Gastric adenocarcinoma with peritoneal carcinomatosis (PC) is therapy resistant and leads to poor survival. To study PC in depth, there is an urgent need to develop representative PC-derived cell lines and metastatic models to study molecular mechanisms of PC and for preclinical screening of new therapies.PC cell lines were developed from patient-derived PC cells. The tumorigenicity and metastatic potential were investigated by subcutaneously (PDXs) and orthotopically. Karyotyping, whole-exome sequencing, RNA-sequencing, and functional studies were performed to molecularly define the cell lines and compare genomic and phenotypic features of PDX and donor PC cells..We established three PC cell lines  and characterized them in vitro. The doubling times were 22, 39, and 37 h for GA0518, GA0804, and GA0825, respectively. Expression of cancer stem cell markers  and activation of oncogenes varied among the cell lines. All three PC cell lines formed PDXs. Interestingly, all three PC cell lines formed tumors in the patient derived orthotopic (PDO) model and GA0518 cell line consistently produced PC in mice. Moreover, PDXs recapitulated transcriptomic and phenotypic features of the donor PC cells. Finally, these cell lines were suitable for preclinical testing of chemotherapy and target agents in vitro and in vivoWe successfully established three patient-derived PC cell lines and an improved PDO model with high incidence of PC associated with malignant ascites. Thus, these cell lines and metastatic PDO model represent excellent resources for exploring metastatic mechanisms of PC in depth and for target drug screening and validation by interrogating GAC for translational studies.The online version contains supplementary material available at 10.1186/s13046-021-02003-8. Gastric adenocarcinoma (GAC) is the fifth most common cancer and the third leading cause of cancer-related mortality worldwide . GAC is The resources to study PC are starkly limited. Patient-derived tumor cell lines, patient-derived xenografts(PDXs), and orthotopic models will be indispensable in this respect. There are 38 gastric cancer cell lines in the cancer cell line encyclopedia (CCLE); the majority of these are from Asian patients . HoweverSeveral human GAC cell lines have been used to create orthotopic mouse models of PC –20. HoweHere, we report on three new GAC cell lines established from PC cells from the Western patients. All three patient-derived PC cell lines are highly tumorigenic in the subcutaneous PDX model. Furthermore, whole exome sequencing (WES) and RNA sequencing (RNA-seq) revealed that the PDXs recapitulate the transcriptomic profile and conserve some of the mutational and copy-number variations (CNVs) similar to donor PC cells. Moreover, these cell lines are suitable for studying cytotoxics and target inhibitors. Most importantly, one cell line (GA0518) reliably formed PC in mice within 30 days in the PDO model. Thus, these new cell lines and the PDX and PDO models provide an opportunity to investigate mechanisms of PC and validate therapeutic targets.Three PC cells were collected from volunteering patients with GAC and malignant ascites; and patient clinical characteristics including therapy, tumor types, site of metastases, and histopathology are listed in Supplemental Table 5 cells in 30 μl in 50% Matrigel–phosphate-buffered saline) were injected into the distal stomach wall. We applied an improved orthotopic model to avoid the leakage into the peritoneal cavity . More details are provided in the results. The injection site and PC were monitored by in vivo BLI imaging weekly with intraperitoneal injection of 150 μg/g of D-Luciferin (PerkinElmer).To assess orthotopic tumor growth and PC in mice, we first transduced the cell lines with a lentivirus expressing Luciferase and mCherry for in vivo bioluminescence (BLI) imaging of tumor growth in SCID mice. Luciferase-expressing cells .Data were analyzed using the Student t-test and Fisher exact test (for colony formation and cell migration assays). Other assay results are presented as mean ± standard error of the mean and represent the results of at least three experiments. The significance of differences between groups was judged using a two-tailed Student t-test. Results were considered statistically significant if the More detailed materials and methods can be found in the online The three PC cell lines had different morphologies under the microscope Fig. A. To visDoubling time was estimated during the exponential phase of growth. GA0518 cells had the shortest doubling time, 22 h. GA0804 and GA0825 cells had similar doubling times, around 39 h and 37 h, respectively  with more mutations  staining of several notable CSC makers  in three cell lines Fig. B and itsAs the three cell lines expressed CSC markers in varying proportions, to define the similarity between the cell lines and their corresponding donor PC cells, we determined the activation oncogenic pathways in three cell lines and the corresponding donor PC cell samples by CyTOF. As shown in Fig. We then performed functional in vitro assays in the three cell lines. The colony formation capability was highest in GA0518 (256.67 colonies/1000 cells), followed by GA0804 (105.33 colonies/1000 cells), and then GA0825 (56.33 colonies/1000 cells)  of SCID mice injected with 1 × 104 cells  was injected into the anterior wall, each injection lasting 30 s. After the injection, an alcohol swab was used to sterilize the posterior wall. The stomach was repositioned into the abdominal cavity, and the wound was closed  of mice with GA0518 cells developed bloody ascites within 4 weeks  of 13.39 nM. GA0518 cells were sensitive to RAD001 with IC50 of 28.91 nM but were less sensitive to 5-fluorouracil, with IC50 values of 45.03 nM, and were resistant to erlotinib, at an IC50 of 261.9 nM , and JQ1 . As shown in Fig. We are reporting three new GAC cell lines derived from three patients with PC. All three cell lines were highly tumorigenic and aggressive in PDX and PDO models compared with commercially available GAC cell lines (Snu-1 and MKN45), and GA0518 cells were the most consistent and aggressive. WES and RNA-seq analyses revealed that the transcriptome and phenotypic features of PDXs closely recapitulated those of the donor cells from patients. Importantly, in the improved PDO model, GA0518 cells formed PC including malignant ascites in mice within 4 weeks. These findings suggest that the cell lines and PDO PC model can be reliably used for studying PC metastatic mechanisms and for target validation to advance therapeutics for GAC.KMT2C, CDKN2A, ABL1, and SC3H12A) than GA0825 donor PC cells (with only KMT2C mutation). It has been reported that most gastric cancers in advanced stage have abnormal chromosomes [Chromosomal instability, including abnormal numbers and structural rearrangements, is a prominent feature of cancers. Through cytogenetic analysis, we found that GA0518 has a near-diploid karyotype and cytogenetic complexity. This cell line is highly heterogeneous and comprises at least four cell subtypes, including 40% of cells with a karyotype of 45,X, 20% with 46,XX,− 12,+der , and 10%omosomes , 24 and Similarly, the three cell lines established from PC patients with GAC also had high heterogeneity, with multiple subclones, and one of them had high hyper-diploidy with 91–106 chromosomes . AnotherCD44 is a well-recognized CSC marker in GAC –31 and iWe also improved the surgical technique for the PDO model to avoid PC due to cell leakage in the peritoneal cavity. It has been well documented that the PDX and PDO models from the same source may respond differently to the same treatments . FurtherIn summary, three new GAC cell lines were established from GAC patients with PC from the United States. All three cell lines formed PDXs with high efficiency, and the molecular features of the PDXs mimicked the donor PC cells from patients, making these cell lines highly suitable for validation of targets to be advanced in human trials. Furthermore, GA0518 cells stand out in their performance as a PC metastatic model in our improved PDO model and may provide an advantage for studying metastatic mechanisms, and for target drug screening and validation by interrogating GAC for translational studies.Additional file 1. Supplemental Materias & Methods.Additional file 2: Supplemental Fig. 1. Genetic alteration of three donor PC cells and cell lines. A. Whole-exome sequencing was performed in three donor PC cells IP-013 (GA0518), IP-107-02 (GA0804) and IP-116 (GA0825) directly from patients according to the Materials and Methods. Mutations in three donor PC cells were analyzed by a bioinformatician (S.Z.). B&C. Karyotyping analyses of GA0518 (B) and Snu-1 (C) were performed by the Cytogenetics and Cell Authentication Core at MD Anderson as described in Materials and Methods. Abnormal chromosomal changes were observed in GA0518 (B) and Snu-1 (C). Red arrows indicate some of the chromosomal changes in each cell lines. Supplemental Fig. 2. Expression of CSC markers in the corresponding donor PC cell samples. Expression of several notable CSC markers  in the corresponding donor PC cell samples  was determined by immunofluorescence staining as described in Materials and Methods. Supplemental Fig. 3. Expression of CD44, CD133, and EpCAM was evaluated by flow cytometry in the three new cell lines. Supplemental Fig. 4. Activation of stemness and oncogenic markers in three corresponding donor PC cells by CyTOF. CyTOF was performed in the donor PC cell samples IP-013, IP-107-2, and IP-116 according to the Materials and Methods. Activation of CSC markers (CD44 and ALDH1) and oncogenic pathways such as pS6, p-AKT, and mTOR was analyzed and identified by bioinformatician (R.W). Supplemental Fig. 5. Representative BLI images for GA0804 PDO model A. Representative BLI images from Day 1 to Day 36 in a GA0804 PDO mouse without PC metastasis; B. Representative BLI images in GA0804 PDO mouse with PC metastasis from Day 1 to Day 36. Supplemental Fig. 6. Representative BLI images for GA0825 and MKN45 PDO models. A. Representative BLI images from Day 1 to Day 27 in two PDO mouse models of GA0825 with no PC. B. Representative BLI images from Day 9 to Day 26 in two PDO mouse models of commercial GAC cell line MKN45 with no PC. Supplemental Fig. 7. IC50 of three new cell lines on chemo and target inhibitors. The cell line GA0518 (A), GA0804 (B), and GA0825 (C) were seeded onto 96-well plates with 3000 cells/well. After overnight incubation, cells were treated with the indicated doses of two chemo-5-FU and Docetaxel; and two target inhibitors- Erlotinib and RAD001 for 6 days and then CellTiter96 Aqueous one solution was added to each well (20ul to 100ul culture media) followed by 2 h incubation at 37 °C and absorbance reading at OD 490. GraphPad Prism7 was used for IC50 analysis by following the instruction from GraphPad.com (https://www.graphpad.com/support/faq/how-to-determine-an-icsub50sub/).Additional file 3: Supplemental Table 1. Patients’ characteristics.


