Epithelial–mesenchymal plasticity (EMP), encompassing epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET), are considered critical events for cancer metastasis. We investigated chromosomal heterogeneity and chromosomal instability (CIN) profiles of two sister PMC42 breast cancer (BC) cell lines to assess the relationship between their karyotypes and EMP phenotypic plasticity. Karyotyping by GTG banding and exome sequencing were aligned with SWATH quantitative proteomics and existing RNA-sequencing data from the two PMC42 cell lines; the mesenchymal, parental PMC42-ET cell line and the spontaneously epithelially shifted PMC42-LA daughter cell line. These morphologically distinct PMC42 cell lines were also compared with five other BC cell lines  for their expression of EMP and cell surface markers, and stemness and metabolic profiles. The findings suggest that the epithelially shifted cell line has a significantly altered ploidy of chromosomes 3 and 13, which is reflected in their transcriptomic and proteomic expression profiles. Loss of the TGFβR2 gene from chromosome 3 in the epithelial daughter cell line inhibits its EMT induction by TGF-β stimulus. Thus, integrative ‘omics’ characterization established that the PMC42 system is a relevant MET model and provides insights into the regulation of phenotypic plasticity in breast cancer. Breast cancer, a leading cause of cancer death in women, is recognized as a molecularly heterogeneous disease ,2. BreasEpithelial–mesenchymal plasticity (EMP), which encompasses epithelial–mesenchymal transition (EMT) and its reversal , including hybrid states within this spectrum, is considered an important hallmark of cancer that drives metastasis ,18,19. EEMP studies in cancer cell lines have been widely reported. Therein, EMP is either induced by external stimuli, such as hypoxia , TGF-β , EGF 3232, FGF , or by iThe relationships between different levels of regulation  in the intrinsic endogenous plasticity  seen in several EMP systems has not been systematically studied, although each has separately been implicated in EMP regulation. Here, we interrogate and integrate the molecular portrait of PMC42, a unique human breast carcinoma cell line originally established from the pleural effusion of a breast cancer patient in 1983 . OriginaCells  were obtained from the American Type Culture Collection . PMC42-ET and the derivative PMC42-LA cell line were derived from a breast cancer pleural effusion with appropriate institutional ethics clearance  ,42,43,45TM, Thermo Fischer Scientific, Waltham, MA, USA) and antibiotics penicillin and streptomycin , and maintained at 37 °C, 5% CO2, and 95% humidity. Cells were routinely tested and were negative for Mycoplasma.Cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing glucose (4.5 g/L), L-Glutamine (0.5 g/L) and sodium pyruvate (0.1 g/L) , and supplemented with 10% foetal bovine serum  for 3–4 h. Cells were harvested using trypsin (Corning™ 25053CI) and the pellet was gently treated with hypotonic solution (75 mM KCl) for 40–60 min at 37 °C and fixed in cold methanol/acetic acid (3:1). Two or three drops of suspended cells were applied to glass slides and chromosomes were stained with DAPI and imaged by confocal microscopy . Counting was performed manually in ImageJ. Karyotype assessment via G-band analysis was performed by a commercial genotyping service  and 50 metaphases were analysed per cell line.Genomic DNA was extracted from cells using Bioline Isolate II Genomic DNA kit (Cat: BIO-52067) as per the manufacturer’s instructions. After quantifying the DNA and checking the purity, DNA samples were shipped to GENEWIZ  for whole exome sequencing (WES) and subsequent analysis. Genewiz, Inc.  performed initial quality control assessments and subsequent exome capture using the SureSelectXT HS Target enrichment kit (Agilent). All samples were paired-end multiplex sequenced (2 × 150 base pairs) on the Illumina Hiseq 2500 platform to a median target depth of over 50× WES data have been deposited at NCBI under BioProject ID PRJNA557326.Paired-end reads underwent quality control before alignment to the reference human genome (hg19) using Burrows-Wheeler alignment   and SAMtCRAVAT , a tool specifically tailored to analyze cancer-specific variants , was useTM cDNA Synthesis kit from Bioline. Real time-quantitative PCR (RT-qPCR) was performed using the SYBR Green Master Mix in a ViiA7 Real-Time PCR system (Applied Biosystems) and analysis performed using the QuantstudioTM Real-Time PCR software v1.1 . The primers sequences are listed in Total RNA was extracted from cells using TRIzol (Life Technologies) and subsequent reactions were carried out as per the Bioline Isolate II RNA Micro kit manufacturer’s instructions, and cDNA was synthesized using SensiFASTp < 0.01 were also examined by Ingenuity Pathway Analysis® (IPA) for functional annotation and gene network analysis. The GSVA method from the GSVA R/Bioconductor package was also applied on the gene expression data for the PMC42-ET and PMC42-LA cell lines to score samples against the TGFβ-EMT signature.mRNA transcript abundances for PMC42 cell lines were measured using RNA-seq as previously reported ; data haCells were washed with ice-cold phosphate buffered saline (PBS), and lysed directly in cell lysis buffer containing 4% (w/v) SDS, 10 mM dithiothreitol (DTT), 10 mM Tris-HCl along with Roche compete protease and phosphatase inhibitors . Lysates were sonicated to shear DNA, and protein concentration was quantified using the Pierce™ BCA Protein Assay Kit . On the basis of protein quantifications, each experimental sample was aliquoted into 25 μg samples for processing using the FASP method . DigestiProtein Pilot (V 4.1) software from SCIEX was used for peptide identification. The human protein library was built using the UniProt database  with thCells were lifted with Accutase®  and stained with anti-human CD44-FITC (BD Pharmingen) and anti-human CD24-PB (Exbio) antibodies at manufacturer’s recommended dilutions in 0.1% BSA  diluted in DPBS for 1 h in a rotary shaker at room temperature. Cells were analysed in the presence of propidium iodide (1 µg/mL) using a BD LSRFortessa (BD Biosciences). After doublet discrimination and compensation for spectral overlap, data were analysed by using FlowJo Software (BD Biosciences). For TGFβR2 surface expression, cells were stained with primary antibody  as per manufacture recommended dilutions for 1 h and then with secondary goat antibody for 1 h.TM Delta Surface-150687). During immunocytochemistry, the growth medium was discarded, and cells were washed thrice gently with Dulbecco’s modified PBS . Briefly, cells were fixed in 4% paraformaldehyde ± 0.1% Triton X-100 (depending on the desired permeabilization conditions), rinsed with DPBS, and incubated with primary antibodies at 4 °C overnight. After rinsing again in DPBS, cells were incubated with an appropriate fluorescence-conjugated secondary antibody  were seeded at a density of 20,000 cells per well . The cells were allowed to grow for 24–48 h at 37 °C in 5% CO2, after which the cells were washed and replaced with assay media . The cells were incubated for 1 h at 37 °C in a non-CO2 incubator. Mitochondrial complex inhibitors -phenyl-hydrazone (FCCP) and combined 1 µM rotenone with 1 µM antimycin A) were preloaded in the injection ports. For basal rate measurements, ECAR (Extra Cellular Acidification Rate) and OCR (Oxygen Consumption Rate) measurements were assessed. Experiments were performed in triplicates and the data were normalized by cell number. Collagen -coated Seahorse cell culture plates .ESR1, PGR and ERBB2 gene products, showed substantial variation across the cell lines and revealed two major branches  and the expression of these surface markers was simultaneously assessed by FACS analysis  and the MDA-MB-468 cell line . Compared to MCF-7 and T47D, all other cell lines were mainly constituted by cells with high levels of CD44, and except MDA-MB-468 also showed lower proportions of CD24 expression, consistent with their mesenchymal subgrouping , had a ubgroup; ), had higrouping A,B. +/high/CD24−/low cell populations across PMC42-ET, PMC42-LA, MCF-7 and MDA-MB-468 were also assessed after stimulation of the cells with EGF or TGF-β for 72 h. Both MCF-7 and MDA-MB-468 cell lines exhibited a 10%–20% increase in their CD44high/CD24low proportions after treatment with EGF, but not with TGF-β. PMC42-ET cells, however, could be made potentially stem-enriched with 93%–94% population of cells in CD44high/CD24low state after stimulation with either EGF or TGF-β, which was due to increased CD44 expression , TNFA signalling via NFκb , inflammatory response signature  and hypoxia signature  were observed in PMC42-LA with respect to its parental cell line PMC42-ET using GSEA . Of these, 73 proteins were significantly upregulated, and 61 proteins were significantly downregulated by a factor of 2-fold or more in the epithelial PMC42-LA cells. KEGG pathway analysis indicated that differentially regulated proteins were involved in glycolysis/gluconeogenesis  (p = 0.00073), proteasome  (p = 6.06 × 10−8), protein processing in endoplasmic reticulum (p = 3.06 × 10−6) and carbon metabolism  (p = 0.00039), respectively. The volcano plot that shows the difference in protein levels between PMC42-ET and PMC42-LA also highlights several EMT markers, among which mesenchymal markers, such as VIM and EGFR, were upregulated by 4-fold (p < 2.51 × 10−5) and 2-fold (p < 5.83 × 10−5), respectively, in PMC42-ET cells (p < 0.00066) and 4.6-fold (p < 00093), respectively. IPA also deduced glycolysis (with a z-score of 3.5), aryl hydrocarbon receptor signalling (with a z-score of 3.1) and ILK signalling (with a z-score of 1.6) as significantly upregulated canonical pathways in the epithelial PMC42-LA cell line. Gene network enrichment plot from proteomics analysis indicated a possible role of NFκb complex dysregulation in PMC42-LA cells , including numerical CIN (resulting in aneuploidy) and structural CIN  are inherent in cancer and underpin many of the phenotypic manifestations that contribute to cancer progression. Hence, in order to assess their possible contributions to the spontaneous MET seen in the PMC42 system, we explored whether karyotype differences existed in the PMC42 cell lines using metaphase spreads and karyotypic G-banding.r2 = 0.67 and 0.59, Pearson correlation) , and a negative correlation (r2 = −0.51)  was found between chromosomes 9 and 12, in the PMC42-ET cell line (p < 0.0001)  C. Two ob 0.0001) . Second,p-arm of chromosome 9 is fused with the long arm of chromosome 10, and chromosome 21 has a third copy of its p-arm fused with the long arm of chromosome 7 , TP53, TSC2) and an additional mutated tumour suppressor gene ATM in PMC42-ET only. Eight commonly mutated oncogenes (ARNT (TF), EML4, GNAS, NTRK3 (PK), PER1 (TF), TCL1A (TF), TLX1, TTL) and 2 additional mutated oncogenes TAL1 and IL6ST in PMC42-ET only were also identified for their role in tumorigenesis (SNAI2 (K188N) and SOX3 (R22P) (GSN (R397W), WNT1 (T363K), ITGA4 (R565W) and NID2 (G426E) (ESRP2 (R248S) was found to be mutated in the parental PMC42-ET cell line.WES was performed to probe more deeply the genetic aberrations in the PMC42 system. After applying selective filters for delineating