The SK-PN-DW cell line was established in 1979 and is commercially available. Despite the use of this cell line as an in vitro model for functional and therapeutic studies of malignant primitive neuroectodermal tumor (PNET), there is a lack of complete information about the genetic alterations that are present at the cytogenetic level. Thus, the current study aimed to characterize the cytogenetic profile of this cell line.Routine G-banded chromosome analysis, fluorescence in situ hybridization, and oligonucleotide array comparative genomic hybridization assays were performed to characterize the chromosomal changes in this cell line.The G-banded karyotype analysis showed that the number of chromosomes in this cell line ranged between 36 and 41. Importantly, all cells displayed a loss of chromosomes Y, 11, 13, and 18. However, some cells showed an additional loss of chromosome 10. Additionally, the observed structural changes indicated: a) unbalanced translocation between chromosomes 1 and 7; b) translocation between chromosomes 11 and 22 at breakpoints 11q24 and 22q12, which is a classical translocation that is associated with Ewing sarcoma; c) a derivative chromosome due to a whole arm translocation between chromosomes 16 and 17 at likely breakpoints 16p10 and 17q10; and d) possible rearrangement in the short arm of chromosome 18. Moreover, a variable number of double minutes were also observed in each metaphase cell. Furthermore, the microarray assay results not only demonstrated genomic-wide chromosomal imbalance in this cell line and precisely placed chromosomal breakpoints on unbalanced, rearranged chromosomes, but also revealed information about subtle chromosomal changes and the chromosomal origin of double minutes. Finally, the fluorescence in situ hybridization assay confirmed the findings of the routine cytogenetic analysis and microarrays.The accurate determination of the cytogenetic profile of the SK-PN-DW cell line is helpful in enabling the research community to utilize this cell line for future identity and comparability studies, in addition to demonstrating the utility of the complete cytogenetic profile, as a public resource. Typically, cell lines play a fundamental role in biomedical research, where they are used as in vitro models through which to investigate the mechanisms of disease initiation and progression, drug efficacy and therapeutic outcomes. In addition, they appear to be important in the study of rare or atypical cancers, where primary biological specimens are difficult to obtain. Thus, the importance of results obtained using cell lines is completely dependent upon their reliability and authenticity. In this regard, for decades, the misidentification of cell lines has been a major and significant concern in the scientific community, and significant efforts have only recently been made to address this issue on a large scale , 2. CurrThe initial authentication of any new cell line involves performing a panel of tests that were designed to address issues of inter- and intra-species contamination, tissue of origin, mycoplasma or other microbial contaminants, and genetic stability. However, re-authentication of a cell line after it is received in the laboratory, or prior to its use, has been simplified to a few tests. One of the most common methods used for re-authentication is SRT profiling, also known as DNA fingerprinting. This method is relatively fast and inexpensive. However, it is not able to detect numerical changes or marker chromosomes, and thus has a limited capacity in the analysis of mixed cell populations. Solid tumor cell lines often display complex genetic arrangements, including multiple numerical and structural aberrations with significant variation among different cells of the same tumor . Thus, cPrimitive neuroectodermal tumors (PNETs) are aggressive, highly malignant and small round cell tumors with diverse clinical manifestations. These PNETs are more 4.6 times) likely to arise in children and adolescents, with an incidence rate of 0.62 per million people in the United States. Therefore, PNETs are classified as a rare cancer .6 times . CurrentIn our current study, we analyzed the SK-PN-DW cell line and define the common chromosomal numerical and structural changes, using modern technology, with the intention of providing the comprehensive cytogenetic profile of this cell line as a public resource for the research community who are using this cell line to further study PNET biology. At the same time, we validated a hypothesis that the cell line undergoes structural changes after passage, which may affect its function. This hypothesis requires a large number of follow-up experiments to prove.2 in an incubator. Later, the cells were frozen for subsequent studies.The primitive neuroectodermal cell line, SK-PN-DW, was obtained from the American Type Culture Collection  in 2011, and was grown in RPMI 1640 medium (Corning) supplemented with 12% fetal bovine serum , 1 x Penicillin-Streptomycin (Gibco), and 2 mM-glutamine (Gibco), at 37 °C and 5% COThe cells were collected, in the metaphase stage, by exposing them to colcemid solution  for one hour. The cells were then harvested from the surface of the culture flask through a brief incubation with 0.05% trypsin-EDTA (GIBCO). Next, the harvested cells were treated with 0.075 M KCl hypotonic solution, and then fixed through three incubations with Carnoy’s fixative (3:1 methanol to acetic acid) before being placed on to glass slides. The slides were then incubated at 58 °C for 16 h before staining.G-banding was achieved through a brief exposure of the cells to 0.1% trypsin (w/v) DPBS solution, followed by two rinses with 0.9 M NaCl solution, and subsequent staining with Giemsa stain (EMD). The final images were captured and analyzed using CytoVision software version 7 .FISH analyses were performed using multiple DNA probes that were purchased from Abbott Molecular  and used based on manufacture’s protocols, with minor changes. The whole chromosome painting probes were used for chromosomes 1, 7, 8, 16, 17, 18, 21, and 22 analysis, while centromere probes were used for chromosomes X, Y, 3, 10, 11, 16, 17 and 18 analysis. Locus-specific probes that were designed for the genes EGR1 on 5q31, cMYC on 8q24, IGH1 on 14q32, and EWSR1 on 22q12, in addition to Vysis probe sets LSI 13 on 13q14, and LSI21 on 21q22,13-q22.2 were used. Overall, a total of 200 interphase cells, and 20 metaphase cells were analyzed with each probe. The digital images of specific hybridization signals were processed using CytoVision software version 7 .Array comparative genomic hybridization (CGH) was performed, as has been described previously . BrieflyFinally, the chromosomal anomalies that were detected through routine G-banded chromosomal analysis, FISH, and array CGH were described based on the guidelines in “An International System for Human Cytogenetic Nomenclature (2013)”.In total, 20 cells at the metaphase stage were analyzed. All analyzed cells displayed consistent chromosomal anomalies with a modal number of chromosomes ranging from 36 to 41. Table  and 6a.TFurthermore, conventional two-color FISH analysis was performed using arm-specific probes for chromosomes 13, 18, 21, X and Y. This analysis confirmed monosomy at chromosomes 13, 18 and Y in all cells. The CEP (chromosome enumeration probe) 10 and 11 confirmed monosomy 11 in all cells, while mosaic monosomy 10 in approximately 11.5% of the cells (23/200). The karyotype and array CGH results indicated that there were rearrangements between chromosomes 1 and 7; 8 and 18; 11 and 22; and 16 and 17. Because of this, whole chromosome painting probes were used for chromosome pairs 1 and 7. This confirmed the translocation of chromosome 7 material to the terminal q arm of chromosome 1 Fig. b. In addTo confirm the findings of the routine G-banded chromosomal analysis, we performed array CGH. Through this, we were able to determine the chromosomal origin of the observed DMs, and to detect possible submicroscopic chromosomal imbalances in this cell line. The array CGH results showed loss of complete chromosomes 10, 11, 13, 17, 18, and Y Figs. . MoreoveBased on G-banding, array CGH and FISH analyses, this study has revealed the cytogenetic profile of the SK-PN-DW cell line. The important highlights of these findings are as follows: a 50.4-Mb terminal deletion on the distal chromosome 1q (del q11.2qteqte2.Tablhttp://www.ashg.org/2015meeting/pdf/57715_Posters.pdf?)These results have been communicated by a poster in the conference of ASHG(2015). and 8q24.23–24.3 , respectively. Following analysis of array CGH and FISH results, we finally concluded the existence of a novel rearrangement, del , tFinally, another translocation was observed between chromosomes 11 and 22. Both array CGH and karyptype analyses indicated contrasting information regarding chromosome 22. The array CGH results indicated that chromosome 22 was normal, while Karyotype analysis demonstrated abnormal chromosome 11 and 22. The FISH analysis indicated the following rearrangement, t. Recently, multiple studies have reported a role of this translocation in Ewing Sarcoma , 29. TheAdditionally, we also observed partial monosomy of chromosome 10, with the tumor suppressor gene, DMBT1, located at 10q25.3–26.1 , 34. TheOverall, our study concluded that the continuous culturing of cell lines induces changes in the copy number, and possibly affecting the function of many chromosomes, thus making them unstable and less authentic. Moreover, the authentication of these cell lines using individual analyses, such as karyotyping, array CGH, or FISH alone is not sufficient, as these analyses can yield varying results. Thus, a combination of these techniques should be used for authentication for important research. We analyzed only one cell line (SK-PN-DW) of PNET. Next stage we will continue to analyze the different generation of this cell line and other cell lines using the same methods, and showing more data.


