyl MethAcrylate (PMMA) phantom in order to obtain a homogeneous distribution of dose and respect the charged particle equilibrium conditions. A computed tomography (CT) scanner examination of this phantom was performed with each device containing the irradiated cells. The dose was prescribed in the middle of the volume containing the cells and the calculation was performed with the Anisotropic Analytical Algorithm (AAA). The irradiated plates remained in the incubator outside of radiotherapy treatment session. The non-irradiated culture plates with or without Olaparib were maintained in the same experimental conditions.The cells were irradiated at a daily dose of 2 Gy for one to five days, on a linear electron accelerator with a 6 MegaVolts X-ray beam used for routine treatment in clinic . The number of γH2AX foci was calculated on >10 cellular fields for each condition with ImageJ® software, representing at least more than hundred cells. The results were represented as the mean number of γH2AX foci per nucleus with their standard deviation (s.d.).Cells were treated with 5 and 50 µM Olaparib for 3 h and then were irradiated with a single X-ray dose of 2 Gy. After one hour, cells were fixed in 4% paraformaldehyde, permeabilized in ethanol, incubated with a saturation solution and then with the primary antibody  or the isotype control . Cells were then incubated with the anti-mouse secondary antibody  and with Hoechst 33258 for nuclear counterstaining. Finally, cells were dried, mounted and observed with CytationCells were treated with increasing concentrations of Olaparib (0.1 to 100 µM) for 24 h, 72 h or 120 h, corresponding to concomitant doses of radiotherapy of 2, 6 and 10 Gy, respectively. After each treatment endpoint, cells were fixed with a trichloroacetic acid (TCA) solution for 1 h, washed in water, dried and incubated with a SRB solution in 1% (v/v) acetic acid for 15 min. Again, cells were washed with 1% acetic acid, dried and a 10 mM Tris-base buffer solution was finally added to the wells for 30 min. The absorbance of SRB dye dissolved in each well was determined at 540 nm with Cytation3MV plate reader . Cell survival was calculated for each treatment condition by the comparison of 24 h, 72 h and 120 h aged untreated control wells with the following formula:n > 45 wells ± their standard deviation (s.d.). The results are presented as mean viability of The clonogenic potential of cells following a treatment with irradiation alone or combined with Olaparib  was evaluated after cumulative X-ray doses of 2, 6 or 10 Gy, corresponding to concomitant Olaparib exposures of 24 h, 72 h of 120 h. After each treatment endpoint, cells were recovered by trypsinisation, enumerated and re-seeded into new plates at an adapted concentration. The number of colonies formed was determined after nine doubling times (objective 10X). The Plating Efficiency (PE) corresponding to the cell repopulation factor after each treatment condition was calculated as follows: Then, the survival fraction after each X-ray dose was calculated compared to the corresponding untreated control of each dose (= No X-ray and No Olaparib treatment) with the following formula:n = 5 replicates.The values of the clonogenic survival were expressed as mean survival ± their standard deviation (s.d.) of Spheroids aged of 3 days were treated with 5 and 50 µM of Olaparib for 6, 8 and 10 days, corresponding to concomitant X-ray doses of 2 Gy (1 session), 6 Gy (three successive daily sessions) and 10 Gy (five successive daily sessions), respectively. TM3MV microplate reader  using the cellular analysis algorithm of the Gen 5 software . Results were expressed as the mean spheroid size of each treatment condition (n > 45) with their standard deviation (s.d.). The size of spheroids after each treatment endpoint  was monitored with the CytationMV plate reader . This allowed the determination of the percentage of spheroid metabolic activity as controls calculated as follows: Spheroids from every treatment condition were transferred in a new microplate containing 60 µM resazurin in PBS. The Fluorescence Intensity (FI) corresponding to the amount of resorufin formed after 17 h incubation was quantified in each well with