deleterious mutations within exons, we identified 465 SNVs in PMC42-ET and 475 SNVs in PMC42-LA A,B. We cH7, CFTR E. We alsH7, CFTR F. The TPigenesis ; Table 33 (R22P)  and othe (G426E) . In addiThe estimated copy number ratios of chromosomal segments for PMC42-ET relative to PMC42-LA are shown in FOXD2, FOXE3 and the chromosome 3 region containing genes GADL1, RBMS3, TGFBR2 were completely lost from the PMC42-LA cell line. The specific functions of FOXD2 and FOXE3 genes are yet to be determined, however the role of TGFBR2 in EMT induction is well established  compared to the parental MCF-7 cells, which do [p-value 1.6 × 10−5). Therefore, we hypothesised that the gain in chromosome 13 upregulated the expression of various genes that drives the signalling mechanism of ER in PMC42-LA, causing it to be represented as an upstream regulator in our comparative IPA findings. The results show a high degree of concordance between the relative transcript and proteome abundance across the PMC42 cell lines (r2 = 0.736). The significant genomic differences in the PMC42 cell lines led us to ask whether the changes in chromosome content mediated changes in transcriptome and proteome that could influence phenotype determination. The relative transcriptome and proteome abundance for a given chromosome across the two PMC42 cell lines were computed from logosome 13 C. PMC42-which do , , MDA-MB-468  and MDA-MB-231, SUM-159 . The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), as indicators of lactic acid production during glycolysis and mitochondrial respiration during OXPHOS, respectively, were measured . InteresAlthough the requirement of MET in metastasis is somewhat controversial ,73,74,75GSN (R397W), WNT1 (T363K), SNAI2 (K188N), ITGA4 (R565W), SOX3 (R22P) and NID2 (G426E)  . SNAI2-np-value of < 0.001) , as a major attenuated pathway between PMC42 cell lines. Significant assessment from in vitro studies have highlighted the significance of various cytokines, such as TNF-α [Comparison of transcriptome and proteome analyses across PMC42 cell lines, combined with GSEA and IPA, were performed to gain insights into the biological processes dysregulated within our model system. In our transcriptome studies, the expression of forkhead box A1 (FOXA1) gene, which acts to control transcription of estrogen receptor-regulated genes and repress the basallike features of breast cancer cells ,85,86,87< 0.001) . These ras TNF-α ,93 and gas TNF-α ,93,94. Furthermore, our recent published work also investigated the dynamic interconversions observed between the transitional epithelial and mesenchymal subpopulations delineated by EpCAM profiling in the predominantly epithelial PMC42-LA breast cancer cells. The subtleties of this transition vary in proportion of epithelial and mesenchymal phenotypes as determined from single cells clonal propagation. Differences observed in the functional attributes of the single cell-derived clones further explains the stochastic nature and the intrinsic cellular plasticity in PMC42-LA. Interestingly, the implementation of whole exome sequencing across the EpCAM-high and -low subpopulation indicates that observed intrinsic phenotypic plasticity in PMC42-LA was not attributable to chromosomal instability . Moreovep-value < 0.0001). PMC42-ET has the higher chromosome number, consistent with a relative increase of its transcriptome and proteome abundance (In this pattern of karyotypic differences reflected in the genomic copy number alterations across the two cell lines, we observed that the allele fractions had significantly deviated in the PMC42-LA daughter cell line (bundance C, a broabundance . The gaibundance C. Strikibundance C. Thus, bundance . Differehigh/CD24low profile as a reflection of a mesenchymal state [high/CD24low profile as compared to the parental PMC42-ET cells. There are other reports of ambiguity regarding functional aspects of these markers [CD44 and CD24 are considered putative stem cell markers ,97 and eal state ,39,100.  markers , suggestALDH1A3, ALDH3A1 and ALDOC between cell lines. The analysis of metabolic phenotypes emphasises the importance of using bioenergetics profiles to decipher phenotype assessment, rather than drawing conclusions solely from mutational profiling, RNA-seq and/or proteome data [Understanding the mechanistic basis of metabolic alterations and their role in tumorigenesis is currently an area of intense research interest ,105,106.ome data ,108.TGFBR2 gene from the derivative PMC42-LA cell line contributes to a reduction in intrinsic TGFB signalling, and these cells are also refractory to TGF-β-induced EMT, however this pathway did not emerge from GSEA analysis. Comparative RNA-seq also demonstrated the PMC42 system to be an authentic MET model. The inter-data relationships illustrate a high degree of concordance between the relative transcript and proteome abundance and chromosome copy number variations across the PMC42 cell lines and identified putative targets to reverse MET. The novel findings provide mechanistic insights into how genomic instability and karyotypic variations have led to acquisition of new autonomous clonal karyotype and phenotype, which is still related to, but distinct from the parental cell line. However, further metabolomics and epigenetics studies might be more compelling to add into the paradigm to determine implications for mesenchymal to epithelial plasticity changes. Overall, this investigation provides an example of the heterogenous changes that may occur in expanding cancer populations and provides evidence for the levels on which these changes occur to affect their phenotypic properties.In conclusion, the data we have presented herein underscore profound differences in the PMC42 system by allowing for a comprehensive integration of whole exome sequencing, RNA-seq, proteomics and karyotyping data. The identification of novel somatic mutations and indels provide insights into the differences between the two cell lines at genetic scale that potentially drive the phenotypic differences from the genomic level. The loss of


EGFR mutational status as a model for genetic profile. Clonogenic assays of EGFR mutant (n = 6) and wild-type (n = 9) non-small cell lung carcinoma (NSCLC) cell lines were performed independently by two oncologists. Clonogenic survival parameters SF2, SF4, SF6, SF8, mean inactivation dose (MID), D10, D50, α, and β were obtained using the linear quadratic model. The differences in the clonogenic survival parameters between the EGFR mutant and wild-type cell lines were assessed using the Mann–Whitney U test. As a result, for both datasets, the p values for SF2, SF4, D50, α, and α/β were below 0.05, and those for SF2 were lowest. These data indicate that a genetic profile of NSCLC cell lines might be predictive for their radiation response; i.e., EGFR mutant cell lines might be more sensitive to low dose- and low fraction sized-irradiation.In the era of precision medicine, radiotherapy strategies should be determined based on genetic profiles that predict tumor radiosensitivity. Accordingly, pre-clinical research aimed at discovering clinically applicable genetic profiles is needed. However, how a given genetic profile affects cancer cell radiosensitivity is unclear. To address this issue, we performed a pilot in vitro study by utilizing  Radiation therapy is one of the most important treatments for cancer. In the clinical practice of definitive radiation therapy, the doses required for clinical tumor control vary widely even for tumors arising in the same anatomical sites. For example, an integrated analysis of 50 single-institution studies performed by Okunieff et al. revealed that the dose required for 50% clinical tumor control ranged from 21.4 to 90.3 Gy for breast cancer, 50.4 to 83.4 Gy for supraglottic cancer, and 24.3 to 64.4 Gy for cervical cancer .EGFR mutant and wild-type cell lines by non-parametric two-sided Mann–Whitney U test. Correlation of the survival data between datasets was examined by Spearman’s rank order test. Differences were considered statistically significant at p < 0.05. Analyses were performed using Prism8 .Clonogenic survival parameters were compared between


Allogeneic hematopoietic stem cell transplantation  is the most effective treatment for various hematological malignancies such as refractory or relapsed leukemia and marrow failure states. The therapeutic benefits of allo-HSCT are mainly derived from an anti-tumor or graft-versus-leukemia (GvL) effect that is mediated by allo-reactive T cells in the donor graft. However, the same donor T cells could also cause graft-versus-host disease (GvHD) by recognizing not only recipient leukemic cells but also healthy tissues and organs as foreign and damage them. Current treatments of GvHD mainly focus on the immunosuppression of the donor T cells via pharmacologic prophylaxis or donor T cell depletion . Given tin vivo administration of JAK1/JAK2-specific inhibitors should significantly reduce GvHD while preserving GvL. As we previously reported in 2012, our group was the first to demonstrate that the administration of ruxolitinib (RUX), a JAK1/JAK2 inhibitor, to MHC-mismatched murine allo-HSCT recipients phenocopies the reduced GvHD potential of IFNGR KO T cells [Accordingly, we have shown that the genetic deletion of interferon gamma receptor (IFNGR) or its downstream chemokine receptor CXCR3 in donor T cells results in donor T cell trafficking away from GvHD organs, thus decreasing the severity of GvHD while maintaining robust engraftment and GvL [ T cells . Of note T cells . Thus, win vivo administration of anti-IL6R antibody to recipient mice are sufficient to completely prevent GvHD [Another major cytokine receptor signaling mediated by JAK1/JAK2 is the interleukin-6 receptor (IL6R) signaling. Therefore, we hypothesized that blocking both IFNGR and IL6R signaling would result in complete prevention of GvHD while potentially preserving GvL. We have demonstrated that genetic deletion of IFNGR in donor T cells plus the ent GvHD . In addient GvHD . Our datent GvHD -5 (FigurGiven the similarity in structures and JAK inhibition profiles of RUX and BARI, understanding the mechanisms underlying the superiority of BARI to RUX will further help guide the rational design of future randomized clinical trials to validate the potential benefit of JAK1/JAK2 inhibitors for the prevention and treatment of GvHD. While both BARI and RUX equally inhibit GvHD-inducing Th1/Th2 cell differentiation and CXCR3 expression, BARI’s superiority to RUX in prevention of GvHD is partially due to a robust upregulation of regulatory T cells by preserving IL2R-JAK1/JAK3-STAT5, and reduction of antigen presenting cells’ allo-reactivity , 8. FurtIn addition to its potent role in GvHD prophylaxis, our data also show that BARI treats ongoing GvHD effectively . Thus, iOur data also show that BARI preserves and even enhances GvL effects . This efAlthough BARI completely prevents and treats GvHD while enhancing the GvL effects in allo-HSCT models, it can inhibit other cytokine/growth factor receptor signaling pathways that are mediated by JAK1 or JAK2, which might result in undesired side effects. Thus, these potential side effects call for the development of more targeted approaches that specifically block IFNGR and IL6R.


