Glioblastomas (GBM) exhibit intratumoral heterogeneity of various oncogenic evolutional processes. We have successfully isolated and established two distinct cancer cell lines with different morphological and biological characteristics that were derived from the same tissue sample of a GBM. When we compared their genomic and transcriptomic characteristics, each cell line harbored distinct mutation clusters while sharing core driver mutations. Transcriptomic analysis revealed that one cell line was undergoing a mesenchymal transition process, unlike the other cell line. Furthermore, we could identify four tumor samples containing our cell line-like clusters from the publicly available single-cell RNA-seq data, and in a set of paired longitudinal GBM samples, we could confirm three pairs where the recurrent sample was enriched in the genes specific to our cell line undergoing mesenchymal transition. The present study provides direct evidence and a valuable source for investigating the ongoing process of subcellular mesenchymal transition in GBM, which has prognostic and therapeutic implications.The online version contains supplementary material available at 10.1007/s00018-022-04188-3. Glioblastoma (GBM) is the most common primary malignant brain tumor and is one of the most aggressive cancers . DespiteEpithelial to mesenchymal transition, an EMT process, is a highly controversial term in GBM tumorigenesis. In gliomagenesis and its progression, a non-classical EMT-like or glial to mesenchymal transition is proposed to be linked to the mesenchymal nature of the neural cells during the neurodevelopmental process . AnotherHere, we isolated and established two distinct cell lines derived from the same GBM tissue sample. These cell lines have genetically developed into different cell lines by subculture. We compared and analyzed the genomic and biological signatures of the two cell lines and found that one of the cell lines exhibits mesenchymal transition characteristics, unlike the other. Furthermore, we reanalyzed publicly available single-cell RNA-seq data to identify analogous clusters to our case and found four similar GBM samples with occult clones of mesenchymal transition. We also found recurrent GBM samples showing evolution of mesenchymal transition clones characterized by upregulation of the EMT genes identical to the cell line which was not identified in the initial sample. These findings provide direct evidence to support the subcellular evolution of mesenchymal transition in GBM, which can be utilized in future investigations into intratumoral heterogeneity and the mesenchymal transition process.2 cell culture flasks. The tumor cells were initially cultured in Opti-MEM medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) (O5). Cultures were maintained in RPMI 1640 medium containing 10% heat-inactivated FBS (R10). Initial passages were completed when abundant tumor cell growth was observed, and consecutive passages were done every 1 or 2 weeks. During passaging, adherent cells were recovered by pipetting by treatment with trypsin while growth was subconfluent. Differential trypsinization was used to obtain a pure tumor cell population when stromal cell growth was noted in the initial cultures. Cultures were maintained in humidified incubators at 37 °C with 5% CO2 and 95% air. The established cell lines used in the present study are available at the Korea Cell Line Bank (https://cellbank.snu.ac.kr/). The cell lines were maintained in opti-DMEM medium supplemented with 20% fetal bovine serum (GIBCO).An IRB-approved written informed consent was obtained from the patient to use the samples and to establish cell lines for research purposes . Tissues were freshly frozen in liquid nitrogen immediately after resection, and white blood cells (WBCs) were extracted from whole blood. Both tissue and WBC samples were then stored at − 80 °C until later use. The cell lines were established using the core area of the fresh tumor tissue just after resection. Solid tumor tissue was carefully minced with scissors and isolated into small mixtures by pipetting. Appropriate amounts of delicate tumor tissue fragments were seeded into 25 cm3 cells/well in 96-well plates and cultured for the designated times. The absorbance of the plates was measured using a microplate reader (Molecular Devices) at a wavelength of 450 nm.Cell proliferation assays were performed with EZ-Cytox (Daeillab Service) on cells seeded at 1 × 102 for 14 days. The cell colonies were fixed and stained with 0.05% crystal violet-methanol-acetic acid solution after 14 days. Stained colonies were scanned and score was calculated.To grow colonies, cells were seeded in 6-well plates at 1000 cells/well density and incubated at 37 °C in an atmosphere of 5% COhttp://rsb.info.nih.gov/ij/) . BP value < 0.05 and Log2 fold change 11/5 ≥ 2. Then functional clustering of the upregulated genes in subclone #11 was done using DAVID v6.8 [Extracted RNAs from the samples were checked for quality control with Bioanalyzer. Library preparation was conducted using a Lexogen Quantseq 3’ mRNAseq library prep kit. Single-end mRNA sequencing was performed with Illumina Nextseq500 at ebiogen, Korea. Generated FASTQ files were mapped to the reference genome (hg19) with Bowtie2. Normalization of the count was done with EdgeR and batch effects were removed with the help of the R package limma (doi: 10.1093/nar/gks042 and 10.1093/nar/gkv007) , 17. We VID v6.8 . For theWe downloaded GBM single-cell RNA-seq data from four recently published studies , which included a total of 71 samples from 65 patients . Initialt test. Kaplan–Meier curve analysis was used to analyze patient survival time. The differences with P values < 0.05 were considered statistically significant.The results were analyzed using IBM SPSS Statistics software . Data are expressed as the mean ± SE. Statistical significance was determined using Student’s The primary culture of GBM cells was performed from the freshly harvested cancer tissue from a 76-year-old female GBM patient. The radiological and histological characteristics of the original tumor showed typical features of GBM . A noteworthy radiological and gross finding was that the cortical direction of the tumor abutted upon the dura and disrupted the leptomeningeal membrane . The tumor was highly proliferative (Ki-67 index 68.7%) and showed positive glial fibrillary acidic protein and vimentin by immunohistochemistry . Its genetic features include wild-type IDH1/2, a TERT promoter mutation (C250T), and wild-type ATRX. Chromosomal abnormalities of Epidermal growth factor receptor (EGFR) gene amplification and chromosome 9p21/p16 locus deletion were revealed by fluorescence in situ .TERT promoter mutation in both cells, implying they were both cancer cells descended from a common ancestor  of the original tumor and cell line, subclone #11 showed higher enzymatic activity of telomerase and TERT expression compared with subclone #5  profile in the SNU-4210 cell line with its subclones and the original GBM tissue using whole-exome sequencing (WES) data. To track the clonality across the tissue sample and cell lines with temporal information, we took advantage of the SuperFreq algorithm to cluster the mutational changes according to the prevalence of their variant allele frequency. A total of 10 clusters across the samples were identified Fig. a. The coWhen we analyzed the subclone-specific mutations, none of those mutations were found to reside in any of the previously reported GBM driver genes Fig. c. The fuP value < 0.05), and genes within the regions showing copy number gains in chromosome 20 for subclone #11 were upregulated  compared to subclone #5 (Table S3). Functional clustering of upregulated genes in subclone #11 compared to subclone #5 showed significant enrichment on EMT and cell cycle-related gene ontology clusters  in subclone #11 . Pearson correlation analysis of the overall gene expression profile of the subclones showed a 78% positive correlation with the parent tumor, a 98% positive correlation with the parent cell line, and a 92% positive correlation with each other Fig. a. Howeve#11 Fig. d. We cou#11 Fig. e. These Based on previous observations that identified mesenchymal-related molecular signatures for GBM tumors and stem cells , and ourhttps://www.gsea-msigdb.org/gsea/msigdb/) for all of the paired longitudinal samples and then calculated the gene expression difference between the recurrent and initial samples. We found 10 pairs that showed significantly high expression for most of the EMT genes in the recurrent samples, suggesting that the mesenchymal transition process was the key factor driving the recurrence of these pairs. We then investigated the same subclone #11 specific genes that we used for the scRNA-seq analysis to see if any of these 10 paired samples had upregulation of the subclone #11 specific genes. Indeed, we were able to find 3 samples among the 10 displaying overexpression of a subset of subclone #11 specific genes . We first extracted the normalized gene expression values of the Hallmark EMT gene set from the MSigDB database Below is the link to the electronic supplementary material.