Urothelial carcinoma (UC) accounts for > 90% of canine tumors occurring in the urinary bladder. Toceranib phosphate (TOC) is a multi-target receptor tyrosine kinase (RTK) inhibitor that exhibits activity against members of the split kinase family of RTKs. The purpose of this study was to evaluate primary UC tumors and UC cell lines for the expression and activation of VEGFR2, PDGFRα, PDGFRβ, and KIT to assess whether dysregulation of these RTKs may contribute to the observed biological activity of TOC.Transcript for VEGFR2, PDGFRα, PDGFRβ, and KIT was detected in all UC tissue samples and UC cell lines. The Proteome Profiler™ Human Phospho-RTK Array Kit (R & D Systems) provided a platform to assess phosphorylation of 42 different RTKs in primary UC tumors and UC cell lines. Evidence of PDGFRα and PDGFRβ phosphorylation was present in only 11% or 33% of UC tumors, respectively, and 25% of UC cell lines. Treatment of UC cell lines with TOC had no significant impact on cell proliferation, including UC cell lines with evidence of PDGFRβ phosphorylation.Phosphorylation of several key RTKs targeted by TOC is present in a small subset of primary UC tumors and UC cell lines, suggesting that these RTKs do not exist in a state of continuous activation. These data suggest that activation of RTKs targeted by TOC is present in a small subset of UC tumors and UC cell lines and that treatment with TOC at physiologically relevant concentrations has no direct anti-proliferative effect on UC cells.The online version contains supplementary material available at 10.1186/s12917-021-03027-0. Canine urothelial carcinoma (UC) represents 1.5% to 2% of all canine neoplasia and accounts for ≥ 90% of urinary bladder tumors in dogs. The majority  is reporGiven the proclivity of UC tumors to develop in the trigone region of the urinary bladder, adequate surgical excision remains challenging and the majority of UC tumors are considered inoperable . AlternaIt is well-established that alterations in processes involved in tumor growth, progression and metastasis are mediated by ligand-receptor interactions involving receptor tyrosine kinases (RTKS) and downstream signaling molecules. Given the role of RTKs in the control of numerous cellular processes  and the importance of aberrant RTK signaling in cancer, RTKs have emerged as important targets for the development of anticancer therapies. Toceranib phosphate (TOC)  , Maltese (N = 1), Shetland Sheepdog (N = 1), West Highland Terrier (N = 1), with the remainder being mixed breed dogs (N = 4). The tumor locations most frequently involved the bladder trigone (N = 4), the apex (N = 4), and the dorsal bladder wall (N = 1) with evidence of prostatic (N = 1) and urethral (N = 4) involvement on ultrasonographic imaging. None of the patients had evidence of locoregional metastasis at the time of diagnosis and none had evidence of distant metastasis. All of the patients were presented with clinical signs which included stranguria, dysuria, hematuria, and/or pollakiuria. All of the patients received therapy with a non-steroidal anti-inflammatory  with 6/9 patients receiving some form of cytotoxic chemotherapy. Additionally, 3/9 patients received therapy with toceranib phosphate  tumor samples were collected from 9 canine patients that were presented to the Ohio State Veterinary Medical Center (OSU-VMC). The mean age was 10.3 years . The patient breeds included the Scottish Terrier  and established canine UC cell lines (N = 5). Transcript for PDGFRa, PDGFRb, VEGFR2, and KIT was detectable in all of the primary UC tumor samples and UC cell lines  accounts for over 90% of canine malignancies occurring in the urinary bladder and despite aggressive therapies including partial cystectomy, radiotherapy, and systemic chemotherapy, long term outcomes for dogs remains poor. While clinical signs often improve following systemic medical therapy, objective response rates are noted in 18–36% , 25 of tWhile transcript for PDGFRα, PDGFRβ, VEGFR2 and KIT was detected in all tumor samples and UC cell lines, the pattern of expression was variable. Furthermore, while mRNA was present in UC tumor tissues and UC cell lines, the presence of RTK phosphorylation for these RTKs was present in only 11–33% of tumor specimens and UC cell lines suggesting that activation of these RTKs is present in only a small subset of UC cells. Importantly, the absence of RTK pathway activation for PDGFRα, PDGFRβ, VEGFR2 and KIT in the majority of UC tumors and UC cell lines suggests that signaling through these pathways is unlikely to drive the aggressive behavior of UC cells. Prior studies  have demOf note, evidence of RTK phosphorylation was variable in primary UC tumor samples evaluated in this study. This variability may be attributed to tumor tissue heterogeneity as the samples were not microdissected and thus included tumor cells, associated stromal cells within the tumor microenvironment and to a lesser degree, necrosis. The presence of cell types within the tumor microenvironment may confer presence of growth factors and other cytokines that influence the phosphorylation status of RTKs which may otherwise not be activated in UC cells. Additionally, primary tumor tissues were collected from patients that received various chemotherapy treatments prior to sample procurement. It is therefore possible that prior treatment with intravenous chemotherapeutic agents, toceranib phosphate, and/or NSAIDs may have affected the expression and phosphorylation profile of the RTKs of interest in this study population. To further dissect the potential influence of RTK pathway activation on UC cell behavior, we profiled RTK activation in established UC cell lines which represent a model system without the confounding influence of tumor stromal and/or immune cells. Importantly, the cells were serum starved for 2 h in order to eliminate the potential for endogenous growth factor influence on the phosphorylation status of the RTKs. We observed that in the primary tumor samples evaluated, the profile of RTK activation in UC cells lines was similar to that found in tumors with evidence of PDGFRα and PDGFRβ phosphorylation present in only 25% of UC cell lines evaluated. While our results from the phospho-RTK arrays showed moderate phosphorylation of the RTKs of interest, it is important to note the possibility of false negative results. The phospho-RTK array used in the present study was originally designed to detect a wide variety of human phosphorylated RTKs and utilizes optimized human antibodies. The array has not been fully validated in canine tissues and therefore, may underestimate the true phosphorylation status of the primary tumors due to a lack of antibody cross-reactivity.Importantly, we evaluated the direct effect of TOC on cell proliferation in UC cell lines showing variable levels of PDGFR activation and found that treatment with TOC did not result in a dose-dependent decrease in cell viability. Small molecule inhibitors such as TOC frequently demonstrate biological activity in cancer cell lines at low nanomolar concentrations and this correlates with physiologically relevant pharmacokinetic profiles for dogs receiving TOC . We failV595E mutation which is present in 75–87% of canine UC tumors [Clinical benefit has been described in dogs with UC receiving TOC and a non-steroidal anti-inflammatory drug (piroxicam) with the majority of dogs (80%) in this study experiencing stable disease . In lighC tumors  has beenC tumors , 30 has , even at micromolar concentrations. Collectively, these data demonstrate that activation of RTKs targeted by TOC is present in a small subset of UC tumors and UC cell lines and that treatment with TOC at physiologically relevant concentrations has no direct anti-proliferative effect on UC cells.The present study provides an initial characterization of the activation and phosphorylation of RTKs in primary canine UC tumors and UC cell lines. We observed that phosphorylation of several key RTKs targeted by TOC is present in a small subset of primary UC tumor samples and UC cell lines, suggesting that many of these RTKs do not exist in a state of continuous activation in UC tumors. Treatment of UC cell lines with TOC had no significant impact on cell proliferation, including those with evidence of PDGFRβ phosphorylation. Furthermore, UC cell proliferation was not significantly altered following treatment of UC cell lines with TOC in vitroKIT ITD mutation in the JM domain, generously provided by Dr. Warren Gold, Cardiovascular Research Institute, University of California – San Francisco, CA, USA) served as a positive control cell line for toceranib phosphate drug treatment assays and was maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco™ Life Technologies) supplemented with 10% fetal bovine serum, non-essential amino acids, sodium pyruvate, antibiotic–antimycotic, GlutaMAX™, and HEPES. All cell lines were cultured in a humidified incubator containing 5% CO2 at 37 °C. The cells were routinely tested for mycoplasma and treated with Plasmocin  as necessary.The canine urothelial carcinoma (UC) cell lines K9TCC-PU-AXA, K9TCC-PU-AXC, K9TCC-PU-NK, K9TCC-PU-Original, and K9TCC-PU-SH were generously provided by Dr. Deborah Knapp . All cell lines were previously established and validated , 32. CelN = 9) were collected from clinical cases treated at The Ohio State University Veterinary Medical Center (OSU-VMC). Owner consent for tissue collection was obtained in all cases in accordance with established hospital protocols and approved Institutional Animal Care and Use Committee protocols (IACUC #2010A0015). Tissue collections were performed by the OSU-VMC Blue Buffalo Clinical Trials Office and Veterinary Clinical Research Shared Resource. Fresh tumors collected at the time of surgery or euthanasia were flash frozen in liquid nitrogen and placed in formalin and processed for routine paraffin embedding for histopathology. Briefly, sections were routinely sectioned at 4–5 µm thickness and stained with hematoxylin and eosin (H&E) on a Leica ST5020 autostainer  using a routine and quality-controlled protocol. All tumor samples were confirmed to be urothelial carcinoma by board certified veterinary anatomic pathologists at OSU-VMC.Primary canine UC tumor samples  and amplification efficiency. All primer sets demonstrated high amplification efficiency (96–98%) with a correlation coefficient (R2) ≥ 0.93  was used to assess relative phosphorylation of 42 different RTKs in primary UC tumor tissue samples and UC cell lines. Briefly, flash frozen tumor samples were pulverized in a frozen mortar and the resulting powder was resuspended in liquid nitrogen and transferred to a 1.5 mL microcentrifuge tube. Upon evaporation of the liquid nitrogen, samples were resuspended in 100–200 μL of complete tissue lysis buffer. Complete tissue lysis buffer was prepared per manufacturer’s instructions using 2.5 mL of Lysis Buffer-17 supplemented with phosphatase and protease inhibitors . Samples were rocked at 4 °C for 1 h, centrifuged for 15 min at 15,000 rpm at 4 °C, and the supernatants collected. Prior to collection, primary UC cell lines were serum starved for 2 h, washed twice with 1X Dulbecco’s Phosphate Buffered Saline (Gibco™ Life Technologies), pelleted, and protein lysates were prepared as described above. Extracted protein concentration was quantified using the Bradford assay BioRad Reagent  and 100 µg total protein lysate was added to each RTK array and developed according to manufacturer’s directions.Canine UC cell lines  were serum starved for 2 h and protein lysates were generated for western blotting, as described previously . 100 µg 3 cells were seeded in 96-well plates and incubated in media overnight. Cells were treated with 0.1% DMSO (control) or increasing concentrations of toceranib phosphate (0.005 µM – 1 µM) for 48 h. The canine C2 mastocytoma cell line which exhibits a predictable dose–response curve following treatment with toceranib phosphate was included in all assays as a positive control for cell proliferation assays. Briefly, 1 × 104 C2 cells were seeded in 96-well plates, incubated overnight in complete medium overnight and treated with 0.1% DMSO (control) or increasing concentrations of toceranib phosphate (0.005 µM – 1 µM) for 48 h at 37 °C. Media was removed at 48 h and plates were frozen overnight at -80 °C. Fluorescence was measured with the UV Spectromax M2 plate reader  with excitation at 485 nm and emission detection at 530 nm. Cell proliferation was calculated as a percentage of the control DMSO wells. All treatments were evaluated in triplicate in three independent experiments.The effect of toceranib phosphate on canine UC cell line viability was assessed with the CyQUANT® Cell Proliferation Assay Kit , as has been described previously . 5 × 103p < 0.05 were considered statistically significant.All experiments were performed in triplicate and repeated three independent times for UC cell lines. Data were presented as mean plus or minus standard deviation. All qRT-PCR data was normalized to endogenous control (GAPDH) and the ∆∆ Ct method  was usedAdditional file 1: Semi- quantitative Assessment of Percentage of Tumor Necrosis. Supplemental Table 1. summarizes the findings and degree of necrosis observed in the primary UC tumor samples. A single tumor sample was comprised of markedly more necrosis than the remaining samples, with >50% of examined tissue consisting of necrotic debris.Additional file 2: Figure S1. Uncropped RTK arrays and western blots. (Fig. ts. Fig. A Phosphots. Fig. A Phosphots. Fig. B Canine