Different cellular uptake, DNA repair rate, and/or increased drug detoxification might be some of the reasons; however, the molecules and signaling pathways responsible for these processes are largely unknown. In the current study, we purified the A2 and B2 from the BLM and tested the cytotoxicities and the molecular mechanisms of each individual compound or in combination with six different cell lines, including a Chinese hamster ovary (CHO) cell line defective in glycosaminoglycan biosynthesis. Our data suggested that glycosaminoglycans might be involved in the cellular uptake of BLMs. Moreover, both BLM and A5 shared similar signaling pathways and are involved in cell cycle and apoptosis in different cancer cell lines.Pingyangmycin is an anticancer drug known as bleomycin A5 (A5), discovered in the Pingyang County of Zhejiang Province of China. Bleomycin (BLM) is a mixture of mainly two compounds (A2 and B2), which is on the World Health Organization’s list of essential medicines. Both BLM and A5 are hydrophilic molecules that depend on transporters or endocytosis receptors to get inside of cells. Once inside, the anticancer activities rely on their abilities to produce DNA breaks, thus leading to cell death. Interestingly, the half maximal inhibitory concentration (IC Both pingyangmycin and bleomycin are used in combination with other antineoplastic agents to treat testicular, ovarian, cervical, head-neck, esophageal, thyroid carcinomas and lymphomas ,2. PingyBLM is a mixture of mainly two components, A2 55%–70%) and B2. The structures of A2, B2, and A5 are shown in 0% and B250 of BLMs vary greatly in different cancer cell lines, ranging from low nM to μM [It is well known that the ICnM to μM . SeveralSince a key target of BLMs is the cellular DNA, it suggests that efficient repair of BLM-induced DNA lesions might be one mechanism explaining the observed difference in cytotoxicity. Indeed, increased DNA repair through prolonged doubling time, evasion of G2/M arrest, and reduced apoptosis are observed , HoweverSaccharomyces cerevisiae, several well studied ABC transporters showed no involvement in the efflux of bleomycin [The increased drug detoxification has been proposed to be the second mechanism responsible for the variable cytotoxicity . The mulleomycin . Thus faA third mechanism suggests that BLM-hydrolase, a thiol protease bleomycin hydrolase (BLH1), can metabolically inactivate BLMs in normal and tumor tissues. However, studies from yeast clearly excluded a role played by this enzyme in bleomycin’s cytotoxicity . More reA fourth and highly relevant mechanism that would largely account for BLM resistance is the limited uptake of the BLMs into cells. It is well known that the cellular plasma membrane limits BLM entry ,21. Thisl-carnitine transporter 2 (hCT2) [l-carnitine transporter 2 (hCT2), which normally transports l-carnitine and polyamine, can also transport the polyamine-containing A5, but not A2 and B2 in cancer cells [Several transporters, such as OCT-1, l-carni2 (hCT2)  and olig2 (hCT2)  are proper cells . More imer cells ,27,28, bProteoglycans are proteins that have one or more covalently linked glycosaminoglycan (GAG) chains. GAGs are linear polysaccharides composed of a hexosamine residue  and an uronic acid residue (glucuronic or iduronic acid), except keratan sulfate, which does not contain uronic acid residues. Moreover, iduronic acid (IdoA) residues are enriched in dermatan sulfate or chondroitin sulfate B but are rare in other types of chondroitin sulfates. The quantity and composition of heparan sulfate/chondroitin sulfate (HS/CS) disaccharide vary, not only from cell to cell, but also from tissue to tissue  because GAGs are the major components of “glycocalyx”, a surface layer of glycans that cover all animal cells. The thickness of the glycocalyx is 50 to 100 times more than that of the cell membrane lipid bilayer ,32. HoweIn the current study, we purified A2 and B2 from BLM and tested BLMs’ cytotoxicities and the underlying molecular mechanisms in six different cell lines, including a unique Chinese hamster ovary cell mutant, defective in GAG biosynthesis (CHO745) , which im/z = 472.18, z = 3; m/z = 707.77, z = 2)   B, which , z = 2) C, which 50 values in the six cell lines were summarized in The cytotoxicity of each pure compound, A2, B2, and A5, the natural mixture of BLM , and the artificial mixtures of A2:A5 (2:1) and B2A5 (1:2) were tested in four cancer and two CHO cell lines. We used human colon cancer cell lines, HCT116, because it does not express hCT2, which is a transporter for pinyangmycin. Therefore, HCT116 has no preference for taking-up A5 over BLM through hCT2 . The humOverall, all BLMs were more toxic to the four cancer cell lines than to the CHO cell lines . Among tIn general, BLMs were more toxic to CHOK1 cells than that of CHO745, which suggested that an inadequate amount of cell surface GAGs in CHO745 made it less susceptible to BLMs’ cytotoxicity, indicating that GAGs might be involved in the BLMs’ cellular uptake.Since A5 was more toxic to all six cell lines tested compared to BLM (A2 + B2), especially to the human lung cancer cell line, A549, and the human colon cancer line, HCT116 , we thenIt is known that BLM induces G2/M cell cycle arrest in cancer cell lines ,39,40. T50 values shown in To this end, both A549 and HCT116 cells were treated for 24 h, either with BLM or A5. Since A5 was more toxic to both A549 and HCT116 cells compared to BLM , we chosSimilar results for G2/M cell cycle arrest were also observed in HCT116 cells, after treating the cells with two different concentrations of BLM or A5 B. At theFurthermore, we examined the effects of BLM and A5 on key proteins, including p21, cdc25C, cyclin B1, Bcl-2, Bax, PARP, involved in DNA damage, cell cycle arrest, and apoptotic pathways, in both A549 and HCT116 cells by Western blotting, which was performed on the whole-cell lysates after the treatments, with either BLM or A5 at two different conditions . p21 and cyclin B1 are indicated in response to DNA damage. The upregulated P21 and downregulated cyclin B1 expressions are associated with cell cycle arrest. Indeed, both BLM and A5 induced p21, resulting in the over-expression and downregulation of the cyclin B1 protein level in both A549 and HCT116 cells at two treatment conditions . cdc25C Bcl-2 and Bax are members of the expanding Bcl-2 family that play key roles in the regulation of apoptosis . Bcl-2 iDecades of research have shown that EGFR forms part of a complex signal transduction network, which is essential for many important cellular processes, including cell proliferation, migration, survival and adhesion . EGFR exWe purified  and systResistance to BLMs’ cytotoxicity and BLM-induced lung fibrosis are two major clinical concerns for both BLM and A5 ,48,49. ICHOK1 cells express cell surface GAGs, whereas CHO745 cells do not ,37. Our 6 cells [It has been reported that cancerous tissues express nearly twice as many GAGs as normal tissues, and both GAG quantity and structures are different for both lethal and non-lethal cancers . Most ma6 cells ,54,55,566 cells  when A54GAGs are well documented endocytosis receptors for different types of molecules and CHOK1 and CHO745 are the cell lines mostly used to demonstrate GAGs as endocytosis receptors . GAGs ar50 data  directed against EGFR, such as gefitinib or erlotinib, are among the first molecular-targeted agents to be approved in the US and other countries, for the treatment of various human cancers with over-expressed or over-activated EGFR. However, cancer patients that have benefited from costly EGFR TKI therapy quickly generate resistance to the drug with 70% known ,59,60 anHuman colorectal cancer cell lines HCT116 and HT29 and human lung cancer cell lines A549 and H1299 were obtained from the type culture collection of the Chinese Academy of Sciences (China); Chinese hamster ovary cell lines CHOK1 and CHO745 were kind gifts from Dr. Jeffrey D. Esko . Chinese hamster ovary (CHO) cell lines (K1 and 745) were described in detail during the past by both Esko  and by uAntibodies for cyclin B1, p21, Bax, EGFR, Bcl-2, PARP, β-Actin, cdc25C, Phospho-EGFR  was from Cell Signaling Technology ; PI/RNase was from BD Biosciences ; BCA protein assay kit was from Beyotime Biotechnology Co. Ltd. . A5 was from Tianjin Tai-he Pharmaceutical Co., Ltd. ; BLM was from Nippon Kayaku Co., Ltd. . All of the other chemicals and reagents were analytical or better grade. Spectramax M5 plate reader was from Molecular Devices ; OLYMPUSCKX41 inverted microscope and cell counting chamber were from Shanghai Qiujing Biochemical Reagents Instrument Co., Ltd. ; Cell culture dishes and 96-well standard plates were from Corning ; 0.22 μm Millipore filter papers were from Whatman ; ZORBAX C18-chromatogram column  and the high performance liquid chromatography (HPLC) system were purchased from Agilent Technologies Co., Ltd. ; the FC500 MPL flow cytometer was from Beckman ; and the mass spectrometer was from Thermo Finnegan .18 . Solvent A was 0.08 M acetic acid (pH 4.3); solvent B was 100% acetonitrile. The gradient used was 5%–20% B in 20 min; the column temperature was 40 °C. The flow rate was 0.8 mL/min, the injection volume was 20 μL, and BLM was monitored at 254 nm. A2 and B2 were collected and then the Thermo Scientific LTQ XL mass spectrometer  with electrospray-ionization was used for examining the purity of A2 and B2 in a positive-ionization mode.BLM hydrochloride injection (15 mg) was dissolved in 250 μL double distilled water, separated and collected by using the Agilent 1260 