ACE2 and TMPRSS2 based on RNA-seq data of the Cancer Cell Line Encyclopedia (CCLE). However, experimentally testing permissiveness towards SARS-CoV-2 infection, we found limited correlation between receptor expression and permissiveness. This underlines that permissiveness of cells towards viral infection is determined not only by the presence of entry receptors but is defined by the availability of cellular resources, intrinsic immunity, and apoptosis. Aside from established cell culture infection models CACO-2 and CALU-3, three highly permissive human cell lines, colon cancer cell lines CL-14 and CL-40 and the breast cancer cell line CAL-51 and several low permissive cell lines were identified. Cell lines were characterised in more detail offering a broader choice of non-overexpression in vitro infection models to the scientific community. For some cell lines a truncated ACE2 mRNA and missense variants in TMPRSS2 might hint at disturbed host susceptibility towards viral entry.The SARS-CoV-2 pandemic is a major global threat that sparked global research efforts. Pre-clinical and biochemical SARS-CoV-2 studies firstly rely on cell culture experiments where the importance of choosing an appropriate cell culture model is often underestimated. We here present a bottom-up approach to identify suitable permissive cancer cell lines for drug screening and virus research. Human cancer cell lines were screened for the SARS-CoV-2 cellular entry factors  Within a few months in the midst of spring 2020 the pandemic was accompanied with a high case fatality rate of up to 20% for vulnerable risk groups  and highnd-maps) , probablnd-maps) –6. Durinnd-maps)  and sincnd-maps) . Re-infend-maps) .While various vaccines are rolled out –12, optiCurrent investigations are mainly limited to the VERO-E6 African green monkey cell line, human lung cancer-derived CALU-3 and human colon carcinoma cell line CACO-2. Using non-human cells to study SARS-CoV-2 bears the inherent risk to draw conclusions from findings that result from species-specific cell properties. The anti-SARS-CoV-2 properties of hydroxychloroquine were found using VERO-E6, while later studies detected no effect in human cells and patients , 20. In ACE2 is shown for other tissues such as myocardial cells, esophagus epithelial cells, and enterocytes, which might be related to non-respiratory symptoms observed in COVID-19 patients [ACE2/TMPRSS2-transfected cell lines such as 293T and HELA cells or non-human material such as African green monkey cells VERO-E6 [Human cancer cell lines are easily available from public collections, characterised in great detail, and offer a great range of tissue origins, expression patterns, growth rates, and functional properties to choose from. They serve as valuable and valid model systems to study different diseases , 25 and patients –33. Many VERO-E6 , 34, 35. VERO-E6 .ACE2 and TMPRSS2 expression within the rich resource Cancer Cell Line Encyclopedia (CCLE) publicly available RNA-seq data [In this study, we have pre-screened and ranked human cancer cell lines of the Leibniz Institute German Collection of Microorganisms and Cell Cultures (DSMZ) for seq data . SelecteACE2 and TMPRSS2 in this data set, which enabled us to identify potential susceptible cell lines prone to SARS-CoV-2 virus entry. At first, we selected for 301 human cell lines readily in stock at the DSMZ cell lines repository within this data set and visualised gene expression of ACE2, which was shown to be the cellular receptor for the viral S protein [TMPRSS2 known to cleave SARS-CoV S protein [ACE2 gene expressing cell lines were selected, originating from diverse tumour origins (NRP1) mRNA expression ACE2 and  origins . Importapression , which hnd KELLY . Since tACE2 and TMPRSS2 expression levels, we performed qRT-PCR on the short-listed cell lines. All selected cell lines exhibited considerable ACE2 mRNA expression according to the RNA-seq data and/or qRT-PCR except for DBTRG-05MG and KELLY, whereas TMPRSS2 mRNA levels were particularly higher in CL-40, CL-34, CL-14, and SW-948  data of 100 human leukemia and lymphoma cell lines . HoweverACE2 and TMPRSS2 were detected in cells from multiple tissues including respiratory tract, esophagus and colon [The presence of the entry factors ACE2 and TMPRSS2 in cancer cell lines of various origins is in accordance with their expression in corresponding primary tissue, where nd colon , 43–45 and colon , organs nd colon .Receptor ACE2 and surface proteinase TMPRSS2 have been shown to play a key role for SARS-CoV-2 cellular entry, however, whether their co-expression is sufficient to allow entry and whether alternative receptors may exist, is still under debate . In ordeNRP1 beside ACE2 and TMPRSS2 expression and the colon carcinoma cell line CL-40 have not been identified before. Despite comparable peak titres, virus replication kinetics differed largely between cell lines, where CAL-51 and CACO-2 showed increase in titres throughout day three postinfection while VERO-E6, VERO-B4, and CL-14 produced highest levels within 24h, while the viral titer for CL-40 remained close to the level of the initial inoculum of SARS-CoV-2  showed no detectable virus replication despite the presence of ACE2 and TMPRSS2 , S2 Fig.RS-CoV-2 , Table 1Therapeutic neutralising antibodies bind to the viral Spike protein and prevent the interaction with ACE2 . HoweverNRP1  between viral expression and infective virus titers in the tested cell lines, probably reduced by high variation in viral infection of the cell lines at detection limit. Hence, these data argue for the absence of abortive virus replication or defects in releasing mature virions in the low-permissive cell lines.In order to test for abortive viral cycles particularly in the non-productive virus releasing cell lines, we compared mRNA levels of the nucleoprotein in each three control, high-permissive, low-permissive, and non-permissive cell lines to viral plaques after 24 hpi . We founACE2/TMPRSS2 expression and virus permissiveness of the cell lines, we seeked for genomic differences in the receptors. Of note, gene expression of different splice variants of the ACE2 mRNA became evident comparing the read numbers of the exons as seen in the CCLE RNA-seq data for MOLM-16, CAL-33, BFTC-905, and CAL-27 , CL-40 (heterozygous), CAL-33 (homozygous), and HDQ-P1 (heterozygous)  , namely ACE2  have not been published as such before. Of 29 selected human cell lines three tested high-level positive permissive for SARS-CoV-2. These selected cell lines are available from the German repository DSMZ. Cancer cell lines have been an integral tool for basic research and drug screening for decades and still are the preferred choice for the vast majority of virus research labs. For SARS-CoV-2 infection experiments, different replication kinetics as well as different degrees of permissiveness can aid answering specific questions and allow different experimental setups, e.g. in compound screening assays. We found permissiveness to SARS-CoV-2 in only 14 out of 29 cell lines pre-selected for the expression of the known entry factor CE2 Figs  and 2. TCE2 Figs , Table 1ACE2 expressing cell lines, we closely examined splice and single nucleotide variants. ACE2 splice variants at the N-terminus, containing eight amino acids critical for binding to the receptor binding domain (RBD) of the spike protein of SARS-CoV-2, are predicted to disturb SARS-CoV-2 attachment [ACE2 gene expression ; reads were split, trimmed and reassigned ; mutatigarReads ; and mutgarReads . Mutatiohttps://www.dsmz.de/collection/catalogue/human-and-animal-cell-lines). Tissue origin, doubling-time, growth properties, cytogenetics and more information on these cell lines can be retrieved from the DSMZ website. Cell lines were grown at 37°C in a humidified atmosphere of air containing 5% CO2. The basic growth media  were supplemented with 10–20% fetal bovine serum . No antibiotics were added to the cultures. All cell lines were free of mycoplasma and other bacterial, yeast and fungi contaminations as tested by PCR and microbiological growth assays [The continuous cell lines are part of the cell culture collection at DSMZ (h assays . The auth assays . The celACE2 (Hs01085333_m1) and TMPRSS2 (Hs01122322_m1) transcripts .Quantitative Real-time polymerase chain reaction (qRT-PCR) analysis for human mRNA was performed to quantify specific transcripts in the cells. Total RNA was extracted from cell line samples using TRIzol reagent . cDNA was generated from 1 μg RNA by random priming using the Biozym cDNA Synthesis Kit . qRT-PCR analysis was performed with the 7500 Real-time System, using commercial buffer and primer sets for the amplification of μg RNA were then reverse transcribed to cDNA applying random hexamers. The primers for the qRT-PCR for the amplification of the SARS-CoV-2 N gene were N_Sarbeco_F (5´-CACATTGGCACCCGCAATC-3’) and N_Sarbeco_R (5’-GAGGAACGAGAAGAGGCTTG-3’) [Viral RNA of infected cell cultures was extracted using spin filter membranes  according to the manufacturer’s recommendations. Briefly, cells were lysed in the presence of carrier RNA and Proteinase K at 70°C for 10 min. Nucleic acids were then bound to the surface of the spin filter membranes, washed and subsequently eluted with RNase-free water. One to five CTTG-3’) .We analysed the transcript of TATA box binding protein (TBP) for normalisation of expression levels using SYBR Green for quantification. Quantitative analyses were performed in triplicate, if not indicated differently, analysed and visualised with R/Bioconductor packages ddCt and ggplot2.Antibodies against ACE2 (ab15348), TMPRSS2 (ab109131), and GAPDH (ab8245) were obtained from Abcam . Western blot samples were prepared as described previously . Proteinμl inoculum and 100 μl total well volume.Infection with SARS-CoV-2 was performed with virus strain Zagreb . If not indicated otherwise, cells were seeded onto 96-well cell culture plates and infected 24–48 hours later with 250 pfu/well using a 10 Quantification of SARS-CoV-2 infectious units was done via a virus plaque assay. Supernatants of infected cells were taken 1–4 dpi, serially diluted, and used to infect confluent VERO-E6 cells on 96-well cell culture plates for one hour. Then, the inoculum was removed and cells were overlaid with cell culture medium  containing 1.5% methyl-cellulose. After 3 days, virus plaques were counted from phase contrast microscopic images taken with a Sartorius IncuCyte S3 at 4x magnification.For neutralisation assays antibody COR-101 and virus were mixed and incubated for 1h at 37°C and applied to cells for 24 hpi. COR-101 for was a kind gift of Corat Therapeutics GmbH .S1 FigOriginal western blots to cropped western blot images in (PDF)Click here for additional data file.S2 FigACE2 expressing, two neuronal , and two oral squamous cell carcinoma  cell lines. The red dotted line indicates the initial inoculum (2000 pfu/ml); the blue dashed line specifies the minimal limit of detection LOD 33.3 pfu/ml. Number of replicates varied between 3-8. pfu: plaque-forming units; mean values with SEM.Virus titers in the supernatant of infected cell lines at 1-4 dpi determined by plaque assay for the top 11-25 (TIFF)Click here for additional data file.S3 FigNeutralising SARS-CoV-2 antibody was applied to the high permissive cell lines CAL-51 and CL-14 and the control cell line VERO-E6. With increasing antibody concentration viral production is decreasing in all three cell lines. The red dotted line indicates the initial inoculum (2000 pfu/ml); the blue dashed line specifies the minimal limit of detection LOD 33.3 pfu/ml. Note the logarithmic scaling for the x-axis.(TIF)Click here for additional data file.S4 FigACE2 for the top 25 ACE2 expressing cell lines plus CALU-3. RNA-seq data were visualised via IGV and cell lines ordered alphabetically.Different exon usage of (TIFF)Click here for additional data file.S1 TableACE2 and TMPRSS2 in the cell lines used in this study. These were identified on the basis of CCLE RNA-seq data Click here for additional data file.