Cytation3n > 16 spheroids) ± their standard deviation (s.d.). The results were presented as mean spheroid metabolic activity of each treatment condition  and 2 µM Calcein-AM  for 45 min. The fluorescence of each fluorophore was then imaged with Cytation3MV plate reader . For the image analysis, same exposure time, LED intensity and gain were programmed for all image acquisitions.All available MDA-MB-231 and SUM1315 transcriptomic data from different studies were collected from the NCBI public dataset GEO DataSet. Accession number, sample and sequencing information are indicated in the p value of <0.1 was the cutoff to select specifically DEG between the two cell lines. This DEGs list was subject as query to Ingenuity Pathway Analysis . IPA permits to infer functional and cellular processes that are modified between these two cell lines using the Ingenuity Knowledge Base. Two statistical indexes  are determined for each prediction. The p-value is calculated by a Fisher exact test and permits to identify significantly enriched pathways. The IPA z-score is the statistical measure of the match between altered genes and the inferred biological function. z-score let to predict the direction of change of a function. Indeed, a cellular function activity is increased when z-score is >2 and decreased when z-score <−2.Fastq files were analyzed on Genotoul Bio-informatic cluster. Input reads for each sample were aligned to the Human (GRCh38) reference genome using TopHat . Two-dimp < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ***** p < 0.00001. The level of interaction between all treatment conditions was determined with a multivariate ANOVA analysis (mixed model), performed with the SEM software . All the data set was statistically compared with a Student’s two-sided test, where significative values are indicated in the graphs as: * p = 10−7) and tripled after 50 µM . After 2 Gy irradiation alone, the number of foci increased up to 19 ± 1.1 (p = 10−26). In presence of the co-treatment, this parameter kept increasing significantly than irradiation alone or Olaparib treatment alone with 23 ± 1.3 and 32 ± 1.6 foci per nuclei  (p = 10−7), and a 1.8-fold increase after irradiation and Olaparib treatment  A. Parallctively) B, with ap = 10−5)B. These The cell survival of both MDA-MB-231 and SUM1315 cell lines was analyzed by an SRB survival test, either after treatment with 0.1–100 µM Olaparib concentrations for 24, 72, or 120 h, or combined with 2, 6 or 10 Gy cumulative irradiation doses. The 2 Gy radiation was prescribed daily. All data are presented in −6 for every parameter. Indeed, in our experimental conditions, the cell survival variance was impacted (i) at 40% by the time of Olaparib exposure, (ii) at 39% by the radiotherapy dose, (iii) at 14% by the Olaparib concentration, and at 9% by the type of cell line. These results suggest clearly that both the duration of Olaparib exposure and the dose of radiotherapy play a major role in the decrease of survival of these TNBC cell lines. Secondly, the multivariate ANOVA analysis allowed to determine the impact of each Olaparib dose with or without irradiation on cell survival  and the strongest parameters influencing this variation were the time of Olaparib exposure (40%) and the radiotherapy dose (38%).The impact of all experimental parameters  on cell survival variance was analyzed in a multivariate ANOVA analysis, performed with the SEM software . The probability of variance influence in the analysis and the percentage of variance explanation were described for each experimental parameter. All parameters explained the survival variance (probabilities inferior to 10−6). For this, the impact of radiotherapy dose and the time of Olaparib exposure were studied on all the data of cell survival whatever the type of cell line, after a treatment with Olaparib alone , radiotherapy alone and Olaparib  combined with irradiation A. The re-6. In contrast, with higher Olaparib concentrations of 50 and 100 µM, cell survival remained relatively similar at 120 h between a treatment with Olaparib alone or combined with 10 Gy radiotherapy, with 36 ± 2 vs. 30 ± 2% for 50 µM and 31 ± 2% vs. 27 ± 2 for 100 µM, with curve effect probabilities