infinity high performance liquid chromatography (HPLC) system. The HPLC conditions were as followings: chromatographic column C2 and 95% air in the incubator. Cells were kept sub-confluent and media were changed every other day. All cells used were between 3 and 30 passages. DMSO was used to dissolve compounds and the final concentration of DMSO in all cell-related experiments was 0.1%.HCT116 and HT29 cells were maintained in McCoy’s 5A media . H1299 and A549 cells were maintained in RPMI-1640 media as described  , A5, A2, B2, A2A5, B2A5  in 200 μL of complete media, and A2 and B2 accounts for 65% and 35% of the total weight of A2A5 and B2A5, respectively. After 48 h of incubation, 20 μL of resazurin  was added to each well. After 16 h of incubation at 37 °C, the fluorescent signal was monitored by using a Spectramax M5 plate reader at 544 nm excitation wavelength and 595 nm emission wavelength. The relative fluorescence unit (RFU) generated from the assay was proportional to the number of living cells in each well. 4 cells/plate). After 24 h of plating, cells were then treated with the BLM or A5 for 24 h with various doses  at 37 °C with 5% CO2 and 95% air in the incubator, and then harvested by trypsinization, fixed with cold 75% ethanol at 4 °C for 24 h and washed with phosphate buffer saline (PBS) twice. The cell pellet was incubated in a solution containing 500 μL PI/RNase at room temperature in the dark for 30 min. The cells were analyzed by using a flow cytometer. HCT116 and A549 cells were plated on 10 cm diameter plates  or artificial mixtures of A2A5 and B2A5 in six different cell lines. Our data suggested that BLMs were more toxic to four cancer cell lines than to the CHO cell lines. For CHO cell lines, BLMs were more toxic to CHOK1 cells (GAG sufficient) than to cells of CHO745 (GAG deficient), indicating that GAGs might be involved in the BLMs’ cellular uptake. Moreover, we demonstrated that both BLM and A5 share the same molecular signaling pathways in conducting their cytotoxicities based on the cell cycling data, the immunoblot analysis of important proteins involved in the cell cycle, apoptosis, and other processes.


This article describes the effect of the oxidative stress inducers Angiotensin II and 6-hydroxydopamine (6-OHDA) on different cell lines. The levels of expression Angiotensin type 1 and type 2 receptors in different dopaminergic cell lines are shown. The data indicate that treatment with Angiotensin II and 6-OHDA increases the production of reactive oxygen species (ROS) and decreases cell viability. NRF2 is a transcription factor induced by ROS. We provide data that NRF2 overexpression increases cell viability in response to oxidative stress inducers compared to control cells, and that these inducers can, both separately and in combination, enhance the expression of NRF2-regulated genes heme oxygenase 1 (Hmox1), NAD(P)H quinone dehydrogenase 1 (Nqo1) and Kruppel like factor 9 (Klf9). Interpretation of these data and additional information is presented in the research article “Angiotensin II induces oxidative stress and upregulates neuroprotective signaling from the NRF2 and KLF9 pathway in dopaminergic cells“   We also22.1Dopaminergic Mes23.5 cell line, dopaminergic N27 cell line and SH-SY5Y cell line were cultured as previously described HA–NRF2, kindly donated by Dr. Donna D. Zhang  was used to infect cell lines. Vectors were transfected into HEK 293 T cells with the corresponding viral packaging plasmids. Viruses were collected from the supernatant and used to infect neuronal cell lines in the presence hexadimethrine bromide . Cells were selected with puromycin  starting 72 h after infection. Stable cell lines were established after 2 weeks of selection and were continuously grown in 5 µg/ml of puromycin afterwards.2.2Cell viability was assessed 24 h after the last treatment: cultures were incubated with MTT  for 4 h at 37 °C. Culture supernatant was then carefully removed and the formazan crystals resulting from cellular dehydrogenases were dissolved in acidic isopropanol and quantified at 570 nm using a plate reader  after gentle shaking. The absorbances were expressed as percentage of control.2.3Nrf2, Nqo1, Hmox1 or Klf9, AT1 or AT2 using Actin as housekeeping gene. Primer sequences are for rat Hmox1 F 5′-TCTATCGTGCTCGCATGAAC-3′, R 5′-CCTCTGGCGAAGAAACTCTG-3′; mouse Hmox1 F 5′-CCGCCTTCCTGCTCAACAT-3′, R 5′-CTGACGAAGTGACGCCATCT-3′; rat Nqo1 F 5′-GCCCGGATATTGTAGCTGAA-3′, R 5′- GTGGTGATGGAAAGCAAGGT-3′; rodent Klf9 F 5′-AGTGCATACAGGTGAACGGC-3′, R 5′-AACGGAACTGCTTTTCCCCA-3′; rat Nrf2 F 5′-CCAGCACATCCAGACAGACA-3′, R 5′-ATATCCAGGGCAAGCGACTC-3′; rodent Actb F 5′-TCGTGCGTGACATTAAAGAG-3′, R 5′-TGCCACAGGATTCCATACC-3’; human HMOX1 F 5′-GAGCCTGAATCGAGCAGAAC-3′, R 5′-AGCCTTCTCTGGACACCTGA-3′; human NQO1 F 5′-AAAGGACCCTTCCGGAGTAA-3′, R 5′-CCATCCTTCCAGGATTTGAA-3′; human KLF9 F 5′-GGAAACACGCCTCCGAAAAG-3′, R 5′-GCCGTTCACCTGTATGCACT-3′; human ACTB F 5′-GGACTTCGAGCAAGAGATGG-3′, R 5′-AGCACTGTGTTGGCGTACAG-3′. Total RNA (without reverse transcription) was used as a negative control. Relative mRNA expression was calculated by using comparative cycle threshold (2−ΔΔCt) method and the relative amount of mRNA was presented in the form of fold change over control.Total ribonucleic acid (RNA) was extracted from cell lines using Trizol . Equal amounts of RNA (2 µg) were reverse-transcribed to complementary DNA with Moloney murine leukemia virus reverse transcriptase  following manufacturer׳s instructions. PCR using real-time iCycler PCR platform  in combination with iQ SYBR Green Supermix  were used to assess expression of 2.4L-cysteine  was used as an antioxidant and control for low ROS generation, and hydrogen peroxide  was used as a control for high ROS generation. Fluorescent intensity was expressed as relative units (RU) calculated as the difference from time 0, normalized relative to control at 15 min.Cellular ROS measurement was determined using 2′,7′-dichlorofluorescin-diacetate (DCFDA). Cultured cells were washed and loaded with DCFDA  for 30 min at 37 °C. Basal fluorescence was measured at this time (time = 0) using a plate reader (Tecan Infinite M200) at excitation 485 nm/emission 520 nm. Fluorescence was again measured 15 min, 30 min, 45 min, 60 min, 75 min and 120 min after addition of different treatments. AngII (100 nM) and 6-OHDA (40 µM) were added at the same time in these experiments. N-Acetyl-2.5two groups were analyzed by Mann-Whitney Rank sum test and multiple comparisons were analyzed with one-way ANOVA or two-way ANOVA followed by Holm Sidak test. Differences at p < 0.05 were considered as statistically significant. Statistical analyses and graph design were carried out with SigmaPlot 11.0  except otherwise stated.All data were obtained from at least three separated experiments, with a minimum sample size of four wells per group and per experiment. Data were expressed as percentage of the control group of the same batch  and expressed as a mean±standard error of the mean (SEM). Data from


Wolbachia suppresses the replication of +ssRNA viruses such as dengue and Zika viruses in Aedes aegypti mosquitoes. However, the range of viruses affected by this endosymbiont is yet to be explored. Recently, novel insect-specific viruses (ISVs) have been described from numerous mosquito species and mosquito-derived cell lines. Cell-fusing agent virus (Flaviviridae) and Phasi Charoen-like virus (Bunyaviridae) persistently infect the Ae. aegypti cell line Aag2 which has been used for experimental studies with both the wMel and wMelPop-CLA strains. Wolbachia was found to restrict the replication of CFAV but not the PCLV infection in these lines. Furthermore, an additional Ae. albopictus cell line (RML-12) which contained either wMel or wMelPop-CLA was assessed. While no infectious +ssRNA or dsRNA viruses were detected, a PCLV infection was identified. These observations provide additional evidence to support that insect-specific, +ssRNA viruses can be suppressed in cell culture by Wolbachia but -ssRNA viruses may not. •Wolbachia suppresses CFAV in Aag2 wMel and wMelPop-CLA cell lines.•PCLV persistently infects the RML-12 cell line.•lbWoLA cachia does not suppress PCLV in Aag2 or RML-12 wMel and wMelPop-Cell lines. Interestus line . Furthermaterial . PCLV, aide CFAV .Wolbachia to Aag2 cells impacted the replication of these viruses, analysis undertaken by wMelPop-CLA for the presence of CFAV and PCLV by RT-PCR. It was suggested by both studies that Wolbachia severely restricts the replication of CFAV. However, upon tetracycline treatment of these lines to clear Wolbachia, contradictory results were obtained where one study suggested CFAV replication could recover after the removal of Wolbachia while the other confirmed clearance of the infection. Interestingly, it was demonstrated that Wolbachia had no effect on the insect-specific bunyavirus (PCLV) known to persistently infect these lines  stably infected with wMel and wMelPop-CLA were assessed for CFAV and PCLV replication both prior to and following tetracycline treatments. CFAV replication was not detected in Wolbachia infected cultures nor was the virus able to be recovered upon passage in the highly permissive RNAi-deficient Ae. albopictus (C6/36) cell line. Conversely, PLCV was detected and isolated from both Wolbachia infected cell lines.To determine if the introduction of se lines . To addr22.1Ae. aegypti cell line Aag2 has been shown to be infected with two insect-specific viruses (ISVs)  . To detey RT-PCR A. While 36 cells . Absence36 cells B.Fig. 1E2.2Wolbachia supressed the insect-specific bunyavirus PCLV