Sebaceous glands are adnexal structures, which critically contribute to skin homeostasis and the establishment of a functional epidermal barrier. Sebocytes, the main cell population found within the sebaceous glands, are highly specialized lipid-producing cells. Sebaceous gland-resembling tissue structures are also found in male rodents in the form of preputial glands. Similar to sebaceous glands, they are composed of lipid-specialized sebocytes. Due to a lack of adequate organ culture models for skin sebaceous glands and the fact that preputial glands are much larger and easier to handle, previous studies used preputial glands as a model for skin sebaceous glands. Here, we compared both types of sebocytes, using a single-cell RNA sequencing approach, to unravel potential similarities and differences between the two sebocyte populations. In spite of common gene expression patterns due to general lipid-producing properties, we found significant differences in the expression levels of genes encoding enzymes involved in the biogenesis of specialized lipid classes. Specifically, genes critically involved in the mevalonate pathway, including squalene synthase, as well as the sphingolipid salvage pathway, such as ceramide synthase, (acid) sphingomyelinase or acid and alkaline ceramidases, were significantly less expressed by preputial gland sebocytes. Together, our data revealed tissue-specific sebocyte populations, indicating major developmental, functional as well as biosynthetic differences between both glands. The use of preputial glands as a surrogate model to study skin sebaceous glands is therefore limited, and major differences between both glands need to be carefully considered before planning an experiment. Lef1) serves as an essential regulatory factor controlling lineage differentiation towards either HF or SG cells .,10.Ctnnb no Lef1 A,B. This1  were significantly increased in late SEB in both tissues (Fdft1) differed significantly between the skin and PGs  and sphingomyelinase , are present in SEB of both tissues. However, our data suggest that sphingolipid synthesis in PGs favors de novo synthesis, as Sptlc1 expression levels were significantly higher in pSEB. The most prominent difference was identified in the salvage pathway, as pSEB showed little to no ceramide synthase (CerS) expression but high expression of sphingosine kinase 1 (Sphk1). Together, these findings indicate that whereas sSEB preferentially produces CER, pSEB favors the production of sphingosine-1-phosphate (S1P) from sphingosine. Besides the crucial contribution of ceramide to the establishment of a functional skin barrier, it was previously linked to suppressing steroid hormone production, which is a major function of the PG [Sphk1 and its lipid product S1P were linked to promoting steroid hormone production [Previous studies implicated CER in intracellular as well as stress-induced signaling responses influencing keratinocyte proliferation, differentiation and apoptosis . In termf the PG ,89,90,91oduction ,94,95,96This study has several limitations. The main limitation of this study is the fact that we solely investigated male murine SEB populations of different tissue origins, which are not fully comparable with human data. Furthermore, we focused our investigations on the expression of lipid metabolizing enzymes without further analysis of lipid production. Comparably detailed analyses of the lipid metabolizing enzymes investigated in this study have not yet been conducted in human SG. As other publications showed significant differences in sebum composition of humans and rodents , it is lThree male wild type C57BL/6 mice at the age of 52 weeks were used for this study. Biopsies were obtained from back skin and preputial glands. Skin biopsies, as well as preputial gland samples, were pooled respectively and subsequently processed for single-cell RNA sequencing, protein lysates or histology.6 cells/mL. Subsequently, gel beads-in-emulsion (GEMs) were generated. GEM generation, barcoding, sample clean-up, cDNA amplification as well as library construction were performed in accordance with the manufacturer’s protocol using Chromium Next GEM Single Cell 3′GEM, Library and Gel Bead Kit v3.1, Chromium Next GEM Chip G Single Cell Kit and Single Index Kit T Set A . Final RNA-sequencing, demultiplexing and counting were performed by the Biomedical Sequencing Facility (BSF) of the Center for Molecular Medicine . Obtaining samples and acquiring cell suspensions by Chromium instrument occurred within 4 h. In total, data of approximately 10,000 cells were obtained.Immediately after obtaining the skin biopsies and preputial glands, tissues were enzymatically digested using GentleMACS whole skin dissociation kit  according to the manufacturer’s protocol. Samples were further processed on a GentleMACS OctoDissociator (Miltenyi). The cell suspension was filtered through 100 µm and 40 µm filters and washed with 0.04% bovine serum albumin  in PBS. Cell viability and concentration was assessed using Acridine Orange/Propidium Iodide (AO/PI) Cell Viability Kit  and analyzed on a LUNA-FLDual Fluorescence Cell Counter (Logos Biosystems). Cell concentrations were adjusted to 0.7 to 1.2 × 10p-value cutoff of 0.01 and a minimum enrichment score of 3 were set for GO terms with molecular function and biological process membership.Bioinformatics analyses were carried out using R , R-studio desktop application and the R-Package “Seurat”  ,100,101.Scd1 , anti Asah1 , anti Sptlc1  or Cers4  primary antibody diluted in 2% BSA in PBS overnight at 4 °C. The secondary antibody was diluted in 2%BSA in PBS. Hematoxylin and eosin staining was performed according to routine laboratory procedures. Images were acquired with an Olympus BX63 microscope  with MetaMorph imaging software.For immunofluorescent staining, tissue samples were formalin-fixed, embedded in paraffin and cut into 5 µm thick sections. Paraffin sections were deparaffinized by heating and ethanol dilution series . Antigen de-masking was performed with Dako TRS citrate buffer at pH6 . Sample sections were incubated with anti


Neuroblastoma is a common childhood cancer with poor prognosis. Prior studies suggest that inhibition of molecules called checkpoint proteins, which normally prevent one’s own immune system from attacking itself, has been successfully used for treatment of multiple advanced adult cancers but has yet to be fully explored in neuroblastoma. Cancer can hijack these pathways to prevent the immune system from recognizing and destroying cancer cells. We investigated checkpoint protein expression in pediatric neuroblastoma and its role in drug resistance. We created drug-resistant neuroblastoma cell lines and compared expression of checkpoint proteins between drug-resistant and parental cell lines. In total, 13 checkpoint proteins were expressed by all cell lines regardless of drug resistance. Although PD-L1 and checkpoint proteins do not necessarily impart drug resistance, they may be potential targets for drug therapy. Benchmarking checkpoint proteins provides the basis for future studies identifying targets for directed therapy and biomarkers for cancer detection or prognosis.Neuroblastoma is a common childhood cancer with poor prognosis when at its advanced stage. Checkpoint molecule inhibition is successful in treating multiple advanced adult cancers. We investigated PD-L1 and other checkpoint molecule expression to determine their roles in drug resistance and usefulness as targets for drug therapy. We developed three doxorubicin-resistant (DoxR) cell lines from parental cell lines. Matrigel in vitro invasion assays were used to compare invasiveness. Western blot assays were used to compare PD-L1 expression. Immuno-oncology checkpoint protein panels were used to compare concentrations of 17 checkpoint molecules both cellular and soluble. PD-L1 and 12 other checkpoint molecules were present in all cell lysates of each cell line without significantly different levels. Three were solubilized in the media of each cell line. PD-L1 is expressed in all DoxR and parental neuroblastoma cells and may be a potential target for drug therapy although its role in drug resistance remains unclear. Benchmarking checkpoint molecules provides the basis for future studies identifying targets for directed therapy and biomarkers for cancer detection or prognosis. Neuroblastoma is a common solid malignancy in childhood that often presents with late-stage disease. Therefore, it accounts for up to 15% of childhood cancer deaths with less than 40% survival despite aggressive therapy . For intA novel method successful in the treatment of chemotherapy-resistant cancers in adults is inhibiting immunomodulatory checkpoint molecules (ICMs), which normally serve to prevent the immune system from reacting against healthy cells ,4,5. HowIn regard to pediatric solid tumors, inhibition of checkpoint molecules as targeted drug therapy is still in the early stages. Although a few phase I/II trials are underway, preliminary studies have demonstrated conflicting evidence in regard to which checkpoint molecules are expressed across the various tumor types ,22,23,24Our goal was to explore PD-L1 expression of cell lines of doxorubicin resistance (DoxR) neuroblastoma to investigate its role as a mechanism for drug resistance. Our prior research with doxorubicin-resistant osteosarcoma suggests a correlation between checkpoint molecule expression and drug resistance . This haThe SKN-SH, SKN-AS, and SKN-DZ cell lines were purchased from the American Type Culture Collection . SKN-SH is a human neuroblastoma cell line derived from brain tissue of a 4-year-old female. SKN-AS is human neuroblastoma derived from brain tissue of a 6-year-old female. SKN-DZ is human neuroblastoma derived from brain tissue of a 2-year-old female .