of 0.02 and 0.0001, respectively. Then, the impact of several Olaparib tested concentration was represented in These results showed that the co-treatment Olaparib and radiotherapy was more efficient than a treatment with Olaparib alone, or radiotherapy alone. Thus, this analysis showed that the beneficial of the co-treatment (Olaparib + RX) compared to Olaparib treatment alone was more important with low Olaparib concentrations. The clonogenic survival of cells treated with Olaparib alone  or concomitantly with radiotherapy  was determined for both MDA-MB-231 A and SUMp > 0.05 for all conditions)  and then increased again after 120 h treatment with 100 ± 3% and 1 ± 0.1%  (p = 0.0006) (p = 0.01) and according to Olaparib concentration with 1 ± 0.8% after 0.5 µM and 6 Gy and 0.2 ± 0.01% after 50 µM and 6 Gy  (p = 0.09). For MDA-MB-231 cell line, the 0.1% DMSO controls after 24 h, 72 h and 120 h incubation presented same clonogenic survivals as the controls with 89 ± 19%, 90 ± 6% and 92 ± 40%, respectively (ditions) Aa,b. Forctively) Aa. These 0.0006) Ab. Thesectively) Ab. In cop > 0.05 for all conditions) (p = 10−5)  (p = 0.75)  Ba. For t = 10−5) Ba. Similctively) Bb. More, = 0.75) Bb.For both cell lines, the co-treatment with low dose of Olaparib (0.5 to 5 µM) and fractioned irradiation (10 Gy) induced very low clonogenic potential inferior to 1% indicating a nearly eradicated repopulation potential of these cell lines. Thanks to preliminary 2D experiments, the long-term cell toxicity of Olaparib was highlighted as well as the interest of using low Olaparib doses (0.5–5 µM) compared to high doses (50–100 µM). Thus, for the tumor-like experiments, the spheroids were treated at long-term (6–10 days) with Olaparib alone (5 µM—low dose and 50 µM—high dose) and without/with fractioned irradiation . For this, (i) the spheroid growth inhibition, (ii) the spheroid viability/mortality profiles and (iii) the spheroid metabolic activity were analyzed . Firstly, for all treatment conditions, no difference in spheroid size and metabolic activity was detected between 0.1% DMSO-treated spheroid controls and control spheroids . Secondl1. Impact of treatment durationp = 10−5) (p = 0.04) (p = 10−30 and p = 10−12) (p = 10−18) and 102 ± 10% to 43 ± 6% (p = 10−10) after 6 and 10 days, respectively  A–C and m = 0.04) D–F was d= 10−12) A–F. In aectively D–I. Sameectively)D–I. p = 10−12) and from 84 ± 10% to 55 ± 2% (p = 0.0008), respectively (p = 10–10) and from 100 ± 12% to 46 ± 1% (p = 10−5), respectively  with or without irradiation, showed a similar significant decrease by the time . For insectively A–F. Simiectively D–I. In addition, for both cell lines, the time-dependant decrease in terms of spheroid growth and metabolic activity was supported by an increase in the number of dead cells (red/yellow markings) within the spheroids, whatever the treatment condition A–C,G–I. 2. Impact of Olaparib concentrationsp = 10−23) and from 59 ± 12% (5 µM Olaparib) to 20 ± 1% , respectively (p = 0.04) and metabolic activity from 55 ± 2% to 46 ± 1% (p = 10−7) after the same treatment condition, respectively  and even induced an increase in spheroid metabolic activity  to 39 ± 4% C,F,I. −6). Additionnaly, whatever the treatment condition, MDA-MB-231 spheroids seemed to be more sensitive to the treatment than SUM1315 spheroids , AURKA , LIG1 , RAD51  and EXO1  were significantly overexpressed in MDA-MB-231 against SUM1315. Conversely, DNAPK , XLF , MRN , PMS2  and PARP3  were overexpressed in SUM1315 cell line compared to MDA-MB-231 cell line  software to infer the functional phenotype of each cell line from their differential transcriptomic patterns C. First,The Triple-Negative breast cancers (TNBC) are very aggressive and have a poor prognostic. TNBC cancer cells exhibit several DNA repair pathway deficiencies, such as deleterious mutations on the BRCA1/2 genes or assimilated “BRCAness” phenotypes. Thus, for this tumour subtype, the use of PARP inhibitors (PARPi), exploiting the concept of synthetic lethality, seem to be very promising ,25,26,27In addition, the inhibition of PARPi has been described to increase the radio-sensitivity of tumour cells exhibiting a BRCAness phenotype ,29,30,31Firstly, the