in co-infected Aag2 cell culture, supernatant obtained from Wolbachia-free Aag2 cell and Aag2 cells infected with wMel or wMelPop-CLA was screened by RT-PCR. In contrast to what was observed with CFAV, PCLV was detected in the Wolbachia-free Aag2 line as well as both Wolbachia infected lines. To further confirm if this was an active viral infection capable of infecting new cells, supernatant harvested from both Wolbachia untreated and tetracycline-treated lines was inoculated onto the permissive C6/36 line for 2 successive passages. PCLV replication was confirmed by RT-PCR in C6/36 supernatant obtained from passage 1 suggesting infectious PCLV was produced in the Aag2 line.To determine if 2.3Wolbachia infected cell lines, Ae. albopictus, Wolbachia-free RML-12 cells as well as RML-12 cells stably infected with wMel and wMelPop-CLA were assessed. PCLV was detected by RT-PCR in supernatant from RML-12 lines regardless of Wolbachia infection  and replication strategies (–ssRNA or dsRNA) in mosquitoes are yet to be widely explored. Recently, ISVs have been isolated in high prevalence from both wild type and laboratory-reared mosquitoes  previously shown to replicate within the parental Aag2 cell line  cells with supernatant harvested from the Aag2 line with or without Wolbachia. CFAV was not detected by anti-dsRNA ELISA from passaged, wMel, wMelPop-CLA or matched tetracycline treated lines. This study and Wolbachia-mediated suppression, Wolbachia densities maintained in the line. However, as it's likely that the CFAV infection was lost prior to both the screening by this study and that of To further assess these interactions between ell line . The repwMel- or wMelPop-CLA-infected Aag2 or RML-12 lines. Consistent with Wolbachia-infected Aag2 cells and in inoculated C6/36 cells.  and vesicular stomatitis virus  was not affected by Wolbachia (wStri) in Ae. albopictus cells while Aedes anphevirus  could be isolated from both wMelPop-CLA and wMelPop-CLATET cell lines. Furthermore, in vivo studies by Wolbachia did not suppress the replication of LACV in co-infected Drosophila melanogaster and more recently Bunyaviridae) was not restricted by Wolbachia in Culex tarsalis. Taken in combination, this provides mounting evidence to suggest differential viral interactions with Wolbachia whereby +ssRNA viruses are susceptible to suppression but –ssRNA viruses may not. However, while Wolbachia restricted CFAV replication, additional investigation should be undertaken in Ae. aegypti mosquitoes to confirm that –ssRNA viruses and +ssRNA viruses which display different tropism and transmission strategies are suppressed by Wolbachia in vivo. The mechanisms underlying viral suppression remain unresolved. Investigations into the interactions of Wolbachia with other virus types may provide information critical to understand the impact of Wolbachia on the mosquito virome as well as mechanisms of viral blocking.Interestingly, this and 4Wolbachia restricts the replication of the +ssRNA virus, CFAV, but not –ssRNA virus, PCLV, in co-infected cell culture. These results support previous studies which have suggested similar interactions  cells were cultured at 28 °C in RPMI 1640 medium (C6/36) or Schneider's Drosophila/Mitsuhashi and Maramorosch 50:50 medium (RML-12) supplemented with 10% foetal bovine serum (FBS) and L-glutamine. Ae. aegypti Aag2, Aag2 wMel and Aag2 wMelPop-CLA cells were cultured in Schneider's/Grace's insect medium (50:50) containing 10% FBS and L-glutamine at 26 °C. Tetracycline treated lines were generated from Aag2, Aag2 wMel and Aag2 wMelPop-CLA by the addition of 10 μg/ml of tetracycline to the maintenance media for 3 successive passages.C6/36 and RML-12  was inoculated onto semi-confluent C6/36 cells and incubated at 28 °C for 5–7 days. Supernatant was harvested and immediately passaged onto fresh C6/36 cells for an additional passage, with the remaining supernatant used for RNA extraction. Monolayers were then fixed using 4% formaldehyde/0.5% Triton X for 4 h at 4 °C. To determine if ISVs were present in other Aedes-derived, Wolbachia-infected lines, RML-12 cells  were assessed (with the exception of tetracycline treatment) by the methods described above. Fixed-cell ELISA was performed using the anti-dsRNA mAb 3G1 as per previously described methods -treatment of Aag2, Aag2  methods . RNA was


Objective The aim of the study was to evaluate the antiproliferative potential of simple phenylboronic acid and benzoxaborole derivatives as well as to provide preliminary insight into their mode of action in cancer cells in vitro. Methods The antiproliferative activity was assessed in five diverse cancer cell lines via the SRB method (sulforhodamine B) or MTT -2,5-diphenyltetrazolium bromide) method after 72 h of treatment. Further studies of the mechanism of action consisted of the influence of the compounds on cell cycle progression and apoptosis induction, which was assessed by flow cytometry, caspase-3 enzymatic activity, fluorescence microscopy and western blot analysis. Results A clear structure-activity relationship was observed for both groups of compounds with several representatives evaluated as highly active antiproliferative agents with low micromolar 18) and 3-morpholino-5-fluorobenzoxaborole (27) exhibited strong cell cycle arrest induction in G2/M associated with caspase-3 activation in an A2780 ovarian cancer cell line. These events were accompanied by a mitotic catastrophe cell morphology and an increased percentage of aneuploid and tetraploid cells. Further experiments indicated that the compounds were phase cycle-specific agents since cells co-treated with hydroxyurea were less sensitive. The observed cell cycle arrest resulted from significant p21 accumulation and was associated neither with cyclin B1 nor β-tubulin degradation. Conclusion Phenylboronic acid and benzoxaborole derivatives were found to be highly promising antiproliferative and proapoptotic compounds with a cell cycle-specific mode of action. The presented data support their candidacy for further studies as a novel class of potential anticancer agents. Camptothecin  was stored as a 1 mg/mL stock solution in DMSO at −20 °C. Cisplatin  was maintained as a ready-to-use stock solution (1 mg/mL) at room temperature.v/v) FBS and 2 mM L-glutamine . The MCF-7 line was cultured in Eagle’s Minimal Essential Medium  supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 1% (v/v) non-essential amino acids, and 8 μg/mL insulin . All culture media contained antibiotics – 100 U/mL penicillin and 100 μg/mL streptomycin . All cell lines were cultured in a humidified atmosphere at 37 °C with 5% CO2 and passaged twice a week using EDTA-Trypsin solution  as a detachment agent (adherent cell lines only).The 5637  cell line was purchased from RIKEN BRC . The A2780 (ovarian carcinoma), A-549  and MCF7 (mammary gland carcinoma) cell lines were purchased from the European Collection of Authenticated Cell Cultures . The MV-4-11 (biphenotypic B myelomonocytic leukemia) cell line was purchased from the American Type Culture Collection . All cell lines were tested for mycoplasma contamination using Venor GeM Classic  with negative results in all cases. All cell lines are maintained at Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences , Wrocław, Poland. The 5637, A2780 and MV-4-11 cell lines were cultured in RPMI-1640 medium w/GlutaMAX®  supplemented with 10% fetal bovine serum . The MV-4-11 culture medium was additionally supplemented with 1 mM sodium pyruvate . The A-549 cell line was cultured in a 1:1 (v/v) mixture of RPMI-1640 and Opti-MEM  supplemented with 5%  were further prepared using a culture medium containing 0.5 mM HU; in samples described as a pretreatment (± ), after 16 h of HU treatment, the culture medium was replaced with a fresh one without HU.For all 96-well plate-based assays, cells were seeded at a pre-optimized density as follows: 1050  ± standard deviation (SD), which was calculated using the Prolab-3 system based on the Cheburator 0.4 software [At 24 h after seeding the cells in 96-well plates , the tested compounds at concentrations ranging from 200 to 5 μM or cisplatin (10–0.01 μg/mL) were added. At the desired points in time, the plates were subjected to the SRB assay , the tested compounds were applied at various concentrations. At the desired time points, the cells were trypsinized, fixed with ice-cold 70% (v/v) ethanol and analyzed for DNA content according to a previously described procedure , using aAt 24 h after seeding the cells in 24-well plates , the tested compounds were applied at various concentrations. At the desired time points, the apoptosis rate was analyzed using a previously described procedure  with a BAt 24 h after seeding the cells on 50-mm petri dishes , the tested compounds were applied at various concentrations. After 48 h of treatment, the cells were washed with PBS  and lysed with RIPA Buffer supplemented with protease and phosphatase cocktails  for 30 min at 4 °C. After centrifuging the samples , the collected supernatant was assessed for protein content using Pierce™ Coomassie Plus  and stored at −80 °C.Cell lysates  were resolved by SDS-PAGE  under reducing conditions and transferred onto a nitrocellulose membrane  using a semi-dry blotting system . The membrane was washed with Tris-buffered saline  and blocked with 5% skim milk in TBS with 0.1% Tween-20 (TBST) for 1 h at room temperature. Subsequently, the membrane was washed with TBST  and incubated with antigen-specific rabbit IgG antibodies  diluted 1:1000 in 5% BSA in TBST . Next, the membrane was washed with