Hydroxy fatty acids are known to cause cell cycle arrest and apoptosis. The best studied of them, 9-hydroxystearic acid (9-HSA), induces apoptosis in cell lines by acting through mechanisms involving different targets. Using mass spectrometry-based lipidomic approaches, we show in this study that 9-HSA levels in human colorectal tumors are diminished when compared with normal adjacent tissue. Since this decrease could be compatible with an escape mechanism of tumors from 9-HSA-induced apoptosis, we investigated different features of the utilization of this hydroxyfatty acid in colon. We show that in colorectal tumors and related cell lines such as HT-29 and HCT-116, 9-HSA is the only hydroxyfatty acid constituent of branched fatty acid esters of hydroxyfatty acids (FAHFA), a novel family of lipids with anti-inflammatory properties. Importantly, FAHFA levels in tumors are elevated compared with normal tissue and, unlike 9-HSA, they do not induce apoptosis of colorectal cell lines over a wide range of concentrations. Further, the addition of 9-HSA to colon cancer cell lines augments the synthesis of different FAHFA before the cells commit to apoptosis, suggesting that FAHFA formation may function as a buffer system that sequesters the hydroxyacid into an inactive form, thereby restricting apoptosis. Colorectal cancer is a frequent cause of death due to late tumor presentation, rapid progression, and metachronous metastases . Althoug0 phase of the cell cycle. At higher doses, 9-hydroxystearic acid (9-HSA) is a strong inducer of cell death by apoptosis. Increased 9-HSA levels may interfere with cell cycle kinetics by interacting with the cdc2 kinase complex and with p21, a potent cyclin-dependent kinase inhibitor controlled by the tumor suppressor protein p53 oleic acid, and analyzed the possible appearance of 9-[2H]HSA by GC/MS and LC/MS at different times. HCT-116 and HT-29 cell lines are well established models for colon cancer with rapid proliferation rates, which offers greater versatility and more controlled conditions for assays than primary cultures of cells isolated from tumors, where cellular heterogeneity may reduce applicability. While both HT-29 and HCT-16 cells readily incorporated 2H-labeled oleic acid into phospholipids, no 2H-labeled HSA could be detected over a wide range of concentrations (up to 100 µM) and times (up to 40 h). Of note, both cells lines contained measurable levels of preexisting ‘endogenous’  9-HSA, both as free fatty acid and in esterified form  with the free radical generators 2,2′-azobis(2-methylpropionamidine) dihydrochloride, Fenton’s reagent (ferrous sulfate plus hydrogen peroxide), or just by exposing the oleic acid in air for at least 1 day. In neither case could 9-HSA be detected, suggesting that significant oxidation of oleic acid to 9-HSA is not attainable non-enzymatically. In addition, our preliminary attempts at establishing whether bacteria present in the gut microbiota make 9-HSA have been unsuccessful to this point.Next, we also examined the possibility that stearic acid, not oleic acid, was the biosynthetic precursor of 9-HSA. We labeled the cells with − ions except for choline phospholipids, which were detected as [M+CH3COO−]− adducts, and were identified by comparison with previously published data [Lipids were extracted according