®) were obtained from Cell Signaling Technology , and monoclonal mouse anti-β-actin (clone AC-15) from Sigma . Secondary goat anti-rabbit immunoglobin G (IgG)-HRP (W401B) and goat anti-mouse IgG-HRP (W402B) monoclonal antibodies were purchased from Promega . Enhanced chemiluminescence reagents were obtained from Thermo Fisher Scientific .Dulbecco’s modified Eagle’s medium (DMEM) and heat-inactivated fetal bovine serum (FBS) were obtained from Fisher Scientific . Penicillin and streptomycin were obtained from HyClone . Doxorubicin was obtained from Sigma . 3--2,5-diphenyltetrazolium bromide (MTT) was purchased from Thermo Fisher Scientific . Rabbit monoclonal antibodies for PD-L1 (clone E1L3N2). Doxorubicin-resistant (DoxR) cells were generated by incubating parental WT cells with incremental concentrations of doxorubicin ranging from 1 nM to 1 µM over a six-month period. Treatment began with 1 nM and was increased to the next 10-fold increment after surviving five consecutive passages. Cells were considered to be resistant after surviving five consecutive passages in 1 µM doxorubicin. Cell viability was determined by the quantitative colorimetric MTT assay according to Roche (previously Boehringer Mannheim) and purchased from Sigma Aldrich  as previously described [All cell lines were maintained in complete media, which consisted of DMEM with 10% heat-inactivated FBS, 100 units/mL penicillin and 100 µg/mL streptomycin and grown in a humidified chamber . Three fields at 40× magnification were counted by light microscopy  for each experiment. Three biologic experiments were conducted . Invasion was reported as the number of cells on the membrane divided by the number on the control membrane (mean ± standard error).Cell invasion was determined and analyzed using a membrane invasion culture system purchased from Fisher Scientific . The number of cells able to invade through a membrane coated with the defined Matrigel extracellular matrix during a 24 h period was compared to the number counted using a control insert with no Matrigel. Cells were seeded at 2.5 × 10Parental and DoxR cells were seeded in complete medium and cultured for 48 h. Cells were lysed using NP40 Cell Lysis Buffer purchased from Thermo Fisher Scientific  with Protease Inhibitor Cocktail obtained from Sigma-Aldrich . Total protein concentration was determined using the bicinchoninic acid assay (BCA) assay from Thermo Fisher Scientific  using the supplied albumin as the analytical standard. Equal amounts of protein were reduced in 1× sample buffer (Laemmli) from Bio-Rad , with 5% β-mercaptoethanol from Fisher Scientific  boiled for five minutes, separated by electrophoresis on 4–20% Mini-Protean TGX Precast Protein Gels obtained from Bio-Rad  and transferred using the Invitrogen iBlot 2 Gel Transfer Device purchased from Thermo Fisher Scientific , onto nitrocellulose membranes via iBlot 2 Transfer Stacks also purchased from Thermo Fisher Scientific . Proteins of interest were identified with specific primary antibodies followed by HRP-conjugated secondary antibodies. Immunoreactive bands were detected by chemiluminescence with image capture on an iBright CL 1500 Imaging System bought from Thermo Fischer Scientific . Three separate Western blot experiments were conducted.Proteins from cell lysates, lysed using RIPA buffer from Thermo Fisher Scientific  in a 10% protease inhibitor cocktail (as above), were tested for 17 checkpoint proteins using the Human Immuno-Oncology Checkpoint Protein Panel purchased from MilliporeSigma . These were chosen based on prior work with doxorubicin-resistant osteosarcoma and the commercial availability of the panel . Similart-tests. For PD-L1 expression in tumor samples across stages, a Kruskal–Wallis test was used. To compare expression of checkpoint molecules between parental and DoxR cell lines (using results from the Human Immuno-Oncology Checkpoint Protein Panel), Wilcoxon rank-sum tests were used. Statistical differences were determined using p < 0.05 via SPSS 26 .For in vitro invasion assays, categorical variables were compared between groups using chi-square tests. For Western blot analysis, differences between parental and DoxR cell lines were assessed using Student’s unpaired Doxorubicin-resistant cell lines were more resistant than their parental cells as determined by a greater than 100-fold difference in half maximal inhibitory concentration (IC50) than their parental, doxorubicin-sensitive, cell lines based on MTT assays, as shown in p < 0.026) as were SKN-AS DoxR cells compared to parental . SKN-DZ DoxR cells were not significantly more invasive than parental cells , as shown in The invasiveness of the SKN-SH, SKN-AS, and SKN-DZ parental and DoxR cell lines was determined using Matrigel invasion assays and compared. For each cell line, the DoxR cells were more invasive than their parental cell lines. SKN-SH DoxR cells were significantly more invasive than their parental cells (fraction of invasion 0.32 vs. 0.09, The PD-L1 protein level from whole-cell lysates was similar between SKN-SH DoxR and parental cell lines as well as SKN-DZ DoxR and parental cell lines but was higher in the SKN-AS DoxR cell line than its parental cell line, as shown in Cell lysates of SKN-SH, SKN-AS, and SKN-DZ DoxR and parental cell lines each expressed 13 out of the 17 checkpoint molecules for which we tested without significant difference between DoxR and parental cells for any checkpoint protein across all cell lines, as shown in This study is the first to explore cellular and soluble ICM protein expression in chemotherapy-resistant neuroblastoma cell lines. We successfully created drug-resistant neuroblastoma cell lines that were more invasive than their parental cell lines serving as a proxy for advanced disease. Next, we evaluated and confirmed PD-L1 expression in each drug-resistant and parental cell line, demonstrating it unlikely plays a role in facilitating drug resistance. Finally, 12 more ICMs were found to be expressed by all neuroblastoma cell lines, three of which were also secreted into the cell culture media. Benchmarking these ICMs may lay the foundation for other studies to explore checkpoint molecules as biomarkers for disease detection or prognosis or for directed drug therapy.The expression of PD-L1 in each parental and DoxR neuroblastoma cell line suggests that the PD-1–PD-L1 pathway may not play a direct role in the development of drug resistance. Rather, these cell lines likely all express PD-L1, as a mechanism of immune escape and possibly indicate advanced disease rather than a mechanism for drug resistance. However, the fact that it is expressed across all cell lines may make it a promising target for directed drug therapy. This particular protein was chosen because there are already commercially available drugs on the market that target the interaction between PD-1 and PD-L1 for adult cancers. Moreover, preliminary studies regarding its expression and inhibition in neuroblastoma have had conflicting results whether PD-L1 is expressed and if its inhibition leads to tumor regression. Aoki et al. initially reported no PD-L1 expression in NB patients, but was contradicted by Chowdhury et al. who reported a high level of PD-L1 expression which positively correlated with a worse survival ,24. SincThe successful treatment of cancer with inhibition of the CTLA-4, PD-1, PD-L1, and CD80/86 pathways in adults was the basis for our exploration into additional checkpoint molecule expression in neuroblastoma. By better understanding the expression of these proteins in the tumor microenvironment and how they control immune suppression may expand their usefulness as biomarkers or targets for drug therapy into the pediatric population. There are multiple advantages to using our drug-resistant and parental cell lines. It preserves our biorepository, it can direct future study of checkpoint pathways, and comparison between parental and drug-resistant groups may identify mechanisms of drug resistance and metastasis.In total, 15 of 17 ICMs were measurable in this study, 13 in all parental and DoxR cell lines. Similar to our results with PD-L1 via Western blot analysis, there were no significantly different levels between the groups when using our checkpoint protein panel as visually demonstrated in the heat map, as shown in Lastly, we measured the checkpoint protein levels in the cell culture media. Molecules can be shed from cancer cells as a result of exosomal or proteolytic cleavage of membrane-bound forms. In regard to checkpoint molecules, this can induce immunosuppression and cancer survival ,47. In oThis study was not without limitations. First, although there appeared to be different expression of PD-L1 in the parental SKN-AS and DoxR SKN-AS cell lines on Western blot, both expressed PD-L1 when measured via checkpoint protein panel, which is more sensitive and specific. The discrepancy may be secondary to the higher-than-expected molecular weight of PD-L1 on Western blot; however, this molecular weight is not inconsistent with other studies using the same antibody . In addiThis is one of the first studies to evaluate checkpoint molecule expression in drug-resistant neuroblastoma. We demonstrated that PD-L1 is expressed across all cell lines without significant change between parenteral and drug-resistant cell lines; therefore, expression may not be related to drug resistance. We also demonstrated that many other checkpoint molecules are expressed by neuroblastoma in the tumor microenvironment and that some of these are secreted into the cell culture media. Therefore, they may still be useful as targets for further directed drug therapy research or as cancer biomarkers.