TBST  and incubated with goat anti-rabbit IgG antibodies conjugated to horseradish peroxidase  diluted 1:1000 in 5% BSA in TBST. Following a one-hour incubation at room temperature, the membrane was washed in TBST , chemiluminescent peroxidase substrate was added  and bands were visualized using the blot imaging system .www.imagej.nih.gov) and GraphPad Prism 7.03.Subsequently, the membrane was washed with 10 mM phosphate-buffered saline with 0.05% Tween  and incubated with mouse anti-β-actin IgG antibodies  diluted 1:2500 in 0.5% skim milk in PBST . After washing with PBST , the membrane was incubated with detection HRP-labeled rabbit anti-mouse IgG antibodies  diluted 1:2500 in 0.5% skim milk in PBST. After a one-hour incubation at 37 °C, the membrane was washed  with PBST, and the signal was developed as previously described. The obtained results were analyzed using ImageJ 1.8.0 , the tested compounds were applied at various concentrations. After 48 h of treatment, the cells were gently washed with PBS, fixed with 4% paraformaldehyde  in PBS , permeabilized with 0.1% Triton X-100  in PBS , labeled with DyLight™ 554 Phalloidin  for 10 min and DAPI  for 5 min, visualized using an Olympus IX81 fluorescence microscope equipped with a XC10 camera and analyzed with CellSens Dimension software .p-values less than 0.05 were considered statistically significant.Statistical analyses were performed using GraphPad Prism 7.03. All results are reported as the mean ± SD. One-way ANOVA analyses with the appropriate post hoc tests described in the figure captions were performed. A series of eighteen phenylboronic acid and nine benzoxaborole structurally diverse derivatives was preliminarily evaluated for antiproliferative activity in five cancer cell lines Table . The act1) was almost completely inactive in all cell lines used, even at concentration as high as 200 μM. Among all PBA modifications introduced at the 2-position, the formyl moiety (4) was the only group that significantly increased the biological activity. Further studies indicated that the substituent position affected the antiproliferative potential of the compound. For example, the introduction of the formyl group at position “4” in 11 resulted in an 4 with the greatest increase observed in the A2780 cell line. Concomitantly, modifications utilizing trifluoromethyl group at various positions (6–8) revealed the 4-position as the most promising. Further studies applying various moieties indicated that the mercapto group (9) was the most potent enhancer of 1 activity among the para-modified derivatives in all cell lines. It should be noted that 9 was the only PBA-derived compound that showed at least moderate antiproliferative activity in the MCF7 cell line and was the most active derivative in the A-549 cell line.Phenylboronic acid  did not influence the compound antiproliferative activity or even lowered it significantly in the A-549 cell line . Simultaneously, fluorine incorporation provided 2-fluoro-6-formylphenylboronic acid (18), which showed high antiproliferative activity in MV-4-11, 5637 and A2780. Excluding the markedly less sensitive MCF7 and A549 cell lines, 18 was the most active PBA derivative tested. It is worth noting the striking influence of the fluorine position in 15–18. Compound 16 with fluorine at the 4-position had almost equal activity to 18, whereas 15 with fluorine at the 3-position showed markedly lower activity, especially in 5637 cells (17 (fluorine at 5-position) was more than 10 times less active in the A2780 cell line and approximately 4 times less active in MV-4-11 and 5637 in comparison to 18.Di-substituted derivatives of 20–23. Moderate activity of unsubstituted benzoxaborole (19) was abrogated when fluorine was present at the 4-, 6-, and 7-positions . However, the presence of that substituent at the 5-position (21) resulted in a pronounced rise in activity towards all cell lines, with the highest 21) was the most active benzoxaborole-based compound tested on the MCF7, 5637 and A-549 cell lines. The benzoxaborole substitution at the 3-position (compounds 24–26) had little (on A2780 and A-549 cell lines) or no effect (on MCF7 and 5637 cell lines). MV-4-11 was the only cell line that responded positively to such modifications. Finally, di-substituted 3-morpholino-5-fluorobenzoxaborole (27) proved to be the most active benzoxaborole-based compound in A2780 and MV-4-11 cell lines. None of the tested compounds showed activity comparable to a widely used cytostatic – cisplatin (CDDP) or an additional reference compound – benzyl isothiocyanate (BITC). However, it should be noted that these reference agents plausibly do not share a common mechanism of action with the tested compounds.An even more notable impact of fluorine position on compound antiproliferative activity was observed among benzoxaborole-derived compounds 18 and 27 as the most active representatives of phenylboronic acids and benzoxaboroles, respectively. The A2780 ovarian cancer cell line was used in all subsequent studies because of its high sensitivity to the tested compounds. First, the influence of the compound on the cell cycle was assessed using a standard, RNAse/propidium iodide-based protocol  significantly increased the percentage of cells in G2/M phase even at the lowest 5 μM concentration used (40.4 ± 8.8% in comparison to 21.0 ± 4.6% in the Ctrl), with highest increase observed for 10 μM (59.12 ± 6.56%). In both cases, the G2/M phase arrest was accompanied by a pronounced decrease in G0/G1 and S phase cells percentage and significant increase in the percentage of tetraploid cells (>4 N) – from 0.9 ± 0.1% in the control to 15.6 ± 2.8% in the sample treated with 18 at a 10 μM concentration. Surprisingly, at a high 25 μM concentration, the cell cycle progression arrest caused by 18 shifted towards significant S phase arrest (53.9 ± 6.3% cells in comparison to 18.9 ± 2.8% in the Ctrl) accompanied by almost complete decay of G0/G1 phase (6.8 ± 1.2% in comparison to 59.2 ± 6.9% in the Ctrl). The tetraploid cell population decreased in comparison to the sample treated with 18 at the 10 μM concentration, but they remained significantly higher than in control samples (5.1 ± 2.8% in comparison to 0.9 ± 0.1% in Ctrl). The 3-morpholin-5-fluorobenzoxaborole (27) influenced the cell cycle by significantly increasing the percentage of cells in the G2/M phase and the number of tetraploid cells when A2780 cells were treated with 10 μM (31.6 ± 5.2% cells in G2/M phase and 5.4 ± 2.1% tetraploid cells) and 25 μM (61.3 ± 3.7% cells in G2/M phase and 8.3 ± 0.6% tetraploid cells), but not with 5 μM. In both cases, the G2/M phase arrest was associated with a significant decrease in G0/G1 cell percentage, with the highest of more than 3.8-times decay in the sample treated with 27 at a concentration of 25 μM (13.2 ± 2.6% in comparison to 50.5 ± 6.9% in the Ctrl).Further studies focused on the basic mechanism of action of 18 at 25 μM concentration (9.7 ± 1.9 – fold caspase-3 activity increase in comparison to the Ctrl). Similarly to the cell cycle analysis, 27 caused apoptosis of a slightly lower scope, with a 5.6 ± 1.8 – fold caspase-3 activity increase with 25 μM.Subsequently, the pro-apoptotic activity of the compounds was assessed after 48 h of A2780 cell treatment by caspase-3 enzymatic activity analysis  [18, even at the highest concentration used (25 μM), which caused S, not G2/M phase cell cycle arrest. Compound 27 induced a slightly less pronounced increase in the p21 protein level, but similarly to 18, a clear dose-dependence was demonstrated. Cyclin B1 level was significantly increased when cells were treated with 18 at 10 μM (1.8 ± 0.3 – fold level increase) and 27 at 25 μM (1.8 ± 0.3 – fold level increase), but its level remained equal to the control sample when 18 was applied at 25 μM, which correlates with its ability to suppress cell cycle progression in S phase. β-tubulin level did not change significantly as a result of the compound treatment regardless of the concentration used.To provide further insight into the compound mode of action, β-tubulin, p21 and cyclin B1 protein levels were assessed by western blot analysis after 48 h of A2780 cell treatment with various compound concentrations   or as a clin B1) . A remar2/M phase, the ability to induce formation of the tetraploid cell population, and the ability of compounds to induce apoptosis was observed. This finding indicated that the compound mode of action was associated with mitotic catastrophe processes, characterized by aberrant mitosis, mitotic cell death, and as a result of aberrant mitosis slippage, the presence of aneuploid and tetraploid cells [A clear correlation between cell cycle arrest in Gid cells . To addr50 assessment at various time points indicated that prolonged treatment was necessary to reveal the high antiproliferative potential , similarly to the cell cycle specific cytostatic – cisplatin  and in contrast to benzyl isothiocyanate (BITC), which had similar IC50 values after 24, 48 and 72 h of treatment , irrespectively of the concentration used (representative results shown). Such a negative effect was not observed when HU co-treated cells were additionally treated with camptothecin (40 ± 5.8 – fold change compared with the untreated control). In the samples that were pre-treated with HU, the ability of 18 and 27 to induce apoptosis was also significantly reduced. Particularly in the case of 27 at 25 μM, which caused a 3.8 ± 0.8 – fold increase in caspase-3 activity in HU pre-treated cells (2.1 ± 0.4 – fold change for HU pre-treated control cells) in contrast