ALK, MYC, and MET, were prioritized as having a likely role in canine OSA. Mutations in eight genes, previously described as human OSA drivers and including TP53, PTCH1, MED12, and PI3KCA, were retrieved in our cell lines. When variants were cross-referenced with human OSA driver mutations, the E273K mutation of TP53 was identified in the Wall cell line and tumor sample. The transcriptome profiling detected two possible p53 inactivation mechanisms in the Wall cell line on the one hand, and in D17 and D22 on the other. Moreover, MET overexpression, potentially leading to MAPK/ERK pathway activation, was observed in D17 and D22 cell lines. In conclusion, our data provide the molecular characterization of a large number of canine OSA cell lines, allowing future investigations on potential therapeutic targets and associated biomarkers. Notably, the Wall cell line represents a valuable model to empower prospective in vitro studies both in human and in dogs, since the TP53 driver mutation was maintained during cell line establishment and was widely reported as a mutation hotspot in several human cancers.Osteosarcoma (OSA) represents the most common primary bone tumor in dogs and is characterized by a highly aggressive behavior. Cell lines represent one of the most suitable and reproducible pre-clinical models, and therefore the knowledge of their molecular landscape is mandatory to investigate oncogenic mechanisms and drug response. The present study aims at determining variants, putative driver genes, and gene expression aberrations by integrating whole-exome and RNA sequencing. For this purpose, eight canine OSA cell lines and one matched pair of primary tumor and normal tissue were analyzed. Overall, cell lines revealed a mean tumor mutational burden of 9.6 mutations/Mb (range 3.9–16.8). Several known oncogenes and tumor suppressor genes, such as  TP53, RB1, DLG2, PTEN, MYC, and MET, were equally mutated in both cOSA and its human counterpart (TP53 pathway were detected in non-sensitive cell lines (Canine osteosarcoma (cOSA) represents the most common primary malignant bone tumor in dogs , 2 and interpart , 8. Morenterpart , 9 and pnterpart –12. Two nterpart , 14. Notll lines .in vitro models will allow the identification of new targets and offer valuable tools in translational medicine  and D22 (ATCC® CRL-6250™) were obtained from American Type Culture Collection.2. The FFPE samples were obtained from the same OSA from which the Wall cell line was established.These were cultured in Dulbecco's modified Eagle's medium  and Iscove's standard medium, supplemented with 10% fetal bovine serum (FBS), 1% glutamine, 100 μg/mL penicillin, and 100 μg/mL streptomycin. Cells were cultured at 37°C in a humidified atmosphere of 5% COGenomic DNA (gDNA) was isolated and purified from cell lines and the FFPE samples  using DNThe isolated DNA and RNA were stored at −20 and −80°C, respectively, until further use.High-quality whole-genome libraries from 10 samples (eight cells lines and two FFPE samples) were prepared using the KAPA HyperPlus Library Preparation Kit . Exome capture was executed using Roche's SeqCap EZ Share Prime Developer Kit  for non-human genomes following the SeqCap EZ HyperCap Workflow User's Guide. Probes for the exome capture were designed on the target enrichment design of 150 megabases developed by Broeckx et al. . The devA single-end sequencing (75 SE) was carried out on a NextSeq 500 platform .https://www.bioinformatics.babraham.ac.uk/projects/download.html) software. Trimmomatic was used to select high-quality reads and remove adapter sequences.Quality control prior to alignment was performed on output from Illumina software and was processed by FastQC v.0.11.9 (https://gatk.broadinstitute.org/hc/en-us/articles/360035894731-Somatic-short-variant-discovery-SNVs-Indels). Briefly, single-nucleotide variants (SNVs) and small insertion and deletions (indels) were identified with the GATK Mutect2 tool (http://dogsd.big.ac.cn/)  using BWA software . To verict2 tool  and filtct2 tool  13, 24), 24https.ac.cn/) . Distrib.ac.cn/) .The detailed WES workflow applied to both canine OSA cell lines and the FPPE samples is summarized in All RNA-seq analyses were performed using conventional RNA-seq analysis tools . DetaileAnnotated variants were subjected to three filtering levels with increasing stringency and designated as follows .Level 1: variants included the totality of exonic-only SNV and indels retrieved from the variant call described above. These were further filtered for number of reads (min. 2), alternate allele frequency (min. 0.05), and each variant's depth of coverage (min. 10). The resulting variants were analyzed to describe the mutational profile of cOSA cell lines.https://cancer.sanger.ac.uk/census) (https://www.intogen.org/search?cancer=OS) . Lev. Lev7). /census) . 5′ UTR ncer=OS)  to identTP53 identified in the Wall cell line and FFPE tumor sample was validated by Sanger dideoxy sequencing on Wall samples gDNA and Penny cell line (negative control). Briefly, two primers (sense 3′-ATGAGGGTGGCTAGGAGTCA-5′) and (antisense 5′-CAGTGCTGGGAAAGAGAGGA-3′) spanning the mutated region were designed by PRIMER3 Express software, and PCR on gDNA was performed using HotStar Taq (Qiagen) at 58°C  for 35 cycles. After evaluation of agarose gel, amplification products were purified by QIAquick PCR Purification Kit (Qiagen).E273K mutation of DNA extracted from eight canine OSA cell lines underwent WES, and an average of 158 million reads per sample (range 143–164) was obtained.The mean depth of reads mapping to the canine reference genome (CanFam3.1) was 187.7 (range 128–219), with over 99% of the targeted exome uniquely aligned. The optimal coverage was achieved for six out of eight cell lines. For each cell line, all reads passed the quality control  ≥ 30. The FFPE samples  achieved a mean depth of 54.71.The median tumor mutational burden of all Level 1 somatic variants was 9.6 mutations/Mb (range 3.9–16.9); in the Wall FFPE sample, the mutational burden reached 17.7 mutations/Mb . On averIn all our cell lines, missense mutations were the most frequently represented somatic coding mutation type, accounting for an average of 38.7% across all exonic variants. Frameshift insertion and deletions were 15.8 and 12.5% of the variants, respectively . In WallThe most common base change identified in all samples was C > T transition . The anaUsing the FidoSNP pathogenicity prediction tool, 50.5%  of all missense point mutations were categorized as deleterious.PDGFRB, PTCH1, and WRN were retrieved in at least three cell lines, whereas oncogenes and tumor suppressor genes, such as TP53, ALK, MYC, and MET, were retrieved in only one cell line each  to 10 genes (Sky) . Overallhttps://portal.gdc.cancer.gov/), four overlapping genes were identified, namely, PIK3CA, KRAS, APC, and NF1, which ranked 2nd, 6th, 10th, and 13th, respectively. At last, four genes were also identified in the Wall FFPE sample but did not overlap those of the corresponding cell line  in known cancer-causing genes in humans.Level 3 included 438 variants coding for 235 genes, regardless of their incidence across the cell line panel . A totalOverall, 190 genes were indicated as having a relevant and documented activity that promotes oncogenic transformation. In particular, 28 were designated as fusion genes, 74 as tumor suppressors, and 63 as oncogenes, and 25 functioned as both. The distribution of putative driver mutations across all cell lines is depicted in About 88% of the SNVs encoding for these genes were categorized as pathogenic.TP53, PTCH1, MED12, and PI3KCA, were identified.When compared to the top 20 cancer driver genes involved in human OSA (COSMIC Cancer Browser), eight genes, encoded by 21 variants, were retrieved in Level 3 genes . Among tTP53E273K mutation (c.818C>T) corresponding to the human TP53E285K mutation hotspot.All putative driver variants were cross-referenced with human OSA driver mutations available on IntOgen. Only the Wall cell line and FFPE tumor sample harbored a putative driver mutation, namely, the This putative driver mutation was further validated in homozygosis in the Wall cell line and tumor sample by Sanger sequencing .RNA sequencing generated over 10 million reads per sample. Quality control and trimming procedures retained the vast majority of the sequences, and unique alignment to the canine reference genome was successful for 86% of the cleaned reads .The normalized gene expression of the osteosarcoma-associated genes described above was then profiled within the same gene across all samples.TP53 gene expression was increased in the Wall cell line, which harbored a hotspot mutation, as well as in D17 and D22, which retained a wild-type gene status. Interestingly, D17 and D22 showed a 9-fold and 5.5-fold increase of MDM2 and MDM4 transcript levels, respectively, compared to the other cell lines. Conversely, the MDM2 transcript level was decreased by 2 to 20 times in the Wall cell line compared to the rest of the cell lines.In particular, MET by 19-fold was observed in the D17 cell line, which harbored a frameshift insertion on this gene, and to the same extent in D22 which retained a wild-type gene status. Notably, genes involved in the downstream MAPK/ERK pathway, such as MAPK1, MEK, and MYC, showed increased transcript levels, although no mutations were detected in WES analysis.Transcriptional upregulation of as PTCH1, MED12, PDGFRB, and PIK3CA, did not show any transcript level change.Conversely, downstream signaling components of mutated cancer-associated genes, such Cancer cell lines are considered valuable models in basic cancer research, drug discovery, and translational medicine , 33, 34.The recent characterization of a large panel of human cancer cell lines with omics technologies has empowered data-driven precision medicine , 35. Desin vivo cOSA pathogenesis and identifying suitable targets for drug discovery. To date, this dataset represents one of the largest explored for a single tumor in dogs.In the present study, we integrated data from whole-exome and RNA sequencing of eight cOSA cell lines to obtain genomic and molecular data recapitulating Specifically, two commercial and six primary cOSA cell lines established and validated in our laboratory were analyzed. The assorted cell lines were previously used in several studies to investigate cOSA pathogenetic mechanisms and response to therapies , 36–38. About exome sequencing, variants were analyzed using three levels of increased stringency: first, describing the mutational profile of each cell line; second, identifying recurrently mutated genes; and third, unraveling putative driver genes having a likely role in cOSA pathogenesis.Overall, the mutational burden in our cOSA cell lines ranged between 3.9 and 16.9 mutations/Mb but was lower than the one described by Das et al. . HoweverConsistent with previous reports in cOSA cell lines and hOSA, the mutational type distribution showed a prevalence of missense mutations, and C>T transitions dominated this mutational spectrum , 39.PDGFRB and MET were retrieved. Both genes are known to play an important role in the development and progression of many canine cancers and were thoroughly investigated in cOSA as well  can be found here: RD and LA contributed conception and design of the study. PB and EM enrolled the canine osteosarcoma cases used for cell line establishment. CG, PM, SI, and RD cultured the cell lines and extracted DNA and RNA from canine osteosarcoma cell lines and FFPE samples. AR performed the NGS sequencing. DG performed the computational analyses. CG performed data post-processing. CG and SI performed data visualization. CG and DG wrote the manuscript. RD edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