The HEK293 cell line has earned its place as a producer of biotherapeutics. In addition to its ease of growth in serum-free suspension culture and its amenability to transfection, this cell line’s most important attribute is its human origin, which makes it suitable to produce biologics intended for human use. At the present time, the growth and production properties of the HEK293 cell line are inferior to those of non-human cell lines, such as the Chinese hamster ovary (CHO) and the murine myeloma NSO cell lines. However, the modification of genes involved in cellular processes, such as cell proliferation, apoptosis, metabolism, glycosylation, secretion, and protein folding, in addition to bioprocess, media, and vector optimization, have greatly improved the performance of this cell line. This review provides a comprehensive summary of important achievements in HEK293 cell line engineering and on the global engineering approaches and functional genomic tools that have been employed to identify relevant genes for targeted engineering. The human embryonic kidney cell line HEK293) was created by transforming human embryonic kidney (HEK) cells with sheared fragments of adenovirus type 5 (Ad5) DNA, immortalizing it. Seventeen percent of the extreme left hand of the Ad5 genome is believed to have been integrated into chromosome 19 of the transformed HEK cells 93 was cr, HEK293FUntil two decades ago, the HEK293 cell line was mainly used for transient, small to medium-scale production of research-grade proteins for scientific and pre-clinical studies. Its desirable characteristics have propelled this cell line into a potential producer of biopharmaceuticals ,12. ThesThe HEK293 cell line is of epithelial origin and adherent in nature. It has been adapted to grow in suspension culture in serum-free or chemically defined media and is currently being utilized for recombinant proteins expression in both adherent and suspension platforms. Adherent platforms include roller bottles ,22,23, mDue to its human origin, HEK293 is better suited for production of biotherapeutics for human use since it produces protein with native post-translational modifications (PTMs). Non-human cell lines, such as the Chinese Hamster ovary cells (CHO), have been shown to implement incomplete humanized PTMs and can introduce potentially immunogenic non-human glycosylation patterns, most notably α-gal and NGNA ,31. PTMsOther human cell lines such as the HT-1080, produced from a fibrosarcoma with an epithelial-like phenotype  and the ® 293 Serum-Free Medium for HEK 293 Cells, and Cytiva’s HyClone SFM4Transfx-293 media.Process and media parameters, such as expression vector, growth media, transfection agent and culture conditions, have been optimized to improved recombinant protein yields from HEK293. Notable innovative approaches include 3D collagen microsphere culture system ; the estSeveral transfection reagents have been optimized for use in transfection of HEK293 with plasmid DNA. For example, the Lipofectamine 3000 reagent by Thermo Fisher Scientific is the superior—but cost-prohibitive—option , while p6/mL for HEK293 vs. 1-2 × 107 for CHO) [Yet, despite these significant improvements in recombinant protein and virus production, this cell line continues to lag behind the CHO cell line in growth capacity  , cultivafor CHO) . ConsequAlthough media and process optimization efforts have resulted in significant improvements in recombinant protein titers, the cell’s inherent biology is the ultimate limiting factor of the amount of recombinant protein that it can produce. The introduction of mutations in chromosomes, such as nucleotide substitutions, gene deletions or insertions, and the overexpression of homologous or heterologous genes can affect the expression of target proteins. Specific genes in cellular pathways involved in protein biogenesis can be temporarily or permanently modified in a targeted manner by using RNA interference, Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9 (CRISPR-Cas9) gene editing systems. Global genetic or strain engineering and whole genome analysis tools, such as random mutagenesis, RNA interference screens, whole genome fluxomic, metabolomic, proteomic, and transcriptomic analyses, have also been employed to identify novel target molecules involved in protein biogenesis. These engineering and analysis strategies are summarized in An efficient approach for the modification of cell lines is targeted engineering of biosynthetic bottlenecks. This approach involves the modification of genes with known effects on protein synthesis, protein folding (including the implementation of PTMs), secretion, and degradation. Typically, inactivating mutations are introduced into genes that negatively impact recombinant protein expression, while genes with a positive effect on recombinant protein expression are overexpressed. Engineering gene targets will be reviewed under the following categories: cell proliferation and apoptosis; central carbon metabolism, glycosylation, protein folding, and secretory pathways. The comparatively long doubling time (~33 h) of HEK293 cells in suspension culture (CHO cell doubling time is 14–17 h) unfavorably affects the rate of recombinant protein production. Cells entering late log and early stationary phases are experiencing decreasing growth rates and ultimately cell death by apoptosis and necrosis . The goaCDKL3), cytochrome c oxidase subunit, Cox15 (COX15) [EIF3I and EIF3C [FGF1) [CDKN) 1B [2C and 1A [CDKN1A, CDKN2C and FGF1, combined with rational vector design and valproic acid treatment resulted in up to 27-fold improvement (from 40 mg/L to 1.1 g/L), in recombinant immunoglobulin G (IgG) expression in a transient system [Proliferation: Growth rate has been modified by either slowing or accelerating the transitions between different cell cycle phases. Increasing the growth rate by overexpressing genes that promote cell cycle transitions, such as the G1/S promoting cyclin dependent kinase like 3 protein ( (COX15) , componend EIF3C , and theC [FGF1) , has sucCDKN) 1B , 2C and C and 1A  has provt system . It is nBAX and BAK [AIF1) genes [XIAP) [NRF2) [BCL2) genes [CrmA in HEK293 cells, conferred protection against apoptosis insults, such as spent medium and Sindbis virus infection, and by the chemotherapeutic reagent etoposide [Apoptosis: Creating cells resistant to apoptosis can potentially increase culture longevity, viable cell numbers, and recombinant protein expression. Apoptosis is under the control of pro- and anti-apoptotic factors whose expression is triggered by nutrient deprivation, low oxygen levels, high osmolarity, or DNA damage. The onset of apoptotic cell death has been effectively delayed in HEK293 cells by down-regulating pro-apoptotic genes, as in the double knock out of effector caspase activators,  and BAK , and the1) genes . Anothers [XIAP) ,63,64, t) [NRF2) , and the2) genes . These mPC) gene [PDK), alongside its activator, the hypoxia inducible factor 1 (HIF1) gene [HEK293 is inefficient at metabolizing glucose ,68,69,70PC) gene ,78,79,80F1) gene . These mIn addition to the production of proteins for therapeutic use, the HEK293 cell line is used for the expression of proteins with proper post-translational modifications (PTMs), such as glycosylation, for structural biology studies . EfficieN-glycosylated proteins [MGAT1) [MAN1A1 and MAN1A2) [MAN2A1 and MAN2A2) and the Alpha--fucosyltransferase (FUT8) [MAN1B1) [MAN1C1), have been knocked out, while those that hydrolyze complex glycans, such as the endo-β-N-acetylglucosaminidase, 8endoT from the fungus Hypocrea jecorina , have been overexpressed [8endoT gene, engineered by Meuris and colleagues [MAN1A1, MAN1A2, MAN1B1, MAN1C1 and MGAT1 genes, produce proteins expressing only high-mannose-type N-glycans [MAN2A1 and MAN2A2 knockout cell line mainly produces hybrid type N-glycans, while the triple MAN2A1, MAN2A2 and FUT8 knockout cell line only produced hybrid type glycans without core fucosylation [Glycosylation: Despite its human origin, challenges exist when expressing glycosylated recombinant proteins in HEK293 for biotherapeutic purposes or structural biology studies, notably the introduction of heterogenous N-glycan glycosylation patterns. Homogenous glycosylation patterns on biotherapeutics are important beyond maintaining protein quality and reproducibility; they also affect folding, intracellular transport, stability, interactions, and clearance ,84. Homoproteins . Consequ [MGAT1) ,87, the  MAN1A2) , alpha-me (FUT8) , the end[MAN1B1) , and thexpressed . Researclleagues  has beenlleagues . Quadrup-glycans . A doublsylation  Sialylatsylation ,92. Thescis/trans isomerases (PPIases), oxidoreductases, protein disulfide isomerases (PDI), and glycan-binding proteins. The X-box binding protein (XBP1), a transcription factor which induces genes involve in the UPR, has also been targeted for engineering. The simultaneous overexpression of the PDI, Protein Disulfide Isomerase Family A Member 2 (PDIA2), the chaperone heat shock protein family A (Hsp70) member 5 (HSPA5), and the XBP1 resulted in improvements of both volumetric and specific gamma retroviral vector production of up to 97% and 92%, respectively [RIC3) [Protein folding and other protein modifications: The folding machinery of producing cell lines is challenged by unusually high quantities of recombinant proteins that are oftentimes not native to the cell. The accumulation of misfolded proteins in the ER may induce the unfolded protein response (UPR), which eventually triggers apoptosis if left unresolved ,94. The ectively . The proy [RIC3) .VKORC1) subunit 1 [CALU) [Gamma-carboxylation (γ-carboxylation) of a cluster of glutamate residues near the amino termini of most clotting factors is required for these proteins to bind Ca++ and function efficiently in blood clotting. The efficiency of this process in HEK293 cells has been increased by overexpressing the rat Vitamin K epOxide Reductase Complex (ubunit 1  gene and1 [CALU) . These eCD9, which enhances exosome biogenesis, [Secretory pathways: Secretion can be limiting factor for host cell productivity, especially in high producing cell lines ,104,105.genesis, . These ea priori hypotheses or knowledge regarding potential bottlenecks in recombinant protein expression. Global genetic engineering approaches typically involve screening a cell line expressing an easily quantifiable reporter protein, such as luciferase or green fluorescent protein (GFP), against an array of gene-specific factors. The screening is followed by evaluation of the effect of individual factors on the expression of the reporter protein. These factors may include RNA species, such as siRNA, miRNA, and shRNA, that bind to and silence homologous RNA sequences, and DNA-binding molecules, such as single guide RNAs(sgRNA) of the CRISPR/Cas9 system, and zinc finger proteins, that bind to and modify DNA in a sequence-specific manner. Global engineering approaches used to query bioproduction in HEK293 cells include high throughput RNA interference screen, omics analysis, randomized zinc finger protein transcription factors, and genome wide CRISPR/Cas9 screens. These strategies are summarized in In addition to the targeting of known genes and pathways, screening and omics analysis approaches have also been utilized in the quest to improve HEK293 culture performance. Such studies employ functional genomic tools to identify novel genes and pathways that limit or enhance the expression of recombinant proteins in HEK293. Identifying genes using this approach obviates the need for RNA interference is a conserved biological process in eukaryotes that regulates the expression of protein