to 6.2 ± 2.0 – fold change in untreated cells.A strong correlation between the compound-induced cell cycle inhibition and apoptosis rate raised the question of whether both events were not only correlated but also dependent. Additionally, the compound ICial Fig. a. The IC S phase . Pre-tre18 and 27 caused a significant increase in G2/M and > 4 N cell percentage, whereas these effects almost completely disappeared when samples were co-treated with HU and were significantly reduced when cells were pre-treated with HU. The described correlation was observed regardless of the used concentrations of compounds (representative results are presented). This phenomenon was most striking for 27, which applied alone at 25 μM caused a massive accumulation of cells in G2/M phase (64.4 ± 1.3% cell percentage) in comparison to the untreated control (22.6 ± 3.5% cells); however, after concurrent usage with HU, its impact was abolished (16.5 ± 2.0% cell percentage in G2/M). Additionally, HU treatment significantly reduced the capability of the tested compounds to induce tetraploidy, e.g., compared with the 16.8 ± 2.5% >4 N cell percentage originally observed for 18-treated cells (at 10 μM), only 3.2 ± 0.3% cells were observed following co-treatment with HU (1.0 ± 0.1% cell percentage in untreated control). Taken together, the above-described observations clearly indicate that phenylboronic acid and benzoxaborole derivatives act as G2/M phase cycle-specific pro-apoptotic agents.The cell cycle analysis confirmed the above-mentioned observation Fig. d, e, f. 1) anticancer properties of phenylboronic acid were studied only by the Plopper group [1 as a cell growth and migration inhibitor, but the required compound concentrations to observe a biological effect exceeded 500 μM in most cases. Recently, moderate anticancer activity of PBA was described in 4T1 murine mammary gland adenocarcinoma and SCCVII squamous carcinoma but also at high concentrations [The first boron-containing anticancer drug was bortezomib – a dipeptide boronic acid that was featured as a strong proteasome inhibitor and approved by FDA for multiple myeloma treatment in 2003. Since then, much effort was undertaken to search for novel boron-containing compounds that are useful in medicinal chemistry, resulting in a large set of bioactive molecules at various stages of development  . In the er group , 37. Thetrations .1 led to highly active phenylboronic acid-derived compounds such as 9, 16 and 18 with low micromolar 9 was observed in all cancer cell lines among all para-substituted derivatives, which indicated that resonance or inductive effects might play an important role in this matter since the mercapto group was the only electron donating group tested. Moreover, compound pKa modulation appeared to be crucial, at least in the case of the di-substituted compounds 15–18, of which the least active was 17 with a pKa = 6.72; the other compounds exhibiting pKa = 5.74, 6.42 and 6.05  were si2/M phases identified as those targeted by 18 and 27.Notable differences were observed in the sensitivity of the cell lines to the tested compounds, with MCF7 breast cancer cells recognized as generally resistant, and ovarian cancer (A2780) as well as leukemia cells (MV-4-11) identified as the most sensitive. The molecular origin of the observed discrepancies remains unknown but identifies biological activity of the compounds as a result of interactions with specific molecular targets rather than their overall toxicity. Moreover, our studies of HU-treated A2780 cells clearly indicated that the compound activity was focused on proliferating cells, with cells in S and/or G21) already approved for the treatment of onychomycosis as the most spectacular example [21), a derivative comprising a single fluorine atom as a substituent.Since the discovery of the sugar-binding properties of benzoxaboroles in 2006 , this cl example . This in example , but fur example , whereas example , similar example . In bothKa lowering from 7.39 and 7.42 for 19 and 23, respectively, to 6.97 observed for 21 was accompanied by a significant increase in biological activity. However, 20 and 22 showed poor antiproliferative potential despite their even lower pKa  [In our studies, we not only observed relatively poor activity of the tested benzoxaboroles in MCF7 but also in some other cancer cell lines (data not shown). Concomitantly, several tested compounds exhibited high antiproliferative activity in A2780 and MV-4-11 cell lines, and, to a lesser extent, the 5637 urinary bladder cell line. Similar to PBA derivatives, the acidity of the compounds appeared to play an important role since pctively) , indicatctively) , which m2/M cell cycle arrest was observed for various cell lines, including DU-145 and PC-3 prostate cancer cells after treatment with 1 [D,D-carboxypeptidase and leucyl-tRNA synthetase (LeuRS) inhibition [21 and some of its derivatives in human osteosarcoma (U2OS) and ovarian cancer (SKOV-3) cells. The apoptotic morphology associated with the ambiguous transcriptional activity of the p21 promotor and no significant impact on the cell cycle was reported [The mode of antiproliferative activity of phenylboronic acids is largely unknown. Inhibition of GTPases by the Rho family in the DU-145 prostate cancer cell line was suggested by McAuley et al. as a plausible mechanism . Additiot with 1 . The antreported .18) and 3-morpholine-5-fluorobenzoxaborole (27) – and the A2780 ovarian cancer cell line were used. A very evident cell cycle arrest at G2/M phase associated with a significantly increased level of p21 protein probably resulted from the binding of p21 to cyclin B1-CDK1 and its inhibition. p21 acts as a cyclin-dependent kinase inhibitor that binds to various cyclin-CDK (cyclin dependent kinase) complexes and regulates cell cycle progression [18 to induce S phase arrest at a concentration of 25 μM. Such activity could also result from p21 binding to the proliferating cell nuclear antigen (PCNA) that is also responsible for S phase arrest [In the present work, we performed a preliminary evaluation of the mechanism of action underlying phenylboronic acid and benzoxaborole derivative antiproliferative activity. The two most potent representatives – 2-fluoro-6-formylphenylboronic acid , a significant increase in cyclin B1 was accompanied by a high level of aberrant tetraploid cells – plausibly a result of failed mitotic death that led to mitotic catastrophe slippage and an increased number of multinucleated cells. Such cells are increasingly susceptible to cell death with every cell cycle progression and eventually undergo apoptosis [Another possible mechanism underlying such strong Glchicine ) or α- alchicine ); howevepoptosis .2 phases. The upstream events caused by 18 and 27 that led to the above discussed image of A2780 cells remain unknown, but based on these preliminary studies, establishment of the exact mechanisms of action of phenylboronic acid and benzoxaborole derivatives should be possible in the future. Cell line features responsible for the specificity of the observed compounds will also be an important part of further studies.In conclusion, we identified phenylboronic acid and benzoxaborole derivatives as potent antiproliferative agents acting as cell cycle arrest and apoptosis inducers. Great possibilities for further modifications makes them a promising new class of anticancer agents. Additionally, the tested compounds were evaluated as phase cycle-specific agents with activity focused on proliferating cells in late S/early G


Dear Editor,Globally, gastric cancer is the most common malignant tumor and the second highest contributor to cancer deaths after lung cancer . Further, expression levels of let-7i were lower in gastric cancer cell lines  as compared to that in normal gastric epithelial cells GES-1, while no significant difference in this respect was observed in the MKN-45 cell lines , and lymph node metastasis . The effects of let-7i restoration on regulation of gastric cancer cell vitality and cell proliferation were assessed by transfecting let-7i mimic or miRNA negative control into two human gastric cancer cell lines, SGC-7901 and MGC-803, which have relatively lower levels of let-7i expression. As expected, ectopic let-7i expression markedly suppressed viability of SGC-7901  as assessed by use of cell counting kits. Furthermore, over expression of let-7i also reduced proliferation of both SGC-7901 , 48 h after transfection, as revealed on colony formation assay. Overexpression of let-7i also reduced invasive and migratory ability of both SGC-7901 . These findings suggest that let-7i reduced cell viability and proliferative ability and inhibited invasive and migratory properties of gastric cancer cells in vitro. SGC-7901 cell lines which stably expressing let-7i and miRNA-control were subcutaneously injected into the dorsal flank of nude mice to evaluate the in vivo effects of let-7i on gastric cancer tumor growth. The result showed reduced tumor volume and tumor weight in nude mice with let-7i mimic injection , foundation of Jilin Province Science and Technology Department (172408GH010234983), and Jilin University-Xinjiang Medical University joint research project.Yue Shi, Zipeng Duan, Xun Zhang, Xiaotian Zhang, Guoqing Wang and Fan Li declare that they have no conflict of interest.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study.All institutional and national guidelines for the care and use of laboratory animals were followed.Supplementary material 1 (PDF 614 kb)Below is the link to the electronic supplementary material.