GATA2, GATA1, and RUNX1 as candidateinducers of the expression of gangliosides and sialylation via regulationof the GTs ST3GAL2 and ST8SIA1.In conclusion, we show that GSL glycan expression levels are associatedwith hematopoietic AML classifications and TF and GT gene expression.Further research is needed to dissect the regulation of GSL expressionand its role in hematopoiesis and associated malignancies.Aberrant expressionof certain glycosphingolipids (GSLs) is associatedwith the differentiation of acute myeloid leukemia (AML) cells. However,the expression patterns of GSLs in AML are still poorly explored becauseof their complexity, the presence of multiple isomeric structures,and tedious analytical procedures. In this study, we performed anin-depth GSL glycan analysis of 19 AML cell lines using porous graphitizedcarbon liquid chromatography-mass spectrometry revealing strikinglydifferent GSL glycan profiles between the various AML cell lines.The cell lines of the M6 subtype showed a high expression of gangliosideswith α2,3-sialylation and Neu5Gc, while the M2 and M5 subtypeswere characterized by high expression of (neo)lacto-series glycansand Lewis A/X antigens. Integrated analysis of glycomics and availabletranscriptomics data revealed the association of GSL glycan abundanceswith the transcriptomics expression of certain glycosyltransferases(GTs) and transcription factors (TFs). In addition, correlations werefound between specific GTs and TFs. Our data reveal TFs  In mammals, GSLs can be classifiedinto three subgroups on the basis of common neutral core structures,namely, gangliosides, (neo)lacto-series, and globosides.7 GSLs play essential roles in cellularprocesses including adhesion, proliferation, differentiation, andrecognition.8 AbnormalGSL expression is associated with the development of many types ofcancers including leukemia.9 A subtypeof leukemia, AML, is a heterogeneous clonal disorder of hemopoieticprogenitors of the myeloid lineage which gives rise to red blood cells(RBCs), several different white blood cells, and platelets.10 AML is the second most common type of leukemiadiagnosed in adults and children, with most cases occurring in adults.The mortality rate of AML in the United States is estimated at 11,400for 2021, accounting for approximately 48% of all leukemia cases.11 AML can be classified into eight subtypes (M0–M7),according to the French–American–British (FAB) classificationsystem which was established in 1976.12 The subtypes are defined based on morphological and cytochemicalcharacteristics of the leukemia cells. Basically, subtypes M0 to M5are all derived from certain immature white blood cells called myeloblasts,M6 AML cells are initiated from an immature form of RBCs (erythrocytes),and M7 AML cells are from immature forms of cells which make platelets(megakaryocytes).Alterations of glycosylation including changes of sialylation and fucosylation  have been describedfor various hematological malignancies for both glycoproteins andGSLs.13 Previousstudies have indicated that certain GSLs play a critical role in thedifferentiation of AML cells.13 For example,the incorporation of GSL GM3 into HL60 cells resulted in the growth inhibition and monocytic differentiationof the cells.14 During the differentiationof monocytic THP-1 cells into macrophages, α2,6-sialylationslightly decreased and GM3 expression increased which correlated directlywith the overexpression of ST3GAL5.15 Theconcerted upregulation of ST3GAL5 and GM3 resulted from the activationof the PKC/ERK pathway during the differentiation of HL60 cells.16 In addition, the decrease of GSL Lc3 expression in HL60 and NB4 was induced by treatment with either all-transretinoic acid which can stimulate the differentiation of cells intothe neutrophil lineage or phorbol 12-myristate 13-acetate which inducedthe differentiation of cells along the monocyte lineage.It is suspected that GSLs, which are presenton the cell surface,might participate in myelopoiesis.Inthis study, we characterized the expression of GSL glycans in19 different AML cell lines belonging to different FAB subtypes. TheGSL glycans were profiled, identified, and quantified using porousgraphitized carbon nanoliquid chromatography hyphenated with massspectrometry via electrospray ionization (PGC-nanoLC–ESI-MS/MS).Overall, a striking diversity of GSL glycan expression was found betweencell lines. In addition, the association of GSL glycan expressionwith GT and TF gene expression and with AML classification was explored.4, Dowex cation-exchange resin (50W-X8), HCl, and ammonium bicarbonate were purchased from Sigma-Aldrich. KOH and 100% glacial acetic acid were from HoneywellFluka . A Sep-Pak tC18 reverse-phase (RP) solid-phaseextraction (SPE) cartridge (50 mg) was obtained from Waters . Ziptip C18 was obtained from Millipore ,and SPE bulk sorbent Carbograph was purchased from Grace DiscoverySciences . TopTip  wasobtained from Glygen Corporation , and 2-propanol waspurchased from Biosolve Chemie . Acetonitrile (MeCN)of LC–MS grade was purchased from Biosolve . Endoglycoceramidase I (EGCase I), 1× EGCase I reactionbuffer, α2–3 neuraminidase S, α1–3,4 fucosidase,α1–2,4,6 fucosidase O, purified bovine serum albumin(BSA), and 10× GlycoBuffer 1 were purchased from New EnglandBiolabs . Iscove’s modified Dulbecco’smedium (IMDM) was purchased from Gibco . PenStrep was obtained from Invitrogen(Thermo Fisher Scientific). Fetal calf serum (FCS) was purchased fromBodinco . The granulocyte-macrophage colony-stimulatingfactor (GM-CSF) was obtained from Cellgenix . Insulinand transferrin were purchased from Sigma .Ultrapure water was generated using a Q-Gard 2 system and used forall preparations and washing steps.Chloroform and trifluoroacetic acid (TFA)were purchased from Merck . Methanol (MeOH) ofultra LC–MS grade was obtained from Actuall Chemical . NaBH2. The AML193 medium was supplemented with 5 μg/mL insulin,5 μg/mL transferrin, and 5 μg/mL GM-CSF, and the mediaof cell lines M07e and TF1 were supplemented with 20 ng/mL GM-CSF.For more information regarding each cell line, see Supplementary Information, Table S-1.AML cell lines were grownat the Department of Immunopathology of Sanquin Research . Briefly, all cells were cultured in IMDM supplementedwith heat-inactivated FCS  and 1% PenStrep at 37 °C and 5% CO17 Briefly, prior tocell lysis, two million cellswere suspended in 200 μL of H2O and vortexed followedby a 30 min sonification at room temperature (RT) in a glass vial.Next, 550 μL of chloroform and 350 μL of MeOH were addedto the suspension. The sample was again briefly vortexed, sonicatedfor 30 min, and incubated for 4 h by horizontally shaking at 1000rpm. After incubation, the sample was centrifuged for 15 min at 3000× g at 20 °C. The upper phase (containingGSLs) was collected, and sequentially 400 μL of chloroform/MeOH(2:1) and 400 μL of MeOH/ H2O (1:1) were added tothe sample. After overnight incubation with shaking at RT, two additionalextraction steps were performed by collecting the upper phase (centrifugationat 3000 × g at 20 °C for 15 min) and adding400 μL of MeOH/ H2O (1:1) to the sample followedby sonication for 10 min before centrifugation. The collected upperphases  were combined into a single vial and evaporatedto dryness with vacuum. The tC18 RP-SPE cartridge was preconditionedby the sequential passing of 1 mL of chloroform/MeOH (2:1), 1 mL ofMeOH, and 2 mL of MeOH/H2O (1:1) over the cartridge. Theextracted GSLs were resuspended in 200 μL using a mixture ofMeOH and H2O (1:1) and loaded on the tC18 RP-SPE cartridgeby passing the sample three times over the cartridge. The loaded cartridgewas washed with 2 mL of MeOH/H2O (1:1) followed by a sequentialelution of the GSLs using 2 mL of MeOH and 2 mL of chloroform/MeOH(2:1). The samples were dried under vacuum in an Eppendorf Concentratorat 30 °C.The cells were processed as describedin a previous study.Rhodococcus triatomea recombinantlyproduced in Escherichia coli was usedto release the glycans from the extracted GSL samples according tothe manufacturer’s instruction with some slight modifications.Briefly, 36 μL of H2O, 4 μL of EGCase I reactionbuffer, and 2 μL of EGCase I enzyme were added to each lyophilizedsample. The final mixture (42 μL) was incubated at 37 °Cfor 36 h. After incubation, the GSL glycans were retrieved using atC18 RP-SPE cartridge, which was preconditioned with 2 mL of MeOHand 2 mL of H2O prior to applying the sample onto the cartridge.The purified GSL glycans were eluted using 500 μL of H2O. The mixture, consisting of the elution and flowthrough, was lyophilizedunder vacuum.EGCaseI from 18 Briefly, GSL glycanswere reduced by adding 40 μL of 1 M NaBH4 in 50 mMKOH to each sample followed by incubation at 50 °C for 3 h. Thereduction mixture was quenched with 4 μL of glacial acetic acid.The RP C18 Ziptips containing 50 W-X8 resin were preconditioned byapplying sequentially 3 × 60 μL of 1 M HCl, 3 × 60μL of MeOH, and 3 × 60 μL of H2O ontothe column. Subsequently, the solution, containing glycan alditols,was loaded, and the column was washed with 100 μL of H2O. The flowthrough and washes were collected and dried under vacuum.To remove residual borate, 150 μL of MeOH was added twice tothe samples during the drying step.Reductionand desalting of the purified GSL glycans were performed as describedin a previous study.2O followed by the addition of either2 μL of α2–3 neuraminidase S from Streptococcuspneumoniae recombinantly expressed in E. coli or 4 μL of α1–2,4,6 fucosidase O from Omnitrophica bacterium recombinantly expressed in E. coli. For α1–3,4 fucosidase  treatment, 5 μLof the pooled sample, 11 μL of H2O, and 2 μLof 10× glycobuffer 1 were mixed followed by addition of 2 μLof 10× BSA and 2 μL of α1–3,4 fucosidase.Combined enzymatic digestion was performed by adding 2 μL ofα2–3 neuraminidase S and 4 μL of α1–2,4,6fucosidase O or 2 μL of α1–3,4 fucosidase to 6μL of the pooled sample with the addition of 2 μL of 10×glycobuffer 1 and 13 μL of H2O. As a control, theenzyme was replaced with 10× glycobuffer 1. Each enzymatic digestionwas performed twice, and all samples were incubated overnight at 37°C followed by RP-SPE and drying of the purified glycans by vacuumcentrifugation.A pool of glycan alditolsreleased from cell lines Molm13, HL60, ML1, ME1, HEL, PLB985, EOL-1,and AML 193 was used for exoglycosidase treatment. In brief, 5 μLof the pooled sample was added to 2 μL of 10× glycobuffer1 and 13 μL of H2O with 0.1% TFA (v/v) followedby a shaking step for 30 min. PGC SPE clean-up was performed as describedpreviously19 with some slight modifications.Briefly, columns were packed with 