to Bligh and Dyer , and thehed data ,44,45,46n-hexane through them. Neutral lipids were eluted with 5 mL n-hexane/ethyl acetate . FAHFA were eluted with 4 mL ethyl acetate and evaporated under a nitrogen stream. Quantitative isolation of FAHFAs by this method was confirmed by using an authentic 9-PAHSA standard. The FAHFA fraction was redissolved in 50 µL methanol, and 40 µL was injected into an Agilent 1260 Infinity high-performance liquid chromatograph equipped with an Agilent G1311C quaternary pump and an Agilent G1329B autosampler. The column was a reversed-phase C18 column . Total flow through the column was 400 µL/min, with 93:7 MeOH:H2O with 5 mM ammonium acetate and 0.01% ammonium hydroxide as solvent. Total run time was 60 min. The LC system was coupled to an AB/Sciex API 4500 Q-TRAP hybrid triple quadrupole mas spectrometer operated in negative ion mode. Source parameters were as follows: ion spray voltage, −4500 V; curtain gas, 30 psi; nebulizer gas, 50 psi; desolvation gas, 60 psi; temperature, 500 °C. For the quantitative measurement of 9-HSA-containing FAHFA species , the instrument was set to multiple reaction monitoring mode, selecting three transitions for each FAHFA: parent ion → 299.3 (9-HSA), parent ion → 281.3 (9-HSA as water loss) and parent ion → m/z of esterified fatty acid as carboxylate ion. Only peaks with a signal-to-noise ratio higher than five were quantified.FAHFA analysis by LC/MS was carried out as described by Zhang et al. . Brieflyt-test was applied for statistical analysis of the data, with p < 0.05 taken as statistically significant.Data are expressed as means ± SE of the number of independent determinations indicated. Student’s We report in this study that human colorectal tumors contain significantly lower levels of 9-HSA than adjacent normal tissue, suggesting that highly proliferating cells such as those of tumors possess mechanisms to keep 9-HSA at low levels and, in this manner, inactivate an endogenous brake to uncontrolled cell division. Our results add to this concept by demonstrating that colorectal cancer lines make significant amounts of various FAHFA when supplied with 9-HSA. Further, we made the key observation that, while 9-HSA is pro-apoptotic, 9-HSA-containing FAHFA is not. Thus, in addition to its well described roles as anti-diabetic and anti-inflammatory lipids, FAHFA may be produced as an anti-apoptotic strategy to ameliorate the pro-apoptotic effects of hydroxy fatty acids accumulating in cells. In accord with all the above, we demonstrate as well that FAHFA content in human tumor samples is significantly elevated when compared with adjacent normal tissue, clearly suggesting a pathophysiological role, such as preventing the pro-apoptotic effects of 9-HSA. Thus, FAHFA synthesis may occur at proliferative tumor stages where there is a predominance of mitosis over apoptosis and, in opposition, cells may activate FAHFA hydrolysis at times of declining cell death, thus regulating the cell cycle via lipid signaling. The finding that FAHFA molecules are found in tumors shows that this mechanism may work in vivo and, at the same time, provides an interesting starting point from which to consider these lipids as pharmacological targets, focusing on the specific hydrolysis of FAHFA that provides apoptotic hydroxyacids in situ.