DNA double-strand breaks induction further to the co-treatment Olaparib 4 h and 2 Gy irradiation was determined on both models by the analysis of focigammaH2AX increase. Indeed, these proteins are the first recruited in the process of DSBs reparation by the HR system and are usually used to determine the cell sensitivity to DNA damaging agents or radiations ,32,33. FSecondly, the cellular response further to the co-treatment Olaparib/fractioned irradiation was studied on both cell models cultured in 2D by cell survival and cell clonogenic repopulation tests. These experiments highlighted the low-dose efficacy of Olaparib combined with high dose of irradiation, in terms of (i) cell survival decrease and (ii) cell clonogenic repopulation capacity, compared to high dose Olaparib alone or high dose of irradiation alone. Indeed, the use of low-dose Olaparib potentialized the irradiation in terms of clonogenic repopulation decrease with a ratio “Olaparib/Olaparib + 10 Gy” of 100- to 650-fold after 0.5 µM, of 0.8- to 188-fold after 5 µM, compared to only 0.4- to 4-fold after 50 µM Olaparib . In the same way, other in vitro studies also showed a strong cytotoxic effect of the treatment combining hypofractioned irradiation (0 to 6 Gy) and 10 µM of PARPi Olaparib, on cholangiocarcinoma, a very malignant and radioresistant tumor model . Our experiments in 2D cell culture conditions allowed us to choose the Olaparib concentrations (5 and 50 µM) for the treatment on more-predictive tumor-like models. More, the spheroid models allow long-term cultures up to 14 days . Thus, aIn the same way, the combination PARPi/radiotherapy has been studied for the treatment of various types of cancers in preclinical studies and has demonstrated improved efficacy compared to PARPi treatments alone or radiotherapy alone. Indeed, in vivo studies evaluated the impact of Olaparib combined with fractioned radiotherapy sessions (5 x 2 Gy) on bronchial cancer xenografts, leading to delayed tumor growths . An in vFurthermore, the cell ANOVA survival analysis in both 2D and 3D cell culture conditions highlighted the heterogeneous sensitivity between the two cell line models. MDA-MB-231 cell line was more sensitive to the co-treatment than SUM1315. Thereby, a in silico transcriptomic comparison was carried out. These analyses highlighted in MDA-MB-231 the overexpression of AURKA, a gene exhibiting cell cycle regulation function and known as a biomarker of PARPi sensitivity ,37,38,39All these preclinical modeling experiments using the more-predictive and high throughput tumor-like models suggest the perspective of a low dose and long-term Olaparib administration alongside fractioned irradiation for resistant metastatic breast cancers. Other studies on Triple-Negative Breast Cancer cases have to be performed to assess whether AURKA overexpression/amplification could predict the “PARPi and irradiation” sensitivity regardless of the proliferation/stem-cell like status and the BRCA-status. These hypothesis are also to be corroborated on other tumor-like spheroid models that are PARPi candidates.


Isobolographic analysis showed that for both melanoma cell lines, the combination of CDDP and osthole exerted synergistic and additive interactions, while the combination of CDDP and xanthotoxol exerted additive interactions. Combinations of CDDP with xanthotoxin, isopimpinellin, and imperatorin showed antagonistic and additive interactions in two melanoma cell lines. (4) The combination of CDDP and osthole was characterized by the most desirable synergistic interaction. Isobolographic analysis allows the selection of potential candidates for cancer drugs among natural substances.