50 μL of PGC resin slurry followed bya conditioning step by loading 3 × 60 μL of 80% MeCN with0.1% TFA (v/v) followed by 3 ×60 μL of H2O with 0.1% TFA (v/v). After sample loading, the columns were washed with 2× 60 μL of H2O with 0.1% TFA (v/v). Subsequently, the GSL glycans were eluted byadding 2 × 40 μL of 60% MeCN with 0.1% TFA (v/v). The two elution fractions were combined anddried under vacuum.Lyophilized GSL glycans were reconstitutedin 40 μL of H2O. A volumeof 1 μL of the sample was loaded onto a Dionex Ultimate 3000nanoLC system equipped with a Hypercarb PGC-trap column  and a HypercarbPGC nanocolumn . The separation platform was coupled to an amaZon speedion trap MS . Buffer A consistedof 10 mM ammonium bicarbonate and buffer B of 60% MeCN in 10 mM ammoniumbicarbonate. The separation of GSL glycans was conducted over a linearnanoLC gradient of solvent B from 1 to 65% in 80 min at a flow rateof 0.4 μL/min. After analysis, the column was washed with 85.5%MeCN in 10 mM ammonium bicarbonate for 10 min. MS spectra were acquiredwithin a mass to charge ratio (m/z) of 340–2000 in negative ion mode with a target mass of thesmart parameter setting at m/z 1200.The glass capillary voltage, dry gas temperature, dry gas flow, andnebulizer gas were set at 1000 V, 280 °C at 5 L/min, and 3 psi,respectively.Prior to MS analysis, the purified GSL glycanalditols were resuspended in 20 μL H22 and general glycobiology knowledge about the biosynthetic pathwaysof GSLs from a KEGG database.23 In addition,exoglycosidase digestion was used to further confirm specific linkages.Extracted ion chromatograms (EICs) were generated by extracting thetheoretical mass of GSL glycans of the observed singly and doublycharged species using the first three isotopes. Peaks were manuallyevaluated and automatically integrated using the Data analysis software(version 5.0). The peak area-under-the-curve was obtained to integrateeach peak of individual glycans if the signal-to-noise ratio was ≥6.Relative quantification was calculated on the total area of all GSLglycans within one sample normalizing to 100%.The GSL glycan structureswere assignedon the basis of the known MS/MS fragmentation patterns in negativeion modeTable S-2). For further data analysis and visualization,the packages “ggplot2”, “tidyverse”, “mixOmics”,24 “devtools”, “MASS”,“lattice”, “BiocManager”, “tidyHeatmap”,and “complexUpset” were used in “R” software(version 4.0.5). In addition, rcc and cim functions of the “mixOmics”package were used for canonical correlation analysis and clusteredimage mapping in which the colored block indicates the correlationbetween glycosylation features and relevant gene expression data extractedfrom the Cancer Cell Line Encyclopedia (CCLE) database, obtained bynonstrand specific RNA sequencing using the large-scale, automatedvariant of the Illumina TruSeq RNA sample preparation protocol.25Glycans wereassigned into different glycosylation features . To reduce the complexity of each GSL glycomic profile per cellline and define technical variability of the workflow used in ourstudy, the single mass spectrometry average composition (MSAC) wascalculated  from relative abundances.27 The low technical variability of the workflow is illustrated bythe clustering of the two technical replicates of each cell line ina principal component analysis (PCA) .To characterizestructural features of GSL glycans in 19 AML celllines, GSLs were extracted from the cell lines followed by glycanhead group release and analysis by PGC-nanoLC-ESI-MS/MS.21 Known diagnostic ions such as 0,2A-type and 2,4A-type were used to characterize C-4 substitution of theGlcNAc and Glc residues, respectively.22 Blood group and Lewis antigens were defined among the GSL glycansbased on the presence of specific D and A diagnostic fragment ions. The subtypes M2 and M4 revealed thehighest diversity, showing 9 and 18 unique GSL glycans, respectively,and most of the unique glycans expressed in M4 belong to (neo)lacto-series.These glycans carried blood group H or Lewis B/Y antigens . However, LewisA/X antigens were more broadly expressed in the M2/4/5 and 7 subtypes.Furthermore, three types of gangliosides and two kinds of (neo)lacto-seriesglycans were found across all subtypes, for more details see the SupplementaryInformation, Figure S-4 and Table S-5. Globoside glycans were present atlow abundance in most AML cell lines but were relatively abundantin the cell lines THP1 (32%), TF1 (17%) and M07e (20%) . As shown in Figure S-5). In contrast, higher expression ofgangliosides as well as globosides including SSEA-3 was found in theglycan profile of cell line M07e. Interestingly, N-glycolylneuraminic acid (Neu5Gc) was detected in most of the AMLcell lines .A high variation was observed in the GSL glycomic profilesbetweenthe 19 AML cell lines as exemplified in Table S-2). Variability in the loadings plot was assessedto elucidate which glycosylation features drive the separation inthe PCA model.PCA was performed todiscover possible associations between GSLglycosylation traits and the AML classification of the cell lines.For this, GSL glycans were grouped based upon the composition andspecific structural features  and sialylation,including α2,3-sialylation and glycans occupied with Neu5Gc. The expression levels of gangliosides,sialylation, α2,3-sialylation, and Lewis A/X antigen in AMLcell lines can be found in the Supplementary Information, Figure S-6. A similar glycosylation profile withadditional high expression of gangliosides with GalNAc was observedfor the M3 subtype cell line NB4, which is the only M3-classifiedcell line in our panel. Cell lines belonging to the M5 subtype  are mainly grouped inthe bottom region of the PCA scores plot because of the higher expressionof (neo)lacto-series, sialyl-Lewis A/X, and Lewis A/X lacto-series expressionof M5 cell lines vs 44.7% for the other 16 AML celllines . In contrast, cell line U937 – albeit also of the M5 class– showed very low expression of (neo)lacto-series with highexpression of gangliosides, and cell line THP1 indicated a specificallyhigh expression of globosides. No consistency was found for the M4subtypes with regard to GSL glycan feature expression. However, itshould be noted that a uniquely high expression of blood group H-typeantigens was found in the ML1 cell line. Two cell lines belongingto subtype M2 (PLB985 and HL60) also show high (neo)lacto-series expression, notably accompanied by high expressionof Lewis A/X type antigen  . HL60 has a highexpression of α2,6-sialylation while the third M2 cell line,Kasumi1, appears to have a distinct profile from the other two, revealinga high abundance of gangliosides, sialylation, and α2,3-sialylation.The M07e cell line, the only M7-classified cell line in our panel,shows specifically high expression of sialyl-Lewis A/X antigens togetherwith higher expression of globosides as shown in As shown in 31 To explore the associations between the expressionof GSL glycans and the expression of a preselected set of genes includingGTs involved in GSL biosynthesis and TFs involving the differentiationof the blood cells, the transcriptomic data of the same AML cell lineswere extracted from the CCLE.25 The selectionof hematopoietic TFs was based on their key roles in the regulationof the process of blood cell differentiation and maturation and theirdysregulation involved in the development of AML.35 Canonical correlation analysiswas performed between expression of GSL glycan traits as listed in B3GNT5 expression, a gene that codes for theonly enzyme that drives (neo)lacto-series biosynthesis (r = 0.38). Likewise, the relative abundance of globosides correlatedwith A4GALT gene expression coding for the firstenzyme specific for the globoside biosynthetic pathway (r = 0.38). A subtle correlation was found between the gene that isinvolved in the ganglioside series, B4GALNT1 (r = 0.21) and the expression of gangliosides. The expressionof the fucosyltransferases FUT7 and FUT9 encoding genes correlated positively with the fucosylated LewisA/X antigen  andα1,3/4-fucosylation .Similarly, a positive correlation was found between the α2,3-sialyltransferase encoding gene ST3GAL2 and α2,3-sialylation (r = 0.24) and between ST8SIA1 and sialylation (r = 0.30) andgangliosides (r = 0.32).Cellular glycosylation signaturesare known to be largely determined by GSTs on the one hand, and variousTFs on the other hand, as indicated by various studies using celllines as model systems, with glycosylation changes often reflectingcellular differentiation.GATA1, GATA2, RUNX1, and MECOM showed positivecorrelations with gangliosides  and sialylation . Meanwhile, GATA1 presented strong positive correlations with ST3GAL2 and ST8SIA1 , In addition, high positive correlations were foundbetween MECOM with ST3GAL2 (r = 0.75) and ST8SIA1 (r = 0.85) as well as between RUNX1 with ST3GAL2 (r = 0.40) and ST8SIA1 (r = 0.40) . These findings revealed that GATA2, GATA1, MECOM, and RUNX1 might play parts in the high expression of sialylation within theM6 subtype by regulation of corresponding GTs ST3GAL2 and ST8SIA1.To elucidate the potential regulationof GSL glycan expression,the correlations between GSL glycosylation features as listed in CEBPA (r = 0.14) and MYB (r =0.22), showed a moderate positive correlation. Interestingly, B3GNT5 responsible for production of (neo)lacto-series exhibitedstrong positive correlations with CEBPA (r = 0.54) and MYB (r =0.39), respectively . Additionally, TFs CEBPA and MYB showed positive correlations with FUT7  which were positivelycorrelated with the Lewis A/X antigen structure and α1,3/4-fucosylationas shown above. Thus, our in-depth analyses show that several relationsexist between the expression of specific GTs and TFs and the biosynthesisof GSL glycans.For the (neo)lacto-series, theTFs, 36 The structural and functional classificationof GSLs is mainly based on the glycan part. Little is known aboutthe expression and regulation of GSLs in AML cells because isolationand characterization of the GSLs is a challenging task due to theheterogeneity and isomeric complexity present in the glycan constituents.In this study, a PGC-nanoLC-ESI-MS/MS platform was employed to detect,identify, and quantify the glycan head group of GSLs extracted fromAML cell lines. The broad specificity enzyme EGCase I was used forreleasing GSL head groups, allowing to profile all human GSL seriesin a single analysis.37 In total, 79 GSLglycans were detected and characterized. An overall high diversityin GSL glycan profiles was found for the 19 AML cell lines.GSLs are involved inmany cellular processes including cellularinteraction, differentiation, signal transduction, and oncogenesis.38 In line withthese reports, we found associations between gene expression and GSLexpression in 19 AML cell lines. The expression of (neo)lacto-seriescorrelated positively with the expression of B3GNT5 which encodes the key enzyme involved in the biosynthesis of (neo)lacto-seriesGSLslacto-series GSLs in the inhibitionof immune recognition.39 Accordingly, onemay speculate about the potential immunosuppressive roles of (neo)lacto-seriesGSLs in AML. Meanwhile, both globosides and SSEA-3, which is a surfacemarker of human embryonic stem cells40 andhuman-induced pluripotent stem cells,41 showed positive correlations with A4GALT whichencodes the corresponding GT responsible for the biosynthesis of globosideglycans. Similarly, the expression of B4GALNT1 encodingfor a key enzyme in ganglioside biosynthesis positively associatedwith ganglioside levels.Previous studies indicated that incomplete biosynthesis and neosynthesisare the two main mechanisms that underlie aberrant glycan expressionin hematological malignancies because of the alteration of transcriptionof GTs and/or glycosidase gene(s).riesGSLs4 and 5. 