coding genes at the post-transcriptional level. This process, which is mediated by two types of small RNA molecules—microRNA (miRNA) and small interfering RNA (siRNA)—is being exploited to selectively silence specific genes for functional studies. High throughput RNAi, which utilizes this process on a larger scale, has been proven to be a fast and unbiased tool for identifying genes involved in cellular processes such as cancer, cell survival, and recombinant protein expression. In this approach, a specific property of the targeted cells is measured while the cells are exposed to a library of microRNA or siRNA. HIPK1) gene was determined to be the most significantly involved in improving recombinant protein expression stimulated by this miRNA. The knockdown of the HIPK1 gene using siRNA resulted in a 3.2-fold increase in FLuc expression and a 2.3-fold increase in GPC3 expression [HIPK1 gene was knocked out by using CRISPR/Cas9 genome editing had up to a 4.7-fold improvement in FLuc expression, while the stable overexpression of miRNA-22-3b resulted in a 2.4-fold improvement in luciferase activity [High throughput micro-RNA (miRNA) screens: Micro RNAs are small, non-coding RNA molecules found in most organisms that function via base-pairing with complementary sequences within mRNA molecules, silencing gene expression. In a 2015 study, our group carried out a mimic microRNA (miRNA) screen to identify miRNAs that can potentially improve HEK293 cell line performance, using the neurotensin receptor as the reporter protein. Cells were screened with a library comprised of 875 human miRNA mimics that resulted in the identification and validation of hsa-miR-22-5p, hsa-miR-18a-5p, hsa-miR-22-3p, hsa-miR-429, and hsa-miR-2110 as potential candidates for improved recombinant protein expression. The treatment of cells with each of these miRNAs led to improvements in expression of membrane neurotensin receptor (NTSR), cytoplasmic firefly luciferase (FLuc), and secreted glypican −3 hFc-fusion protein (GPC3), ranging from 1.1- to 3-fold . In follpression . A stablactivity . High throughput small interfering RNA (siRNA) screens: Small interfering RNA (siRNA) is a class of double-stranded non-coding RNA molecule, typically 20–27 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. These molecules interfere with the expression of specific genes through complementary nucleotide sequences by degrading mRNA after transcription, thereby preventing translation. Synthetic siRNA molecules that silence almost every gene in the genome have been developed for the human and other relevant genomes. Lwa and colleagues executed a targeted high throughput siRNA screen using a panel of 153 siRNA pairs that targeted 153 representative genes involved in the unfolded protein response (UPR), transcription, and cell cycle regulation using HEK293 cells transiently expressing the secreted Gaussia luciferase (Gluc), and gene hits confirmed in erythropoietin (EPO) expressing cells. The KCTD2 and CEBPG genes whose knockdown negatively affected the expression of Gluc were identified as potential engineering targets. Their overexpression enhanced transient production of both Gluc and EPO by 2- to 4-fold in HEK293 cells . INTS1, HNRHPC, OAZ, CASP8AP2 and PPP2R1A consistently improved the expression of at least two reporter proteins tested by up to 72% [OAZ1) resulted in a 5-fold improvement in luciferase production and a 2.5-fold improvement in transient specific secreted alkaline phosphatase (SEAP) production [CASP8AP2) gene resulted in a 7-fold increase in specific luciferase production and a 2.4-fold increase in transient specific SEAP production [On a much larger scale, our group performed a high throughput genome-wide siRNA screen where 21,585 genes were individually silenced with six different siRNAs each in HEK293 cells constitutively expressing the Fluc reporter gene and confirmed in cells expressing secreted GPC3, neurotensin receptor type I and the serotonin transporter. The knockdown of p to 72% . In folloduction , and knooduction . Genome-wide CRISPR/Cas9 screens have emerged as a powerful tool for performing large-scale loss-of-function screens, with low noise, high knockout efficiency, and minimal off-target effects. This approach utilizes the CRISPR-Cas9 gene editing system, coupled with libraries of single guide RNAs (sgRNAs), which are designed to target every gene in the genome. The main advantages of genome-wide CRIPSR screens over RNA interference screens is the ability to achieve highly efficient and complete protein depletion  without S. cerevisiae and secretory alkaline phosphatase (SEAP) from HEK293 cells. By co-transfecting a plasmid-encoding SEAP with randomly chosen plasmids from a ZFP-TF library, and screening individual clones for SEAP production, they identified a single ZFP-TF fused to an activator which improved SEAP production 5-fold, and two ZFP-TF fused to repressors which improved SEAP expression 2- to 3.5-fold. The activator-fused ZFP-TF acted synergistically with either repressor-fused ZFP-TF. These results were reproducible in other human cell lines [ZFP-TF libraries are libraries of zinc finger proteins (ZFPs) with distinct DNA-binding specificities fused to either a transcriptional activation or repression domain that activate or repress specific genes. ZFP-TFs have been screened for various phenotypic changes, such as drug resistance, thermo-tolerance or osmo-tolerance in yeast, and differentiation in mammalian cells . Park anll lines .FABP5) gene and the highly upregulated (66-fold) resistin-like β (RETNLB) gene are associated with the reduced glucose uptake of the producer cell line. Additionally, a pyruvate carboxylase (PC) flux was present in the producer, but not in non-producer cultures. PC overexpression has been previously employed to improve the metabolic efficiency of HEK293 and CHO producer cell lines [XBP1), HSPA5 and HERPUD1, suggesting that the UPR (unfolded protein response) was activated and protein folding capacity may limit recombinant protein production in this cell line. Heterologous expression of XBP1 has previously been shown to increase the specific therapeutic antibody productivity of CH-DG44 cells in inoculum suspension cultures [Another strategy to identify engineering targets for improved expression is omics analysis, which includes genomic, transcriptomic, metabolomic, proteomic and fluxomic. Identification of relevant genes and pathways is done by comparing the omics of homologous (or sometimes heterologous) cell lines exhibiting disparate production characteristics to uncover differentially expressed genes, abundant proteins and consumed or secreted metabolites (both in quantity and rate of consumption/secretion), that could provide insight into potential production bottlenecks. In 2012, Dietmair and colleagues performed a multi-omics study in which they compared the transcriptome, metabolome and fluxome of a stable HEK293 cell line producing a heavy chain variable region fused to the Fc region of human IgG (dAb-Fc), with the non-producing parental cell line . Fluxomill lines ,128. Tracultures , and wasegulated -fold resIn another transcriptomic analysis study, Rodrigues and colleagues compared two human cell lines of different genetic backgrounds, producing a murine leukemia virus-based vector- a high producer Te Fly Ga 18 with a low producer 293 FLEX . Analysis of metabolic pathways showed upregulations in branched chain amino acid catabolism, de novo nucleotide synthesis, polyamine biosynthesis, glutathione mediated detoxification, and folate transformations, in the high producer cell line. By modifying the identified pathways using media manipulation , it was possible to increase the specific productivity of the virus-based vector by up to 600% in the low producing 293 FLEX cell line .In a 2020 proteomic study, Lavado-Garcia and colleagues compared the proteome of HEK293 when growing without transfection, when transfected with an empty plasmid, and when transfected with Gag coding gene producing Gag VLPs, to identify bottlenecks in growth, transfection and VLP production. Identified bottlenecks in transfection and growth included pathways downregulated in transfected vs. untransfected cells, such as lipid biosynthesis, intracellular protein transport, glycosphingolipids metabolism, calcium regulation, oxidant detoxification, xenobiotic metabolism, peptidase activity and DNA detoxification pathways. Endocytosis, late endosomal-related pathways and members of the endosomal sorting complex required for transport (ESCRT) , and neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) proteins), which were downregulated in VLP producing vs. empty plasmid- transfected cells, were identified as bottlenecks in VLP production . NEDD8, NEDD4L and CIT), and the glycosphingolipid precursor enzyme UDP-Glucose Ceramide Glucosyltransferase (UGCG) in HEK293 cells resulted in respective 1.5-, 3.3-, 2.9- and 2.4-fold improvements in VLP production, accompanied by a 51-61% increase in budding efficiency, and a 17% increase in transfection efficacy. In addition, shRNA Knockdown of the Gag-binding protein 2’,3’-Cyclic Nucleotide 3’ Phosphodiesterase gene, CNP, which inhibits viral replication, resulted in a 2.7-fold increase in VLP production and a 36% increase in budding efficiency [In a follow-up study carried out by the same group, overexpression of members of the endosomal sorting complex, , in which the N-acetyl-glucosaminyltransferase I (GnTI) gene has been knocked out and lacks complex N-glycans, and its derivative cell line HEK293SGGD (293SGlycoDelete (RRID:CVCL_6E38), which expresses a heterologous Golgi endoglycosidase for secretion of de-glycosylated glycoproteins. In addition, treatment of cells with small molecule inhibitors such as kifunensine, that targets α-mannosidase I and swainsonine, that blocks α-mannosidase II, results in relatively simple and chemically uniform glycans [Among the targeted engineering strategies discussed in this review, glycosylation is the most critical. Obtaining homogenous glycosylation patterns is practically impossible without genetic manipulation or chemical treatment of the cell line . Two HEK glycans . ®, 2014; ELOCTATE®, respectively) in HEK293-H, the Human-cl rhFVIII (NUWIQ®) produced in HEK293F, the Glucagon-1-like peptide (GLP-1) Fc fusion protein  produced in HEK293 EBNA, while the currently withdrawn drotrecogin alfa (XIGRIS®) was produced in the parental HEK293 cell line [The majority of the currently approved therapeutics are being produced in derivative cell lines, underscoring the relevance of their unique properties; the recombinant FVIIIFc and recombinant FIXFc  produced in HEK293 cell line include Rabies virus G protein VLP, Influenza virus A/Puerto Rico/8/34 (H1N1) VLP, Hepatitis B virus small surface antigen (HBaAgS) VLP, Human scavenger receptor class B, member 2 (SCARB2) VLP; Lentiviral vectors for gene therapy: Kymriah™ (Lymphoblastic leukemia) and ZyntegloT™ ; AAV gene therapy vectors: Glybera™ (Lipoprotein lipase deficiency), Luxturna™ , and Zolgensma™ ; retroviral gene therapy vectors: Rexin-G™ (Pancreatic cancer), Strimvelis™ (Adenosine deaminase—immunodeficiency), Zalmoxis™ (Leukemia) .The choiThe HEK 293 cell line, along with a few other human cell lines, currently serves as a valuable niche for the production of biotherapeutics that require human PTMs. It is also the cell line of choice for the production of recombinant gene therapy vectors. Whether or not this cell line will become as widely used as the CHO cell line in biotherapeutic production will depend on the continued technological achievements and research investments in further optimizing this cell line.