(1) Cisplatin (CDDP) is used in melanoma chemotherapy, but it has many side effects. Hence, the search for natural substances that can reduce the dose of CDDP, and CDDP-related toxicity, is highly desired. Coumarins have many biological properties, including anticancer and antiproliferative effects. (2) An in vitro 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assay on two human melanoma cell lines (FM55P and FM55M2) examined the antitumor properties of CDDP and five naturally occurring coumarins . The antiproliferative effects produced by combinations of CDDP with the coumarins were assessed using type I isobolographic analysis. (3) The most potent anticancer properties of coumarins were presented by osthole and xanthotoxol. These compounds were characterized by the lowest median inhibitory concentration (IC Cancer, with over 11 million deaths annually, is the leading cause of death in economically developed countries and the second leading cause of death in developing countries .Platinum-based chemotherapeutic drugs are widely used to treat cancer. Cisplatin (CDDP) is used to treat many types of cancers, including ovarian, testicular, bladder, lung, and head and neck cancer . CDDP isAt present, novel structural analogues of platinum, which can reduce the side effects of CDDP and resistance to chemotherapeutics, as well as improve the effectiveness of anticancer activity, are designed, synthesized, and tested ,13. So fAlthough surgical excision is the fundamental treatment for malignant melanoma of the skin, chemotherapy is used as an adjuvant treatment, especially if melanoma undergoes invasion in the form of metastases. In such a clinical situation, chemotherapy for melanoma remains an oncological challenge. Dacarbazine is considered the gold standard regimen for melanoma. However, in the treatment of melanomas, several active agents, e.g., CDDP, vinblastine, vemurafenib, and temozolomid, are also used . Due to So far, the antitumor activities of many combinations of CDDP with compounds of natural origin, including thymoquinone , baicaleH-1-benzopyran-2-ones), widespread in plant species belonging to various families, including Apiaceae, Rutaceae, Asteraceae, Fabaceae, Oleaceae, Moraceae, and Thymelaeaceae [Naturally occurring coumarins are the largest group of benzopyrone derivatives  values for CDDP, OST, XIN, XOL, ISO, and IMP are presented in CDDP and the tested coumarins  inhibited the proliferation of human melanoma cells (FM55P and FM55M2) in a concentration-dependent manner when applied separately . Of note50 values, resulted in a concentration-dependent reduction in cancer cell viability. The test for the parallelism of the concentration–response lines between CDDP and the tested coumarins confirmed that the log-probit lines of these compounds were either nonparallel or parallel to one another  revealed that in vitro interactions were synergistic, additive, or antagonistic, depending on the coumarins used. In both cell lines, the FM55P and FM55M2 interactions of CDDP with ISO and XIN were antagonistic, while those of CDDP with XOL and IMP were additive . In the 50 values of OST oscillate from about 75 μM for human ovarian cancer cells [50 values of XIN range from above 50 µM for human lung and colon cancer cell lines to 46.8 µM for prostate cancer, 44 µM for the melanoma cell line A375, and 37.8 µM for human squamous carcinoma [50 for this compound amounted to 11.92 mg/mL [50 for XOL is about 25 µM for lung cancer, 37.3 µM for prostate cancer, and above 50 µM for the human squamous carcinoma cell line A431 and melanoma cell line A375 [50 values of IMP range from 12.3 µg/mL for central nervous system cancer, 13.7 µg/mL for ovarian cancer, 14.5 µg/mL for melanoma SK-MEL-2 cell line, 16.4 µg/mL for lung cancer, to 19.4 µg/mL for colon cancer [All the tested coumarins have antiproliferative activity in various cancer cell lines. For example, OST induces apoptosis and inhibits proliferation in bile duct cancer , stomacher cells  to 46.2 er cells . The IC592 mg/mL . The IC5n cancer .50 values. It was observed that the most favorable coumarin was OST, offering 50% anticancer effects with the lowest concentrations, while the highest IC50 values were documented for IMP, XIN, and ISO in both melanoma cell lines  and some selected furanocoumarins  to ascertain which of the naturally occurring compounds can be used in the treatment of melanoma. Of note, all the tested coumarins produced antiproliferative effects in both melanoma cell lines, finally resulting in the calculated ICll lines . ConsideThe results of our research indicate that the combination of CDDP and OST is characterized by the most desirable synergistic interaction. The other tested coumarins (belonging to a furanocoumarin