Acute graft-vs.-host disease (GVHD) causes activation of multiple pro-inflammatory pathways in a diverse array of immune cell subsets that are often regulated by micro RNAs (miRs). MiRs are small non-coding RNAs that post-transcriptionally regulate gene expression and thereby impact inflammatory events at multiple levels including the release cytokines, chemokines, signaling by their receptors, cell-cycling, migration, and filament stabilization. Multiple inflammation-related miR-target genes are described, and while some miRs target anti-inflammatory genes other promote inflammation by targeting immunosuppressive genes. Additionally, the same miR can have different effects by preferentially targeting certain genes depending on the cell type and their context, in which the miR is analyzed. Through these diverse effects in various critical immune cell subsets miRs play critical functional role in GVHD and graft-vs.-leukemia effects (GVL). Thus, miRs are potentially attractive targets for the modification of allogeneic immune responses using miR mimics and inhibitors. Additionally, miR-levels in different body fluids could help to guide clinical decision making and function as biomarkers to predict or diagnose GVHD. This Research Topic, authored by experts in the field, describes the pleomorphic function of different miRs as potent regulators of multiple pro- or anti-inflammatory target genes, in various immune cell subsets that affect alloimmunity and GVHD. A better understanding of the miR-biology may help to mitigate GVHD and augment GVL effects and improve the outcomes after allogeneic hematopoietic transplantation.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Changes in expression of components of the DGC or in the O-glycosylation of α-dystroglycan result in muscular dystrophy but are also observed in certain cancers. In mice, the loss of either of two DGC proteins, dystrophin or α-sarcoglycan, is associated with a high incidence of rhabdomyosarcoma (RMS). In addition, glycosylation of α-dystroglycan is aberrant in a small cohort of human patients with RMS. Since both the glycosylation of α-dystroglycan and its function as an ECM receptor require over 18 post-translational processing enzymes, we hypothesized that understanding its role in the pathogenesis of RMS requires a complete analysis of the expression of dystroglycan-modifying enzymes and the characterization of α-dystroglycan glycosylation in the context of RMS.α-Dystroglycan is the highly glycosylated component of the dystrophin-glycoprotein complex (DGC) that binds with high-affinity to extracellular matrix (ECM) proteins containing laminin-G-like (LG) domains via a unique heteropolysaccharide LARGE1 was ectopically expressed in RMS cells to determine its effects on matriglycan modifications and the ability of α-dystroglycan to function as a laminin receptor.A series of cell lines and biopsy samples from human and mouse RMS were analyzed for the glycosylation status of α-dystroglycan and for expression of the genes encoding the responsible enzymes, in particular those required for the addition of matriglycan. Furthermore, the glycosyltransferase LARGE1. Ectopic expression of LARGE1 in these cell cultures restored matriglycan to levels comparable to those in muscle and restored high-affinity laminin binding to α-dystroglycan.Immunohistochemistry and immunoblotting of a collection of primary RMS tumors show that although α-dystroglycan is consistently expressed and glycosylated in these tumors, α-dystroglycan lacks matriglycan and the ability to bind laminin. Similarly, in a series of cell lines derived from human and mouse RMS, α-dystroglycan lacks matriglycan modification and the ability to bind laminin. RNAseq data from RMS cell lines was analyzed for expression of the genes known to be involved in α-dystroglycan glycosylation, which revealed that, for most cell lines, the lack of matriglycan can be attributed to the downregulation of the dystroglycan-modifying enzyme LARGE1, and that ectopic expression of LARGE1 can restore matriglycan modifications and the ability of α-dystroglycan to function as an ECM receptor.Collectively, our findings demonstrate that a lack of matriglycan on α-dystroglycan is a common feature in RMS due to the downregulation of  Dystroglycan is a ubiquitously expressed extracellular matrix (ECM) receptor and plays key roles in the formation and maintenance of many tissues . In