7FUT7 and FUT9 are known for their involvement in the biosynthesis of α1,3-and α1,4-linked fucosylation, which is in agreement with ourfindings that FUT7 and 9 show positivecorrelations with the levels of α1,3/4-fucosylation . Our data imply that the expressionof FUT7 may play a role in the synthesis of Lewis A/X antigens ratherthan sialyl-Lewis A/X. In addition, the finding of a previous studyof increased Lewis A/X antigens on N- and O-glycome in the M2 and M5 subtypes is consistent with ourcurrent result.5 In contrast, blood groupH antigens were detected in cell lines originating from myelocytes/erythrocytes/monocytes.These H antigens are highly expressed in ML1 and ME1 (M4 subtype belongingto myeloblasts). Additionally, Lewis B/Y antigens which are potentialtherapeutic targets due to the high expression in various cancerswere only detected in ML1 and ME1 cell lines .44 Therefore, Lewis B/Y antigen mightbe a potential target for M4 subtype AML. To further corroborate this,AML cells obtained from AML patients with the M4 subtype could beinteresting study objects for glycomics analysis.Fucosylation, which is a nonextendablemodification, plays a criticalrole in the homing of hematopoietic cells to the bone marrow.sylation 4. Of notcmah, Neu5Gcis absent in human glycans.45 However,GSL glycans with Neu5Gc were found in most AML cell lines in thisstudy, including GM3 (Neu5Gc), GD3 (Neu5Gc), GM1a (Neu5Gc), and twoglycans in the (neo)lacto-series. The abundance of glycans with Neu5Gcvaried between cell lines with an average Neu5Gc expression of 2.3%in M6 cell lines vs only 0.8% in the rest of theAML cell lines . Several studies have suggested that increased levelsof Neu5Gc may be attributed to the higher metabolic rate in malignantcells,46 the induction of sialic acid transportersunder hypoxic conditions, and dietary incorporation.49 We suspect that the Neu5Gc found in the studied AML cell lines mayhave been incorporated from the culturing medium or its biosynthesismight originate from the mutation of relevant genes in AML cell lines.Previous studies revealed the expression of Neu5Gc in humans beingassociated with malignant transformation, becoming, therefore, anattractive target for cancer immunotherapy.50 Therefore, the glycans with Neu5Gc may be potential therapeutictargets for M6-stage AML. For a better understanding of the underlyingmechanism of expression of glycans with Neu5Gc in AML cell lines,more studies are needed.Because ofa deletion of the gene 51 Iantigens were first identified on human RBCs and have a reciprocalrelationship with i antigens.52 Adult humanRBCs fully express I antigens and contain only few i antigens, andalteration of expression patterns of I and i antigens has been foundduring oncogenesis.52 In this study, theexpression of I antigens was also found in most of the cell linesbelonging to the subtypes M2, M4, and M5. Interestingly, all the celllines expressing I antigens – except for U937 – wereinitially derived from peripheral blood . In addition, the cell lines HEL92.1.7,KG1, KG1a, TF1, and NB4 clustered well in the scores plot in PCA analysisand were all obtained from bone marrow , which indicates that the expressionof certain GSLs may reflect the origin of the cell lines.A recent study revealed that I bloodgroup antigens (I-branchedglycans), which are initiated by the addition of a GlcNAc to internalresidues of poly-LacNAc chains in β1,6 linkage, played a criticalrole in cancer progression through the regulation of malignancy-associatedadhesive and metastatic activities.34 Accordingly,certain hematopoietic TFs such as CEBPA,53MYB,54 and RUNX1 and the expressionof GATA2 correlated positively with sialylation,α2,3-sialylation, and gangliosides (GATA2 correlated with ST3GAL2 (r = 0.66) responsible for additionof sialic acid to terminal galactose of gangliosides and with ST8SIA1 (r = 0.66) gene expression encodingfor an α2,8-sialyltransferase which acts onsialic acid residue by addition of another sialic acid . Therefore, GATA2 might be involved in the upregulation of gangliosideexpression and sialylation in the M6 subtype. We speculate that poorprognosis of AML might be linked to the high expression of α2,3-sialylation,sialylation, and gangliosides regulated by GATA2 whichis highly expressed in M6-subtype cell lines. Our data point towarda shared effect of certain TFs on different types of glycoconjugates,as GATA2 likewise shows positive correlation withthe total levels of sialylation on N- and O-glycome in AML cell lines.5 Meanwhile, erythrocyte/megakaryocyte-related TF GATA1 and the megakaryocyte-derived M07e cellline . Interestingly, GATA1, MECOM, and RUNX1 expression positively correlated withsialylation .Strong positive correlations were observed for GATA1 with ST3GAL2 and ST8SIA1 , for MECOM with ST3GAL2 and ST8SIA1  as well as for RUNX1 with ST3GAL2 and ST8SIA1 (r = 0.40 and 0.40) . These findings indicate that GATA2, GATA1, MECOM, and RUNX1 may be involved in the high expression of sialylationin the M6 subtype by regulation of the corresponding GTs ST3GAL2 and ST8SIA1. Taking into account the previous findings,TF GATA2 may play an essential role in the regulationof sialylation of various glycoconjugates in M6 cell lines. To confirmthis, knockout, knockdown, and overexpression experiments targeting GATA2 in M6 cell lines assessing the effect on various glycoconjugateswould be informative.5High expression of CEBPA participates in the differentiation of commonmyeloid progenitors into basophils,32 andits mutation is associated with AML.58 Additionally, MYB plays a key role in the hematopoietic system and hasbeen recognized as an attractive therapeutic target for the treatmentof leukemia.59 Both TFs (CEBPA and MYB) are mainly expressed in subtypes M2 andM5 of the AML cell lines which are characterized by high expressionof (neo)lacto-series and Lewis A/X antigens . Our study revealed a modest positivecorrelation for TFs MYB and CEBPA with (neo)lacto-series structures  (B3GNT5 (r = 0.39 and 0.54) . As mentioned above, the Lewis A/Xantigen structure and α1,3/4-fucosylation were positively associatedwith FUT7 (CEBPA (r = 0.84) and MYB (r = 0.31) . The results indicate that the hematopoieticTFs CEBPA and MYB may play criticalroles in expression of GSL glycan structures ((neo)lacto-series, LewisA/X and α1,3/4-fucosylation) within AML classifications M2 andM5 by regulation of the expression of GTs B3GNT5 and FUT7 lacto-series, respectively) positively correlated with theexpression of gangliosides, globosides, and (neo)lacto-series, respectively(ST3GAL2/3/4 positively correlated with α2,3-sialylation andsialylation. Lewis A/X and α1,3/4-fucosylation showed positivecorrelations with FUT7 and FUT9, respectively. Moreover, certain hematopoieticTFs may play essential roles in the association of GSL glycans withAML classifications by regulating the related GTs as shown in In summary, the expression of genes ectively5. In addOur currentwork is fully descriptive in nature, providing a detailedmap of GSL glycan structures in AML cell lines. The multiple, reasonablystrong correlations between glycan species and GT and TF transcriptomicdata lead to new hypotheses regarding the regulation of the GSL repertoirewhich will benefit from experimental confirmation. This will be thesubject of future studies, with implications for cancer biology. Likewise,the analysis of patient materials using a similar approach is warranted,the main challenge being to obtain patient AML cells of sufficientpurity for deriving disease glycomics signatures.GATA2, GATA1, MECOM, and RUNX1 potentially play a role in the high expression of gangliosides,sialylation, and α2,3-sialylation within the M6 subtype viaregulation of GTs ST3GAL2 and ST8SIA1. The hematopoietic TFs CEBPA and MYB may play decisive roles in determining the expression of GSL glycanswithin AML classifications M2 and M5 by regulating the related GTs B3GNT5 and FUT7. Finally, the explorationof expression and regulation of GSL glycans in AML cell lines pavesthe way for future studies on the functional role of such GSL glycans.The enzymes in control of their production may provide novel therapeutictargets for specific AML subtypes.In this study, a striking diversity in expression of GSL glycanswas found between different AML cell lines. The biosynthesis of GSLglycans is regulated by specific GTs, some of which were confirmedat a transcriptional level. Additionally, we found new associationsof GSL glycans with AML classifications. These are potentially generatedby GTs whose expression is under the control of a subset of TFs. Ourstudy shows that the hematopoietic TFs


For decades, various plants have been studied as sources of biologically active compounds. Compounds with anticancer and antimicrobial properties are the most frequently desired. Cruciferous plants, including Brussels sprouts, broccoli, and wasabi, have a special role in the research studies. Studies have shown that consumption of these plants reduce the risk of lung, breast, and prostate cancers. The high chemopreventive and anticancer potential of cruciferous plants results from the presence of a large amount of glucosinolates, which, under the influence of myrosinase, undergo an enzymatic transformation to biologically active isothiocyanates (ITCs). Natural isothiocyanates, such as benzyl isothiocyanate, phenethyl isothiocyanate, or the best-tested sulforaphane, possess anticancer activity at all stages of the carcinogenesis process, show antibacterial activity, and are used in organic synthesis. Methods of synthesis of sulforaphane, as well as its natural or synthetic bifunctional analogues with sulfinyl, sulfanyl, sulfonyl, phosphonate, phosphinate, phosphine oxide, carbonyl, ester, carboxamide, ether, or additional isothiocyanate functional groups, and with the unbranched alkyl chain containing 2–6 carbon atoms, are discussed in this review. The biological activity of these compounds are also reported. In the first section, glucosinolates, isothiocyanates, and mercapturic acids (their metabolites) are briefly characterized. Additionally, the most studied anticancer and antibacterial mechanisms of ITC actions are discussed. Aliphatic GSLs covering more than 50% of glucosinolates are divided into: alkenyl (1–3), hydroxyalkenyl (4–5), and those containing sulfur on II (6–7), IV (8–10), and VI (11) oxidation states ..33) -L-homoserine lactones (3-oxo-C12-HSL) and lasR, genes encoding transcription regulators. The rhl system consists of the pair RhlI/RhlR, which respond to N-butyryl homoserine lactones (C4-HSL) pyridine (b), 6-methoxy-1H-benzo[d]imidazole (c), 5-phenyl-1H-tetrazole (d), or benzo[d]thiazole (e). The sulfides 81a–e were transformed into amines 82a–e, and then were converted into sulfide derivatives of ITCs 83a–e with thiophosgene (34), in good and very good yields. In the next step, compounds 83d–e were oxidized with MCPBA into sulfoxide analogues of SFN 84d–e with good yields  to sulfoxides 85a–c and sulfones 86a–c and then transformed to amines 87a–c and 88a–c using methylamine. In the final step, isothiocyanation of amines 87a–c and 88a–c with thiophosgene afforded ITCs 84a–c and 89a–c in good yields . Among all synthesized analogues, tetrazole analogues 83d, 84d, and 89d were generally the most potent—and significantly more active—than SFN against the tree cancer cell lines  involved in the antioxidant defense in the neuronal cell line. All hybrids produced a significant decrease in ROS production elicited by pro-inflammatory stimulus compared to the rivastigmine, which completely lacks antioxidant activity. The new hybrids were also able to reduce NO release in BV-2 cells, whereas rivastigmine showed no effect. Moreover, the most active compounds 99 and 100 increased the GSH level in the human neuroblastoma cell line SH-SY5Y.Studies on the murine microglia cell line (BV-2) showed that all synthesized ITC hybrids 48) as a desulfurizing agent. Sulfide derivatives of ITCs 28, 128–147, were prepared, in the presence of Et3N, from the corresponding ω-alkanamines 61, 108–127, carbon disulfide (35), and mesyl chloride (48) in 43–65% yields. The key intermediates, the appropriate ω-alkanamines 61, and 108–127, were obtained