group) mostly showed antagonist interactions in combination with CDDP. The exception was XOL, which showed an additive interaction with CDDP and IMP, producing additivity with a slight tendency toward antagonism when combined with CDDP. The results presented here indicate clearly that some of the tested coumarins (OST and XOL) can be used in combination with CDDP because of synergistic and additive interactions. By contrast, ISO, IMP, and XIN should not be recommended as add-on drugs with CDDP. These furanocoumarins produced an antagonistic or additive interaction with a tendency toward antagonism in human melanoma cell lines (FM55P and FM55M2). It should be highlighted that the isobolographic analysis of interaction has been used several times to assess the interactions of various chemical substances with cancer cells ,55,56,57In our study, we used two melanoma cell lines to observe any differences in the antiproliferative activity of the tested coumarins. The first melanoma cell line (FM55P) was derived from the primary tumor of the skin, while the second line (FM55M2) was derived from the metastases of melanoma. We confirmed that CDDP when combined with OST exerts a synergistic interaction in terms of anticancer effects on FM55M2. The combination produced a synergistic interaction in the metastatic cell line FM55M2. Although the combination of CDDP and OST exerted an additive interaction with respect to anticancer effects on the primary melanoma cell line FM55P, this combination can be recommended as a treatment option, especially if we are not sure whether melanoma metastases appear. The synergy observed in the human melanoma metastatic cell line (FM55M2) is very beneficial as a treatment option. By contrast, antagonistic interactions in terms of anticancer effects are not recommended because drugs should be used in higher doses/concentrations to eliminate 50% of neoplasmic cells. In such a case, under clinical conditions, patients should receive higher drug doses, and thus, adverse effects may occur more frequently than expected. Both interactions, synergy and antagonism, may also be explained in light of their molecular mechanism(s) of the antiproliferative effects influencing the cell cycle. Generally, if two drugs synergistically inhibit proliferation, they probably affect various different phases/sites of the cell cycle, finally contributing to faster apoptosis of the affected cells. In oncology, this effect is highly desired by patients and doctors. By contrast, antagonistic interaction between two anticancer drugs is not favorable, because one of the drugs, in its anticancer activity, probably competes with and reduces the effects produced by the second drug. Another explanation is also possible while considering the fact that one drug can affect different phases of the cell cycle. In such a case, one of the drugs used in the mixture stops/blocks the cell cycle, making the second drug ineffective. If the first drug switches the cell cycle off, the second drug cannot exert its anticancer action, especially if its molecular mechanisms are associated with the transition of cancer cells to another phase of the cell cycle. Thus, one drug can block the activity of the second drug, making the mixture less active than particular drugs when used alone. XIN and ISO are the coumarins that produced antagonistic effects in both melanoma cell lines, primary and metastatic melanoma. XOL and IMP produced additive interaction in both cell lines, primary and metastatic melanoma.The results presented here (indicating that various coumarins produce various interactions in various cell lines) are a good example illustrating that the screening test can select the most active anticancer agents among the naturally occurring coumarins tested, which can be useful in the treatment of melanoma. This screening among the five naturally occurring coumarins allows us to find the most promising agent, OST. We are fully aware of the fact that translation of the results from this in vitro study to clinical conditions is not so fast as one would expect, but it provides us with the hope that confirmation of this synergistic interaction in other human melanoma cell lines brings us a new option for