skeln called matriglycan, which has been reported only on α-dystroglycan and binds with high-affinity to ECM proteins that contain LG domains. In humans, mutations in any of the genes coding for the glycosyltransferases involved in this process can lead to a spectrum of syndromes known as secondary dystroglycanopathies. These conditions are characterized by an absence or reduction in the matriglycan modification on α-dystroglycan and by muscular dystrophy with or without brain and eye defects.Similarly, loss of dystroglycan function has been associated with human pathologies, including muscular dystrophy with or without various degrees of brain and eye defects . Dystrogmdx) and α-sarcoglycan, demonstrated that primary defects in the DGC are sometimes associated with the formation of rhabdomyosarcomas (RMS) [mdx) or α-sarcoglycan, 21% (32/150) and 5% (4/80) of mice developed such tumors, respectively. Mdx-derived tumors display both cellular and molecular characteristics of embryonal rhabdomyosarcoma (eRMS) and almost no characteristics of alveolar rhabdomyosarcoma (aRMS).We and others have demonstrated that glycosylation of α-dystroglycan is also compromised in a wide variety of cancers, and that this defect correlates with poor prognosis –16. An aas (RMS) . SpecifiBiochemical evidence supporting the involvement of α-dystroglycan in RMS comes from a manuscript reporting on five RMS tumors, which found the expression of α-dystroglycan to be reduced or absent while that of β-dystroglycan was normal . ParalleLARGE1 in RMS cells restored both the matriglycan modification of α-dystroglycan and the ability of α-dystroglycan to bind laminin, demonstrating that the loss of LARGE1 expression causes the lack of matriglycan and a loss of high-affinity laminin binding in RMS.In the study described here, we expand on the above-described investigations by describing a functional role for dystroglycan in RMS, and begin to elucidate the molecular mechanism underlying its altered function. Analysis of a microarray panel containing eRMS and aRMS tissue revealed that matriglycan levels on α-dystroglycan are reduced in both sample types. Similarly, analysis of cell lines derived from human and mouse RMS shows that in a subset of these cell lines, α-dystroglycan lacks matriglycan, and that this is accompanied by reduced expression of the glycosyltransferase LARGE1. Lastly, we found that ectopic expression of All experiments were repeated in the laboratory three times. Data reported are representative.2 at 37 °C. Experiments were performed at or before passage 10.Primary cells from genetically engineered mouse aRMS samples were cultured as previously described , 21. ThePOMT1, POMT2, POMGNT1, POMGNT2, B3GALNT2, POMK, LARGE1, LARGE2, B3GNT1, Fukutin, FKRP, ISPD, and TMEM5 were cloned into replication-deficient adeno-viral (Ad5) vectors. Adenovirus was generated by the Viral Vector Core at the University of Iowa. The generation of Ad5 constructs was described previously [The human genes that code for dystroglycan, Total RNA was extracted from cells in culture using the RNeasy isolation kit (Qiagen). First-strand complementary DNA (cDNA) was synthesized from total RNA using the AMV reverse transcriptase (Roche) and a combination of random hexamers and polyA primers, according to the manufacturer’s instructions. Each of the target genes was amplified in real-time from cDNA using oligonucleotides specific to that gene (sequences and conditions available upon request), with 28S-RNA, Rpl27, and Rpl4 used as the normalization controls. cDNA levels were determined using SYBR green in a MyiQ rt-PCR detection system (BioRad). All samples were run in triplicate.g for 15 min. The solubilized supernatant was added to 200 μl of wheat germ agglutinin (WGA) slurry (Vector Labs) and the samples were rotated overnight at 4 °C. The pelleted WGA beads were washed three times with 1 ml TBS containing 0.1% Triton X-100. After the final wash, 250 μl of loading buffer (loading sample buffer (LSB)) was added to the beads. The samples were heated to 99 °C for 10 min before loading into 3–15% SDS–PAGE gels, and protein was subsequently transferred to polyvinylidene fluoride (PVDF) membranes, which were subsequently blocked in 2% nonfat dried milk in TBST buffer . All antibodies were diluted in the same buffer that was used for blocking. Membranes were washed in TBST buffer. Blots were developed with IR-conjugated secondary