from ω-bromoalkylphthalimides 59 and 105–107 in a standard procedure. Oxidation of sulfides 28, and 128–147 with MCPBA, led to sulfoxide analogues 24 and 148–167, with very good yields , human lung adenocarcinoma (A549), woman breast cancer ER+ (MCF-7), human colon cancer cell line (HCT-116) and human neuroblastoma cell line (SH-SY5Y). All tested compounds exhibited more potent inhibitory against five cancer cell lines than SFN. The IC50 for the most active ITCs are presented in All synthesized ITCs 151 with the IC50 value 2.05 μM. ITC 161 had the strongest inhibition against A549 with IC50 5.64 μM, and ITC 155 possess significant cytotoxicity for MCF-7 with IC50 3.3 μM. Derivative 152 against HCT-116 had an IC50 value of 2.06 μM, and isothiocyanate 157 against SH-SY5Y ITC had an IC50 value 2.79. Based on the tested ITCs 28, 24, 148–167, it was found that compounds with sulfanyl moiety 135 showed weaker inhibitory effects than most derivatives; however, compound 168 with a sulfone group showed a higher inhibitory activity against all cancer cell lines than SFN. It may indicate that replacing the sulfoxide group with a sulfone group results in higher biological activity. The studies on anticancer mechanisms examined on the HepG2 cancer cell line showed that SFN, as well as model ITC 155, could induce the S or G2/M phase cycle arrest, and 155 has more potent inhibitory activity than SFN. Moreover, ITC 155 exhibited greater induction of apoptosis upon treatment than SFN. ITC 155 presented a time- and dose-dependent activation on the Nrf2 transcription factor. Moreover, 155 acted as a more potent Nrf2 inducer than SFN.For HepG2, the most active was ITC 24) and its analogue erucin (28) via the tandem Staudinger/aza-Wittig reaction. Thus, the starting 4-bromo-butanol (169) was transformed using a standard procedure into azido thioether 170. Then, this compound, in the reaction with triphenylphosphine and carbon disulfide, was converted into erucin (28) in an 81% yield. SFN (24) was obtained by oxidation of erucin (28) with MCPBA in 77% yield (Meijer and co-workers  synthesi7% yield .P. aeruginosa show that SFN and erucin strongly inhibit quorum sensing (QS) and virulence (biofilm formation and pyocyanin production). The assays on P. aeruginosa and in E. coli strongly indicate that SFN and erucin effectively bind LasR, resulting in inhibition of QS activation at concentrations that can be found in broccoli.Studies on R)-SFN ((R)-24) pyrimidines-2-amine from ribociclib as CDK9 pharmacophore, and analogues of SFN with alkyl chains of different lengths as targeting CSC pharmacophore. Briefly, the key N-Boc amines 462a–e and 463aa–de were converted into the final ITCs 464a–e and 465aa–de after prior deprotection of the amino group by trifluoroacetic acid, followed by the reaction with carbon disulfide and N,N’-dicyclohexylcarbodiimide   . The sam and 471 .464a–e, 465aa–de, 469, 470a–g, and 471 were evaluated regarding inhibition activity against CDK4, CDK6, and CDK9, with ribociclib as the positive control. Some compounds showed high potent activity against CDK9 as well as high selectivity for CDK9. The inhibition of tested ITCs was similar or higher than inhibition of ribociclib. In particular, ITC 470e with an 8-isothiocyanatooctanamide linker, exhibited high enzymatic inhibition (IC50 = 11 nM vs. IC50 of ribociclib = 197 nM). ITC 470e was tested against several tumor cell lines, including NSCLC, breast cancer, hepatocarcinoma, cervical cancer, leukemia, and lymphoma, using CCK8 assay and SFN as positive controls. ITC 470e had the highest activity against NSCLC cell lines, especial A549 and H1299, with IC50 values less than 0.5 μM. For comparison, IC50 of SFN on A549 was more than 10 μM and IC50 of ribociclib was 7.455 μM. For other cell lines, ITC 470e exhibited lower anti-viability activity. These results demonstrated good cellular selectivity of ITC 470e. Additionally, studies on A549 and H1299 showed that ITC 470e dose-dependently inhibited the cell cycle at G2/M phase and induced apoptosis of A549 cells in a concentration-dependent manner. Moreover, ITC 470e decreased the formation of colonies in the two NSCLC cell lines. In vivo studies on H1299 xenograft mouse models showed that ITC 470e displayed potent anti-tumor activities.All ITCs P. aeruginosa. Briefly, etherification of phenol 472, followed by amino group deprotection with TFA and a two-step reaction with carbon disulfide and molecular iodine, afforded ether-linked isothiocyanates 473 and 474 in low yields  with no effect on bacterial growth.Perez and co-workers  synthesiw yields . A LasR 2S donors with the isothiocyanate group. For this purpose, the starting N-Boc-glycoconjugates 475a–b were deprotected with TFA, followed by the reaction of amino derivatives 476a–b with an excess of carbon disulfide, triethylamine, and hydrogen peroxide to afford final, fully acetylated ITCs 478a–b, with low yields. On the other hand, deacetylation of O-acetylated amines 476a–b with sodium methanolate, followed by the isothiocyanation of amines 477a–b, performed in the same manner as above, gave isothiocyanate glycopyranosides 479a–b in 21% and 22% yields, respectively . Studies showed that glucose-derivative 479a was totally ineffective at inhibiting cell viability. However acetyl-galactosamine-derivative 478b exhibited a marked cytotoxicity against AsPC-1 tumor cells in a concentration-dependent manner with a pIC50 value of 4.45 ± 0.02. ITC 478b efficiently released H2S intracellularly with desirable slow kinetics and inhibition of cell cycle progression G0/G1 phase.ITCs 481a–c were obtained in good yields from amines 480a–c in a two-step reaction with carbon disulfide and triethylamine used as a base, followed by desulfurization with acyl chloride (He and co-workers  designedchloride .481a–c showed higher anti-tumor effects in vitro than dihydroartemisinin (DHA) against human glioblastoma U87 cells. The most active was ITC 481b (IC50 = 7.41 ± 1.56 μM) (IC50 DHA = 118.95 ± 12.39 μM). ITC 481b reduced the viability of glioblastoma multiforme (GBM) in a concentration-dependent manner. ITC 481b also inhibited migration and induced apoptosis in U87 cells. Pyknosis and nuclear shrinkages were observed in ITC 481b-treated cells. Additionally, caspase 9 and cytochrome c were induced in U87 cells treated with ITC 481b and cleaved caspase 3 was increased. On the other hand, the anti-apoptotic protein Bcl-2 was downregulated, and pro-apoptotic protein BAX was upregulated.All ITCs N-acetyl cysteine. Both isothiocyanate functional groups in diITCs were separated with unbranched alkyl linkers containing three and four carbon atoms (484 and 485). DiITCs 484 and 4825 were obtained from the corresponding diamines 482 and 483 in a two-step procedure with carbon disulfide and triethylamine as a base, followed by the reaction with HBTU as desulfurizing reagents  than Gram-negative . Studies on wide selections of Gram-negative as well as Gram-positive strains showed that the most active were diITCs 492 and 493, which, on Gram-positive strains, such as Streptococcus, Staphylococcus, and Bacillus, exhibited high activity (MIC = 1–2 μg/mL). DiITCs 492 and 493 presented high activity against mycobacteria. The most active were against M. bovis and M. tuberculosis (MIC = 2–4 μg/mL) than against nontuberculous mycobacteria. DiITC 492 efficiently inhibited the growth of the pathogens, such as Candida albicans or Candida glabrata, for which MIC < 1 μg/mL.The growth-inhibitory activity of synthesized diITCs The described above methods of the synthesis of SFN and its bifunctional analogs are presented in 2) is dominant. The next often used method is the tandem Staudinger/aza-Wittig reaction, and the least frequently used is the two-step method using a desulfurizing agent. Another group involves phosphorus analogues of SFN (P-SFN) with phosphonate, phosphinate, or phosphine oxide moiety with aliphatic or aromatic substituents. For this class of compounds, both the tandem Staudinger/aza-Wittig reaction and the method using a desulfurizing agent were equally frequently used. The reaction with CSCl2 for synthesis P-SFN was used less frequently. Carbonyl and amide analogs of SFN were synthesized by all three methods. The synthesis of ether-linked analogs of SFN was completely performed by the two-step methodology with a desulfurizing agent, while the synthesis of diisothiocyanates was also performed with the desulfurizing agent and thiophosgene. The tandem Staudinger/aza-Wittig reaction, to the synthesis of ether-linked analogs of SFN and diisothiocyanates, was not used.Based on the 75, 80d), heterocyclic (84d), or aliphatic  (152) substituents result in increases of the biological activities of analogs of SFN on different cancer cell lines, such as Malme-3M, MCF-7, or A549. For phosphorus analogs of SFN (P-SFN), the presence of phosphine oxide moiety with methyl substituents (191) does not affect biological activity. However, replacement of the methyl sulfinyl group by phosphonate moiety with aliphatic (298) or aromatic (407) substituents or phosphinate moiety (372) results in an increase in the anticancer activity on LoVo and LoVo/DX cancer cell lines. Carbonyl or amide analogs of SFN (419 and 455) can by characterized by biological activities similar to SFN, but on the other hand, for some analogs , biological activity could be higher than for SFN. The presence of the second isothiocyanato group (484) also results in an increase in the biological activity on LoVo and LoVo/DX cancer cell lines.Based on There are many examples describing the synthesis of SFN and its natural or synthetic difunctional analogues, with methyl sulfinyl group replaced by sulfinyl, sulfanyl, sulfonyl, phosphonate, phosphinate, phosphine oxide, carbonyl, ester, amide, ether, or a second isothiocyanate group. The most common strategies involve the use of thiophosgene as a thiocarbonyl transfer reagent, utilization of a carbon disulfide/desulfurizing agent system (dithiocarbamates approach), or use of the tandem Staudinger/aza-Wittig reaction (triphenylphosphine/carbon disulfide system). In the first two approaches, primary amines are utilized as substrates; the last comprises azides as starting materials. The choice of method depends on the availability of starting substrates and sensitivity of functional groups present in the substrates to the reaction conditions. Several ITCs are characterized by high biological activity , usually higher than parental SFN. Which modifications of the structure of SFN seem to have important impacts on the biological activities of the new analogues of SFN.As SFN is biologically active, natural ITC is found in cruciferous vegetables, and it is non-toxic—it has been selected for phase I and II clinical trials, where it is administered in the form of an extract or broccoli sprouts. The results of these studies are promising and indicate which SFN, in the future, may be considered as an anticancer drug. The situation is different for synthetic analogs of SFN, which have been described in this review, having sulfinyl, sulfanyl, sulfonyl, phosphonate, phosphinate, phosphine oxide, carbonyl, ester, amide, ether, or a second isothiocyanate group. The vast majority of these compounds have only been tested for anticancer or antibacterial activity in vitro, and a few analogs, additionally in vivo, on mice or Zebrafish. None of the synthetic analogs of SFN described in this review qualified for clinical trials. This is likely due to the toxicity of these compounds in higher doses, often seen in in vitro or in vivo studies. SFN, which is a natural product, does not show toxicity, even in higher doses. Another reason is the financial constraints of many research teams.One interesting solution would be to synthesize a conjugate—a combination of SFN and a drug or peptide, with anticancer properties, characterized by no (or low) toxicity. This conjugate could separate, in cancer cells, on two drugs, causing increased anticancer activity.