Background: Cutaneous squamous cell carcinoma (cSCC) incidence continues to rise with increasing morbidity and mortality, with limited treatment options for advanced disease. Future improvements in targeted therapy will rely on advances in genomic/transcriptomic understanding and the use of model systems for basic research. We describe here the panel of 16 primary and metastatic cSCC cell lines developed and characterised over the past three decades in our laboratory in order to provide such a resource for future preclinical research and drug screening. Methods: Primary keratinocytes were isolated from cSCC tumours and metastases, and cell lines were established. These were characterised using short tandem repeat (STR) profiling and genotyped by whole exome sequencing. Multiple in vitro assays were performed to document their morphology, growth characteristics, migration and invasion characteristics, and in vivo xenograft growth. Results: STR profiles of the cSCC lines allow the confirmation of their unique identity. Phylogenetic trees derived from exome sequence analysis of the matched primary and metastatic lines provide insight into the genetic basis of disease progression. The results of in vivo and in vitro analyses allow researchers to select suitable cell lines for specific experimentation. Conclusions: There are few well-characterised cSCC lines available for widespread preclinical experimentation and drug screening. The described cSCC cell line panel provides a critical tool for in vitro and in vivo experimentation. Cutaneous squamous cell carcinoma (cSCC) is a common and growing problem, with over 45,000 cases per year in the UK in 2015 ,4. AlthoA total of 16 cell lines were derived from 11 patients with cSCC attending dermatology outpatient clinics. Five of these 11 patients were immunocompetent individuals  and six of these 11 patients were immunosuppressed male organ transplant recipients (OTR) aged 51–87 years and 45–67 years, respectively, at the time of biopsy . One celCell morphology representative of nine of the 16 lines is shown in Fourteen of the 16 cell lines were investigated in xenograft models. The occurrence and growth rate of tumours developing after subcutaneous injection varied between cell lines A. PalpabFifteen STR loci plus amelogenin were genotyped to give tumour-specific profiles . These pWhole exome sequencing (WES) was performed on the 15 lines, for which matched normal samples were available (GSE98780) . Our res−4), chromatin modification , RNA splicing , and the mRNA metabolic process , were significantly overrepresented for these 58 genes, highlighting the potential involvement of these pathways and processes in driving or significantly contributing to cSCC metastasis.The non-silent mutations identified were used to construct evolutionary trees for the two isogenic cell line sets, primary cSCC IC1 and its metastasis IC1MET, as well as for the three related (MET1/MET2/MET4) and two unrelated lines (PM1/T9) derived from the single immunosuppressed transplant recipient male patient A. A majop < 0.0001). In contrast, the proportion of other mutations became less abundant. In particular, there was a >10-fold increase in A > G/T > C transitions during the tumour progression, representing more than 20% of all late mutations for both series (https://cancer.sanger.ac.uk/cosmic/signatures), which often consist of A > G/T > C substitutions, became more dominant after the tumours are fully established and during the tumour progression. Although signature 7 (UV light exposure) remained the most dominant signature throughout, its influence became important after the full establishment and during the progression and metastatic stages.We next investigated the mutational signatures (with respect to transitions and transversions) for early and late mutations, and aimed to identify any significant differences between them. As shown in h series B. This sp-value ≤ 0.01), including known cancer drivers .We then explored the methylation characteristics of six cSCC cell lines  using genome-wide DNA methylation microarray. The cSCC lines were hybridised to the same chip with three normal human keratinocytes (NHK) to account for possible batch effects. Genome-wide methylation profiles reflected the original histologies (cSCC vs. NHK) and also differentiation status subtypes of cSCC based on Pearson’s correlation . Cell liWe then explored the potential enrichment of subsets of differentially methylated genes. Gene-set enrichment analysis revealed significant enrichment in several canonical pathways including MAPK, JAK-STAT, insulin signaling and the p53 pathway. P53 is known to play a critical role in cSCC oncogenesis, and genes involved in p53 signaling, IGF1, IGFBP3, CASP8, SERPINB5, were identified as being differentially methylated. This suggests that the genome-wide methylation profile retained by stable cell lines maintains the original tissue features and reflects the methylation changes in processes inherent to cSCC onset and progression.We describe for the first time a unique panel of 16 cSCC lines which have been extensively characterised in terms of clinical and histological phenotyping; genotyping by STR DNA profiling, exomic sequencing, copy number alteration analysis and phylogenetic analysis; in vitro cell morphology, growth, transwell migration, organotypic invasion; and in vivo tumourigenicity. This panel includes multiple isogenic lines and lines which represent the spectrum of tissue sites and histologies, and is a unique resource for preclinical cSCC research. Indeed, to date, a few of these lines have been used individually in multiple collaborative research efforts .There are few well-established cSCC cell lines. An online search using the cell line catalogue databases ATCC, the European Collection of Authenticated Cell Cultures (ECACC) and the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) revealed that the only SCC lines listed are of head and neck origin. As far as we are aware, the only cutaneous SCC line widely available commercially is A431. This line is of vulval origin ,9,10 andCancer-derived cell lines lack the critical influence in vivo from the tumour microenvironment, but this can be mimicked in organotypical cultures and subcutaneous xenografts. We present the optimal lines from our series, which produce early xenografts and highlight the range of individual tumours from multiple mice in each of the key lines. This illustrates that the optimal line for consistent rapid tumour growth for drug and therapeutic screening appears to be IC1MET. The remarkable conservation of the pathological changes found in the primary tumours is shown in the photomicrographs. Particularly striking are the hyperkeratotic lesions from a field of actinic dysplasia . As the xenografts are in SCID mice, this clearly provides a permissive environment for tumour development, which allows the intrinsic pattern of tumour differentiation to develop. However, in our published studies of organotypical cultures using keratinocytes and fibroblasts from patients who develop cSCC due to the rare genetic disease recessive dystrophic epidermolysis bullosa (RDEB-type VII collagen deficiency), RDEB cancer-associated fibroblasts (CAFs) increased the adhesion and invasion of both tumour (including IC1) and non-tumour keratinocytes . It woulThere have been a number of potential problems in the use of cSCC cell lines derived from tissue collections or by multiple partnering labs using those cell lines for experimentation. For example, analysis of 122 HNSCC lines  authenticated only 85 unique cell lines by STR DNA profiling within this collection and highlighted misidentification and cross-contamination as common problems 3, where L is length and W is width. The time points which ended tumour sampling were determined by the point at which the tumours reached the size requiring sacrifice of the animal (800 mm3) or developed ulceration.Female SCID Balb/c mice or male CD1 nu/nu mice were subcutaneously injected in the right flank with 4 × 10DNA was extracted from cultured cells and blood using the Qiagen DNA Mini kit  according to the manufacturer’s instructions. Quantitation was performed using the double-stranded DNA-specific Qubit dsDNA BR assay kit in conjunction with the Qubit 2.0 fluorometer . Short tandem repeat (STR) genotyping was performed to authenticate the unique identity of each cell line. Sixteen loci distributed across the human genome were amplified using the AmpFLSTR Identifiler PCR Amplification Kit followed by capillary sequencing on the 3730xl DNA Analyzer . For SNP 6.0 analysis, DNA samples from cultured cells and paired non-tumour blood samples were hybridised to the microarrays according to the manufacturer’s protocols . Exome sequencing was performed by Oxford Gene Technology (OGT), using Agilent SureSelect All Exon v5  for exome capture. Briefly, 1 µg DNA from each sample was used to prepare the sequencing library through shearing of the DNA followed by ligation of sequencing adaptors. Sequencing was performed on the Illumina HiSeq platform . Paired-end sequencing (2 × 100 bp) was carried out using HiSeq sequencing instruments.The WES data generation and full analytical procedure has been described previously in detail (accession number GSE98780) .Two independent approaches were applied to the copy number analysis of WES data. First, to generate single nucleotide polymorphism (SNP) and indel variant genotyping information, the tumour-normal pair was processed against the reference genome using the VarScan2 germline variants calling method. The minimum coverage for identified sites was 10 reads for both tumour and normal. Next, the logR and BAF  files were created based on the tumour-normal pair genotyping information, with the depth information normalised by dividing the depth of each variant by the median depth across all variants. The ASCAT R package  was thenhttp://evolution.genetics.washington.edu/phylip.html). The non-silent mutations were used for this analysis. The mutations in the tree trunk were defined as “early” events, and the mutations in the branches after the tree split were “late” events.For two patients where multiple tumour cell lines were generated and sequenced, evolutionary trees were constructed on the basis of distance matrix among normal control and multiple cell line samples, using the neighbour-joining algorithm  implemenA total of 6 cSCC lines characterised within this manuscript were used for the detection of differentially methylated genes. Three early-passage primary normal human keratinocytes derived from 3 different participants served as a normal control for comparison. DNA was extracted as described above and bisulfite modification was carried out as the following: 500–1000 ng of DNA yield from each sample was modified with bisulfite using the EZ DNA Methylation Kit  according to the manufacturer’s instructions. Methylation detection was performed using the Illumina Infinium HumanMethylation27 BeadChip. This platform detects the methylation status of 27,578 CpG sites spread across 14,495 genes by sequencing-based genotyping of bisulfite-converted DNA. Eventual methylation scores  were generated for each site with BeadStudio software  and raw background-corrected values were used for further analysis. The methylation assay was performed according to the manufacturer’s instructions. Briefly, bisulfite-converted DNA was amplified, fragmented and hybridised to the chip arrays, followed by imaging with the Illumina BeadArray reader. Image processing and intensity data extraction were performed according to Illumina’s instructions. All analyses were carried out using Bioconductor and R.We present a definitive analysis of a unique set of cSCC lines that can be used as a model of skin squamous carcinogenesis for in vivo and in vitro studies. We provide details of their extensive characterisation, including clinical, phenotypic, genotypic, in vitro and in vivo properties. In addition to the research reported to date using these cell lines, we anticipate that this unique resource will continue to provide researchers with a powerful preclinical model for future investigations of the molecular drivers of cSCC progression, the identification of novel therapeutic targets and, ultimately, the development of more effective treatment strategies for this common skin cancer.