the treatment of metastatic melanoma in the future, and this study can contribute to bettering our knowledge on melanoma treatment. More advanced studies are required to confirm whether OST can be used as an add-on drug in the treatment of melanoma in in vivo preclinical studies.The antitumor activity of various naturally occurring compounds belonging to flavonoids, alkaloids, polyphenols, glycosides , and carotenoids has been demonstrated earlier ,19,20,21It should be stressed that in this study, we did not determine the cytotoxicity of the tested coumarins on normal  cells. However, some reports revealed that IMP, ISO, XIN, XOL, and OST do not exert cytotoxic effects on normal skin cells ,59. ThusAnother fact should be emphasized while explaining the rationale of combining coumarins with CDDP. It is thought that the resistance of cancer to chemotherapy is one of the major causes of treatment failure and is responsible for over 90% of deaths in cancer patients receiving traditional chemotherapeutics and/or novel target drugs . The oveCell culture. Primary malignant melanoma cells FM55M2 and FM55P were purchased from the European Collection of Authenticated Cell Cultures  and cultured in RPMI 1640 medium  containing 10% fetal bovine serum  and 1% penicillin/streptomycin  in a 37 °C incubator with 5% CO2. The cells grew to 80% confluence.Cell treatments. Cell viability was determined using the MTT assay. Cisplatin  was dissolved in phosphate buffered saline (PBS) with Ca2+ and Mg2+. The examined coumarins, osthole (OST), xanthotoxin (XIN), xanthotoxol (XOL), isopimpinellin (ISO), and imperatorin (IMP) , were dissolved in DMSO as stock solutions. The drugs were dissolved to the respective concentrations with culture medium before use. PBS and DMSO had no effect on cell proliferation. Briefly, the FM55M2 and FM55P cells were seeded on a 96-well plate at a density of 104 cells/well and treated with different concentrations of CDDP and five coumarins  for 72 h. MTT assay. Inhibition of cancer cell proliferation was evaluated by an MTT assay. After treatment with the examined drug and naturally occurring coumarins, the cells were incubated at 37 °C for 3 h with 10 μL of MTT solution . Then, 100 μL of stop solution  was added to dissolve the crystals in each well. Following a 12 h incubation, the optical densities were determined at 570 nm with a microplate spectrophotometer . Each treatment was performed in triplicate, and each experiment was repeated 3 times.Isobolographic analysis. Isobolographic analysis is a statistical method allowing the characterization of pharmacodynamic interaction between drugs and chemical substances. This method was performed as described previously [50 values for the drugs administered alone, median additive inhibitory concentrations for the mixture of CDDP with one of the investigated coumarins  at the fixed ratio of 1:1 (IC50 add) were calculated, as described earlier [50 exp values for the mixture of CDDP with each of the studied coumarins (at the fixed ratio of 1:1) were determined based on the concentrations of the mixtures of CDDP with one of tested coumarins, inhibiting 50% of cell viability in the melanoma cell lines (FM55P and FM55M2) measured in vitro by the MTT assay. Log-probit analysis was used to determine the experimentally derived IC50 and IC50 exp values for CDDP and the tested coumarins  when the drugs were administered alone or in combination for the fixed ratio of 1:1 [50 exp values for the mixture of CDDP with the tested coumarins and the theoretically additive IC50 add values was statistically verified by using the unpaired Student’s t-test, as recommended elsewhere [eviously ,67,68. Feviously . The teseviously ,67,70. F earlier . The expo of 1:1 . The difIn conclusion, the synergistic interaction of CDDP with OST observed isobolographically in the human metastatic melanoma cell line (FM55M2) is worthy of recommendation for further intensive investigations to reveal the molecular mechanism(s) of action involved in this interaction. On the other hand, the antagonistic interaction or additive interaction with a tendency toward antagonism between CDDP and ISO, IMP, and XIN should be explained in further in vitro experiments.