Precision onco-nutrition (PON) is the use of specific nutrients and dietary factors to enhance cancer treatment efficacy and to improve the prognosis for long-term survival. PON requires integration of an understanding of nutrient metabolism with knowledge of the signaling pathways characteristic of each molecular subtype of cancer which can be targeted to improve treatment and control efficacies. Herein we report differences among n-3 fatty acids on the growth and survival of established breast cancer cell lines. Docosahexaenoic acid (DHA) is shown to have greater anticancer activity than eicosapentaenoic acid (EPA). Moreover, we show that a penultimate metabolite of one pathway by which DHA is metabolized inhibited the growth of common molecular subtypes of breast cancer via differential effects on identified canonical signaling pathways and the functions within the cell that they regulate. These findings provide a knowledge base for juxtaposing molecular subtype targeted treatment strategies with the adjuvant use of specific n-3 fatty acid metabolites as an example of precision onco-nutrition for the management and control of breast cancer.In vivo evidence of heterogeneous effects of n-3 fatty acids (N3FA) on cell signaling pathways associated with the reduced growth of breast cancer has been reported and is consistent with the expectation that N3FA will not exert uniform effects on all molecular subtypes of the disease. Similarly, available evidence indicates that many metabolites of N3FA are synthesized by mammalian cells and that they exert metabolite-specific biological activities. To begin to unravel the complex relationships among molecular subtypes and effects exerted by specific N3FA metabolites on those pathways, proof-of-concept experiments were conducted using cell lines representative of common molecular subtypes of human breast cancer. N3FA differed in anticancer activity with docosahexaenoic acid (DHA) having greater anticancer activity than eicosapentaenoic acid. 4-oxo-docosahexaenoic (4-oxo-DHA), a penultimate metabolite of 5-lipoxygenase mediated DHA metabolism, induced dose-dependent inhibition of cell number accumulation with apoptosis as a primary effector mechanism. Interrogation of protein expression data using the Ingenuity Pathway Analysis (IPA) bioinformatics platform indicated that 4-oxo-DHA differentially impacted six canonical pathways and the cellular functions they regulate across common molecular subtypes of breast cancer. This included the endocannabinoid pathway for cancer inhibition that has not been previously reported. These findings provide a rationale for juxtaposing molecular subtype targeted treatment strategies with the adjuvant use of specific N3FA metabolites as an example of precision onco-nutrition (PON) for the management and control of breast cancer. Decades-long efforts to identify individual nutrients and dietary factors associated with differences in risk for cancer have evolved to the elucidation of food patterns associated with changes in cancer risk ,2,3,4,5.From the time of the publication of one of the first most comprehensive reviews of diet, nutrition, and cancer, there has been continuous interest in the role of the amount and type of dietary fats in the development of cancer at multiple organ sites . The worBy shifting the paradigm in the field of N3FA and breast cancer from risk reduction to PON, i.e., from cancer prevention to cancer control, the existing data can be viewed in a new light. Thus, the evidence that has emerged represents a rich resource for developing anticancer metabolites for use in precision oncology. Those findings include: (1) that parent N3FA, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) are metabolized to active intermediates , (2) thaHuman breast cancer cell lines of BT474, MCF7, SKBR3, MDAMB231, and MDAMB468 were obtained from American Type Culture Collection . Primary antibodies used in this study have previously been reported by us for use in Western blot probing of both rat mammary gland and mammary tumors as well as human breast cancer cell lines . Table S2CO3 was added to DHA, 4-oxo-DHA, EPA, or LA at 5 mg DHA/mL 0.05 M Na2CO3. The vials were flushed with N2 and then vortexed extensively. The materials were calculated to make 2.5 mM FA-BSA as a complex at the FA:BSA = 3:1 mole of ratio was needed. DHA (MW:328.5) of 5 mg in 1 mL 0.05 M Na2CO3, BSA :15% Solution. The FA-BSA complex was sterile filtered under a sterile cell culture hood, aliquoted, and stored at −80 °C.Docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and linoleic acid (LA) were purchased from Sigma Aldrich . 4-oxo-docosahexaenoic was synthesized as previously reported ,12. PrioFive human breast cancer cell lines were used in this study: BT474, MCF7, SKBR3, MDAMB231, and MDAMB468. They represent 5 subtypes: luminal A (MCF7), luminal B (BT474), post-EMT  (MDAMB231), human epithelial growth factor receptor 2 (HER2) over-expressing (SKBR3), and basal-like (MDAMB468). Basal-like breast carcinomas are characterized by high expression of basal cyto-keratins, low or absent expression of estrogen receptor, progesterone receptor, and HER2/neu, and expression of epidermal growth factor receptor (EGFR). MDAMB231 and MDAMB468 are classified as triple-negative/basal-like although MDAMB231 has a mesenchymal-like phenotype.2 in DMEM and F-12 medium  (1:1 ratio) containing 10% fetal bovine serum . The next day, cells were provided fresh medium at doses of 0, 25, 50, 100, and 150 µM with DHA, 4-oxo-DHA, LA, or EPA. On days 1, 3, and 5 after exposure, cells were fixed with 1% glutaraldehyde, replaced with phosphate-buffered saline (PBS), and stored at 4 °C. At the end of an experiment, all the plates were stained with 0.02% aqueous crystal violet for 30 min and rinsed with deionized water. After re-dissolving the bound crystal violet in 70% ethanol, the absorbance was determined at 590 nm using a Spectromax Plus Microplate Spectrophotometer System .Cell lines were grown at 37 °C in a humidified incubator containing 5% COn = 6000) were suspended with 100 uL of DMEM and F-12 medium (1:1) ratio containing 10% fetal bovine serum and seeded into flat-bottomed 96 wells. Twenty-four hours after initial seeding, cells were allowed to grow in either the control medium or the same medium supplemented with 25 μM 4-oxo-DHA for 3 days in a 5% CO2 incubator at 37 °C. This dose of 4-oxo-DHA was chosen because it induced a similar degree of inhibition of cell number accumulation across these three cell lines after 3 days of treatment. The cell proliferation was determined by a proliferation assay kit according to the standard manufacturer’s protocol . Absorbance was measured at a 450-nm wavelength with a reference wavelength of 650 nm using a SpectraMax M5 Microplate Reader . The experiments were performed in seven replicates and repeated three times. Cells . To minimize subjective bias, the analyst was blinded to the identity of the treatment sample. At least 200 cells were evaluated per sample. Live and dead apoptotic cells were identified by nuclear condensation of chromatin stained by acridine orange and ethidium bromide, respectively. The quantification of apoptotic cells was calculated as a percentage of the total number of cells counted. The experiments were performed in triplicate and repeated three times.The induction of apoptosis in cultured cells was determined morphologically by fluorescent microscopy after labeling with acridine orange and ethidium bromide, as described by Duke and Cohen . Floating for 10 min at 4 °C and supernatant fractions were collected and stored at −80 °C. Supernatant protein concentrations were determined by the Bio-Rad protein assay . Western blotting was performed as described previously [Three cell lines, BT474 , SKBK3 (Her2 over-expression), MDAMB468 (TN) were treated with 4-oxo-DHA at 25 µm for 3 days and lysed at 4 °C in the lysis buffer . The lysates were centrifuged at 7500× eviously . BrieflyFor statistical analysis of data resulting from Western blotting, the actin-normalized scanning density data obtained from the ChemiDoc scanner using Quantity One (Bio-Rad) were first rank transformed. This approach is particularly suitable for semiquantitative measurements that are collected as continuously distributed data, as is the case with Western blots. The ranked data were then subjected to the non-parametric Kruskal Wallis rank test. Ratio data were computed from the scanning units derived from the densitometric analysis, i.e., the arbitrary units of optical density for variables stated, and then the ratios were rank transformed and similarly evaluated. All analyses were performed using Systat statistical analysis software, version 13.As an independent approach to validating which proteins may mediate the biological effects of 4-oxo-DHA, we used orthogonal projections to latent structures for discriminant analysis (OPLS-DA), a supervised, class-based method where class membership is assigned to samples and used to elicit maximum data separation. Visualization of OPLS-DA Scatter plots of the first two-score vectors for each model were drawn based on Hotelling’s multivariate T2, to identify outliers that might bias the results of OPLS-DA. For OPLS-DA, class separation was shown as the first predictive score plotted against the first orthogonal score to visualize the within- and between-class variability associated with the first principal component. S-plots were constructed to identify influential proteins in the separation of breast cancer cell lines. S-plots based on the first principal component show reliability (modeled correlation) plotted against feature magnitude (loadings or modeled covariance). If proteins have variation in correlation and covariance between classes, this plot will assume an S-shape (giving the plot its name), with heavily influential features separating from other features at the upper right and lower left tails of the feature cloud within the model space. Only proteins influential in distinguishing among breast cancer cell lines are reported. All analyses were done using SIMCA-P+ v.12.0.1 . p-value and FDR value. The resulting data were exported to an Excel file and uploaded to IPA  for further analysis. Phosphorylation Core Analysis was performed in Ingenuity Pathway Analysis (version 68752261) for each cell line. The canonical pathways feature was utilized and a z-score ≥ 2 was the cutoff for a statistically significant activated pathway while z ≤ 2 was the cutoff for a statistically significant inhibited molecule. Comparison analysis across cell lines was used to show predicted activation or inhibition of diseases and biological functions based on z-score. The analysis match feature was used to query potential drug treatments best predicted to work in concert with DHA treatment.For bioinformatic analyses, samples were grouped by experimental conditions, and protein expression data was normalized and log-transformed. Statistical analysis was