Mature T-cell lymphomas (MTCLs) represent a heterogeneous group of aggressive non-Hodgkin lymphomas comprising different entities. Anthracycline-based regimens are considered the standard of care in the front-line treatment. However, responses to these approaches have been neither adequate nor durable, and new treatment strategies are urgently needed to improve survival. Genomic instability is a common feature of cancer cells and can be caused by aberrations in the DNA damage response (DDR) and DNA repair mechanisms. Consistently, molecules involved in DDR are being targeted to successfully sensitize cancer cells to chemotherapy. Recent studies showed that some hematological malignancies display constitutive DNA damage and intrinsic DDR activation, but these features have not been investigated yet in MTCLs. In this study, we employed a panel of malignant T cell lines, and we report for the first time the characterization of intrinsic DNA damage and basal DDR activation in preclinical models in T-cell lymphoma. Moreover, we report the efficacy of targeting the apical kinase ATM using the inhibitor AZD0156, in combination with standard chemotherapy to promote apoptotic cell death. These findings suggest that DDR is an attractive pathway to be pharmacologically targeted when developing novel therapies and improving MTCL patients’ outcomes. Nodal T-cell lymphomas (TCL) are a rare and aggressive subgroup of lymphoid malignancies, accounting for 10–15% of non-Hodgkin lymphomas (NHL). According to the latest World Health Organization (WHO) classification, they comprise several entities, such as angioimmunoblastic T-cell lymphoma (AITL), anaplastic large T-cell lymphoma (ALCL), and peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), which is the most common subtype including all the TCL cases that still fail to be categorized . AnthracIn recent years, several efforts have been made by us and others to elucidate the genetic features of TCLs, reporting a broad range of copy number alterations (CNAs) and structural variations (SVs) rather than point mutations ,7,8, thuHere, we report for the first time the characterization of intrinsic DNA damage and basal DDR activation in preclinical models in T-cell lymphoma. Additionally, we provide evidence that targeting the apical kinase ATM in combination with CHOEP is effective in promoting apoptotic cell death.We first explored whether T-cell lymphomas as well as solid tumors and some hematological malignancies are characterized by intrinsic DNA damage, a feature of genomic instability. The presence of γH2AX and 53BP1 foci, well-recognized markers of DNA damage ,27, was We then assessed whether the presence of intrinsic DNA damage was associated with constitutive activation of DDR signaling pathways. To this aim, we studied the expression of activating post-translational modifications of several DDR players including ATM, Chk2, Chk1, and KAP1 before and after 3 h treatment with increasing concentrations of CHOEP and with 20 µM etoposide alone, which is well known to activate DDR in human cells ,32. As sTaken together, these data indicate that in vitro models of T-cell lymphoma show intrinsic DNA damage and constitutive activation of the DDR signaling pathways, despite some degree of heterogeneity observed in the analyzed cell lines.As we have shown that the DDR signaling cascade is basally activated and gets further triggered in response to chemotherapy, we hypothesized that targeting the DDR could block the possible attempts made by the cell exposed to CHOEP to repair the induced DNA lesions, thus leading to augmented apoptosis. For this purpose, we took advantage of a recently developed inhibitor, AZD0156, that targets the apical kinase ATM  and is b50 range: 0.55–2.3 µM) (20 (50 (not shown) doses of AZD0156 with no significant changes detected among untreated and AZD0156-treated cells.First, we exposed cell lines to increasing concentrations of AZD0156 (range 1 nM–500 µM) for 48 h and observed a dose-dependent reduction in viable cells in all cell lines, even though sensitivity to AZD0156 was heterogeneous A. The on–2.3 µM) . The dec–2.3 µM) B. This w µM) (20  and IC5020 CHOEP (defined in our previously reported study [20 AZD0156 (p < 0.05)  A. Consis < 0.05) B. Import < 0.05) C.ATM and/or TP53, we performed targeted sequencing analysis. As expected, all cell lines bear pathogenic TP53 alterations but no ATM mutations. ATM is mutated only in JURKAT cell line, but we were unable to define a clear inactivating effect of the detected alterations  (p: ns)  A, with a 0.0001) B. As exp (p: ns) . The oth (p: ns) A.To conclude, we confirmed the efficacy of ATM inhibition in potentiating CHOEP effects using another ATM inhibitor, KU-55933, widely used in in vitro studies.We tried to titrate KU-55933 in our cell lines, but in a range of concentrations between 16 nM and 16 μM, we observed a reduction in cell viability of 10–20%. Cell viability was significantly reduced when we reached higher concentration (100 μM–200 μM) (data not shown). As KU-55933 has been administered to cell lines at 10 μM to inhibit ATM kinase activity in other studies ,36, we d20 CHOEP, and we observed a significant reduction in cell proliferation J recombination, which expose cells to high levels of DNA damage. For this, in recent years, several inhibitors of DDR proteins—including ATR, DNA-PK, PARP, Chk1, and WEE1, administered alone and in combination with other agents—have been preclinically tested . Consistn status .In the present study, we report encouraging preclinical data describing the benefits of ATM inhibition in promoting CHOEP-induced cell death in preclinical models of T-cell lymphoma using two different chemical compounds—AZD0156 and KU-55933. Despite the presence of basal DDR activation and intrinsic DNA damage observed in all the cell lines included in this study, their response to AZD0156 and AZD0156–CHOEP treatment was heterogeneous, further confirming the heterogeneity of the pathology.Interestingly, we observed a reduction in γH2AX foci upon combined treatment with AZD0156 and CHOEP because this serine is directly phosphorylated by ATM. Nonetheless, 53BP1 foci formation was not impaired by AZD0156 addition to CHOEP in our models. Thus, the decrease in γH2AX foci is not suggestive of the absence of DNA damage, but rather of dysfunctional DDR signaling caused by ATM inhibition. Consistently, DNA damage is not efficiently repaired, leading to apoptotic cell death, as in The AZD0156 inhibitor is currently clinically tested in advanced solid tumors alone or in combination with olaparib and irinotecan. Moreover, recent preclinical studies reported AZD0156 capability to enhance the genotoxic effects of olaparib and radiation in preclinical models of different solid tumors ,25. In aGiven alone, AZD0156 does not induce cell cycle perturbations in malignant T cells, as observed in solid tumors ,25. NoneIn recent years, first-line regimens built on a CHOP-like backbone have been studied, but none, including the one combining the histone deacetylase (HDAC) inhibitor romidepsin with CHOP (Ro-CHOP), significantly improved the survival of patients affected by PTCL , thus suTP53 mutations, suggesting that AZ0156 is effective independent of TP53 mutational status. On the contrary, we did not observe ATM putative inactivating mutations. Nonetheless, consistent with the fact that mutations have been reported in the ATM gene in TCL patients [ATM.The in vitro models of T-cell lymphoma employed in this study are characterized by pathogenic patients ,42, addiNotably, our in vitro data suggest that suboptimal concentrations of the ATM inhibitor are not detrimental for healthy T lymphocytes. Nonetheless, we cannot rule out the possibility that the combined treatment with AZD0156 and CHOEP could cause adverse effects in humans. Preliminary data from the phase I clinical trial aimed at assessing efficacy and tolerability of AZD0156 in combination with olaparib reported minor toxicities in about 40% of patients enrolled in the study. However, hematologic toxicities were observed when higher doses of both drugs were used . Thus, pKARPAS-299 (ALCL), SUP-T1 (lymphoblastic TCL), HH (cutaneous TCL), HD-MAR-2 (T-cell leukemia), and JURKAT (T-cell leukemia) were purchased from DSMZ–Leibniz Institute . OCI-Ly12 cell line (PTCL-NOS) was a kind gift from Dr. Leandro Cerchietti  . Cells w50) were determined upon 48 h of chronic exposure, as the concentration of drug able to reduce 50% of cell growth.All drugs  were purchased from Selleck Chemicals . CHOEP was prepared as described in [Upon treatment, cells were transferred on glass slides using a cytospin centrifuge (5 min at 500 rpm). Glass slides were dried overnight at room temperature, then fixed with 2% paraformaldehyde and permeabilized with PBS 0.2% Triton X-100. Saturation was performed in PBS 5% BSA 0.2% TWEEN 20. Primary antibodies used were γH2AX #A300-081A  and 53BP1 #NB100-305 ; secondary conjugated antibody used was Alexa Fluor 488 #A11034 . At the end, nuclei were counterstained with DAPI . Images were acquired with a Nikon Eclipse E1000 fluorescence microscope equipped with a DSU3 CCD camera, using a 100× magnification objective, as previously described . In eachCell viability, cell death, mitochondrial membrane potential, and cell cycle were studied by flow cytometry as previously described . BrieflyCell lysates, SDS-PAGE and Western blot were performed as described in . AntibodGenomic DNA was extracted using the Nucleospin Tissue kit  and quantified using Qubit 2.0 and the Qubit DNA HS Assay kit . DNA was run on the Oncomine Comprehensive Assay (OCA) Plus targeted panel of 1.7 Mb  with an AmpliSeq-based enrichment. All library preparation was performed manually according to manufacturer’s instructions (MAN0018490). Multiplex PCR amplification was conducted using 20 ng of DNA as input. Purified libraries were quantified with real-time PCR with the Ion Library TaqMan™ Quantitation Kit . The 50 pM libraries were pooled and loaded onto Ion 550™ Chips  according to manufacturer’s instructions (MAN0017275) and prepared for sequencing using the Ion Chef™ System . Sequencing was performed using the Ion Gene Studio S5 Sequencer .https://opencravat.org (accessed on 14 February 2022)) and the Ion Reporter™ Software . Variants were classified using ClinVar (available online: https://www.ncbi.nlm.nih.gov (accessed on 14 February 2022)), cBioPortal ), and dbSNP databases. Variants categorized as neutral/benign and variants with a frequency >0.00001 in the population (not pathogenic) were considered as single nucleotide polymorphisms (SNPs) and were thus excluded.For the analysis, data were initially processed using Ion Torrent Suite Software™ , and variant calling was performed using the Variant Caller plugin. Variants were filtered for coverage greater than 40 reads, frequency greater than 5%, quality value greater than 30, and coverage depth greater than 500X. Resulting variants were annotated using the OpenCravat tool  of independent experiments. GraphPad Prism 9 software  was used to perform graphs and statistical analyses. Specifically, one-way ANOVA test was used to calculate significance of cell viability, cell death, and TMRE assays. Tukey post hoc test was applied. Two-way ANOVA followed by Bonferroni posttest was used to calculate the significance of cell cycle experiments. For γH2AX and 53BP1 foci enumeration,