performed to generate non-phosphorylated, phosphorylated, and ratio  expression values each with associated p < 0.05). On the other hand, DHA showed the greatest inhibitory effects at concentrations ≤ 100 µM with dominant effects at 3 and 5 days of exposure, primarily in BT474 and MDAMB231 (p < 0.01). These data provided a rationale for focusing on DHA.Breast cancer cells representative of prevalent molecular subtypes were used to determine the comparative effects of DHA and EPA on cell number accumulation, that is the net effect of proliferation and death on cell number as a function of concentration and time of exposure. Linoleic acid (LA), an n-6 fatty acid, was used as a negative control and all fatty acids were bound to bovine serum albumin to avoid nonspecific detergent effects of free fatty acids. To facilitate the integration of a large amount of data and to permit the visualization of the effects of fatty acid dose and duration of exposure on the kinetics of cell number accumulation, values were computed as a percent of control and were graphed using an area under the curve (AUC) function. LA had little effect on cell number accumulation; however, at concentrations above 100 µM, cell number decreased rapidly, most likely because of nonspecific toxicity due to increased levels of free fatty acids being released from albumin into the media a. Effectp < 0.001  was minimally affected. Collectively these data are consistent with the induction of apoptosis via the intrinsic pathway although they suggest that the magnitude of induction may be overestimated by the apoptotic index. Under dose and exposure conditions of 4-oxo-DHA that reduce cell number accumulation by 50% (25µM for 3 days) in BT474, SKBR3, and MDAMB468 cells, indicators of cell proliferation and apoptosis were assessed, and cell lysates were Western blotted for proteins involved in the regulation of these processes. For assessment, the results of 8 replicate, independent experiments for each cell line at 0 or 25 µM 4-oxo-DHA were evaluated statistically. Rates of apoptosis induction  and suppression of cell proliferation  were statistically different across cell lines, although this “snapshot in time” needs to be interpreted with caution since there are recognized biases in these estimates. To complement these indices, the levels of proteins involved in these processes was determined by Western blotting . SpecifiThese data were subjected to advanced multivariate regression techniques using orthogonal projections to latent structures discriminant analysis (OPLS-DA) to determine if these differences were sufficient to distinguish among cell lines; complete separation was observed a. The apCollectively, these analyses indicated that apoptosis induction is the driver accounting for inhibition of cell number accumulation across cell lines. A key question emerged during these analyses: were differences in response to 4-oxo-DHA due to distinctive patterns of deregulated cell signaling characteristic of each cell line? To pursue this question, experiments were extended to signaling pathways affected by PPARγ activation and to pathways that have direct effects on cell survival mediated via the mTOR regulatory network based on work reported in .Ser536, phospho-FOXO-3aThr32, Hif-1α, and SIRT-1. As shown in Ser536, Hif-1α, and GADD153 were lower in BT474 and MDAMB468 but unaffected in SKBR3; whereas, SIRT1 was only affected (reduced) in BT474. Given that a considerable body of evidence indicates that N3FA mediate effects on cell signaling via binding to PPARα, β/δ, and γ, Western blot analyses were conducted to determine levels of PPAR β/δ, and γ. PPARα was not measured since it is primarily expressed in hepatic tissue . PPARγ cIn addition, the same approach as described above was used to determine if these differences were sufficient to distinguish among cell lines; complete separation was observed a. The reThe evaluation of insulin-related signaling involved a large number of proteins in the Akt-mTOR-AMPK–signaling network. A summary of the levels of expression for protein components of the mTOR network (IGF-1R and PI3Kp110) and the activity of key regulatory nodes in the mTOR signaling, measured as the ratio of the phospho-specific residue to total protein is provided . The resThe induction of PPARγ activity and suppression of mTOR activity are expected to modulate de novo lipid biosynthesis which is considered a prerequisite for cell growth. To assess the effects of 4-oxo-DHA on lipid biosynthetic potential, cell lysates were blotted for levels of proteins involved in regulating lipid biosynthesis. The effects of 4-oxo-DHA on these proteins and the activity of acetyl-CoA carboxylation, a key control point in fatty acid synthesis is shown for each cell line . The levp-value, the lower the probability that the association between the protein expression set and the canonical pathway is due to chance. In addition, the direction of the differences in expression between the treatment and the control (magnitude and direction) for each protein component was compared to that tabulated in the IPA knowledge basis  that support the canonical pathways that have been annotated and a z-score was computed. A z-score ≤ 2 indicates the pathway is inhibited and ≥2 that the pathway is activated. If the z-score is between −2 and 2, no prediction of inhibition or activation is deduced. The results of the analysis are shown in All protein expression data, i.e., both phosphorylated and non-phosphorylated, were subjected to log2-fold change computational analysis (4-oxo-DHA vs. control) and statistical analysis with false discovery rate (FDR) correction. Those data were evaluated using the IPA Core Analysis algorithm. The components of the protein expression panel were matched for their overlap with the >250 canonical pathways that are annotated in IPA using a Fisher Exact Test with FDR (B-H) correction for multiple comparisons. The smaller the The phospho-proteome data identifies the mTOR signaling network, which integrates signaling effects of AMPK and PI-3 kinase/AKT on mTOR activity as being differentially regulated across BT474, SKBR3, and MDAMB468 even though 4-oxo-DHA suppresses cell number accumulation to a similar extent at 25 µM for 3 days of exposure. Also, a predicted induction of autophagy was observed that also varied in its intensity across cell lines. The non-phosphorylated protein data resulted in the identification of two additional canonical pathways, sirtuin signaling (inhibited) and endocannabinoid signaling associated with cancer inhibition (activated). HER-2 signaling in breast cancer was identified as being inhibited in all analyses in which a prediction was made. The Core Analysis algorithm in IPA also identifies diseases and functions within cells that are consistent with the protein expression data. Those data are shown in heat maps based on the expression of non-phosphorylated and phosphorylated proteins . This daEffects are variable across cell lines. The prediction of effects on cell proliferation (decreased), cell cycle progression (decreased), apoptosis induction, and lipid metabolism are variable but consistent with the data reported . More imGiven the central role of the mTOR signaling network and its differential regulation by cell type in accounting for effects of 4-oxo-DHA as reported , that paThe Analysis Match algorithm in IPA was used to identify published databases from the evaluation of breast cancer cell lines with targeted chemotherapeutics that induced a pattern of expression like that induced by 4-oxo-DHA. Analysis Match compared the pattern of protein expression in the MDAMB468 breast cancer cell line, which is representative of a poor prognosis molecular subtype of breast cancer, with more than 100,000 highly curated and quality-controlled human, mouse, and rat disease and oncology datasets re-processed from SRA, GEO, Array Express, TCGA, and LINCS. This “analysis-to-analysis” matching is based on shared patterns of Canonical Pathways, Upstream Regulators, Causal Networks, and Diseases and Functions. IPA computed a z-score for the match of the “query” signature against the signatures of all other analyses. There are many examples in the literature documenting how a detailed understanding of the chemical species produced during the metabolism of a nutrient or dietary factor within mammalian systems has led to the development of pharmaceuticals that specifically target a cellular function and/or disease process. Prominent examples include natural and synthetic metabolites of vitamin A and vitamin D, but many other examples exist ,19. Beca®, a drug used in the treatment of disorders in lipid metabolism including cardiovascular disease. However, EPA and DHA are now commercially available as single-agent formulations. Accordingly, the data reported indicate that DHA  and two steroid hormone receptor-negative molecular subtypes that vary in Her2/Neu expression status. SKBR3 over-expresses Her2/Neu; whereas, MDAMB468 is a triple-negative cell line. Both molecular subtypes have a poor prognosis. The experiments were performed on cells after three days of exposure to 4-oxo-DHA at a dose of 25 µM, which inhibited growth by 50 percent . The levAs discussed in preceding paragraphs, given the rationale underlying the proteins that were chosen for analysis based on the work reported in , the facGiven that canonical signaling analysis in IPA identified the inhibition of HER-2 signaling in breast cancer, the Analysis Match function in IPA was used to identify databases in the public domain that have been annotated in the IPA knowledge database for patterns of expression consistent with the effects of 4-oxo-DHA on MDAMB468, a poor prognosis molecular subtype of breast cancer with metastatic potential. That analysis was filtered for datasets in LINC in which targeted breast cancer treatment agents were evaluated. The molecular targets and therapeutic agents investigated are summarized in Herein, the focus has been on one metabolite of DHA in a series of proof-in-concept data-driven experiments to provide a foundation for PON. The use of this in vitro approach was purposeful in that the number of DHA metabolites that should be investigated is significant  and coulIn summary, the evidence reported is consistent with anticancer effects of 4-oxo-DHA mediated primarily via induction of apoptosis via the intrinsic pathway. Breast cancer molecular subtypes vary in responsiveness to 4-oxo-DHA, and canonical pathways not previously associated with N3FA activity appear to participate in the inhibition of cancer cell accumulation in culture. The in vitro approach coupled with bioinformatics as reported herein can contribute to the identification of key N3FA species and the molecular targets by which they can be coupled with therapeutic strategies that collectively enhance breast cancer treatment efficacy and improve the prognosis for long term survival. This is the goal of precision onco-nutrition.