antibodies  and scanned using an Odyssey infrared imaging system . α-Dystroglycan and β-dystroglycan core proteins were detected using antibody AF6868, and matriglycan was detected using antibody IIH6 [Proteins were extracted from cultured cells using lysis buffer . Insoluble material was removed by centrifugation at 12,000×ody IIH6 , 23. Ele2, 1 mM CaCl2, pH 7.6) containing 3% bovine serum albumin (BSA) followed by incubation with laminin overnight at 4 °C in LBB. Membranes were washed and incubated with anti-laminin antibody  followed by Licor dye-conjugated donkey anti-rabbit IgG.Ligand overlay assays were performed on membranes using mouse Engelbreth–Holm–Swarm (EHS) laminin (Sigma-Aldrich) . Briefly2O2, and then reactions were stopped with 2 N H2SO4. Absorbance per well was read at 490 nm by a microplate reader.WGA eluates were diluted 1:50 in TBS and coated on polystyrene ELISA microplates (Costar) overnight at 4 °C. Plates were washed in LBB and blocked for 1 h in 3% BSA in LBB. Mouse EHS laminin was diluted in LBB and applied for 2 h. Wells were washed with 3% BSA in LBB and incubated for 30 min with anti-laminin antibody  followed by HRP-conjugated anti-rabbit IgG . Plates were developed with o-phenylenediamine dihydrochloride and HA previous study showed a loss of fully glycosylated α-dystroglycan in the majority of RMS samples from a small cohort; notably, the expression of β-dystroglycan, with which α-dystroglycan is co-translated, was not significantly altered . Since tThe first sample-set tested was the Children’s Oncology Group Lookback tissue microarray P1542), which includes both aRMS and eRMS subtypes. Nearly all eRMS and aRMS samples that were examined were negative for IIH6 staining, whereas normal skeletal muscle was positive  for functional glycosylation of α-dystroglycan and the expression of various enzymes involved in this process. We found that although the amounts of α-dystroglycan and β-dystroglycan in this line were similar to those in the C2C12 normal muscle cell line and in mouse skeletal muscle  and one from an aRMS tumor (PCB380). All three cell lines produced bands that were detected by the anti-core dystroglycan antibody , which expresses low levels of LARGE1; and Rh41 (aRMS), which expresses LARGE1 at normal levels. Analysis of both cell lines revealed that Rh18 is nearly devoid of functional α-dystroglycan as reflected by a lack of matriglycan and by a loss of activity in the laminin overlay assay  and compared the resulting cells to the parental (non-transduced) line  and the ability of α-dystroglycan to function as a laminin receptor.In epithelium-derived cancers, downregulation of glycosyltransferase expression has been found to underlie abnormal glycosylation of α-dystroglycan , 15, 27.LARGE1 is repressed in many epithelial cancer-derived cell lines, and showed that its ectopic expression reduces the invasiveness of such cells [n onto α-dystroglycan [LARGE1 restores the matriglycan modification in all of the RMS cell lines tested, irrespective of the internal levels of LARGE1, indicates that LARGE 1 restructures the binding of α-dystroglycan so that it functions as an ECM receptor in RMS. Correspondingly, in the RMS cell line Rh18, ectopic expression of LARGE1 resulted in functional glycosylation of α-dystroglycan and conferred its ability to bind laminin, its natural ligand, with high-affinity.We previously reported that ch cells . LARGE1 roglycan , formingroglycan . The facLARGE1 expression in order to orchestrate the remodeling of the ECM in the growing secondary tumor. Although germline mutations in LARGE1 have been reported in a group of congenital muscular dystrophies that also involve brain and eye defects, no increase in the rate of any cancer type, including RMS, has been described in patients. However, these are rare diseases and the number of patients available for such studies is limited. Similarly, higher cancer rates have not been reported in patients who carry mutations in dystrophin or α-sarcoglycan, two other components of the DGC that have been linked to RMS [LARGE1 in these cell lines enables cells to bind laminin and reduces the invasiveness of the phenotype. Recently, two other dystroglycan-modifying glycosyltransferases, LARGE2 and B4GALNT2, were linked with the cancer-associated abnormal glycosylation of α-dystroglycan [With respect to whether defects in the glycosylation of α-dystroglycan might contribute to tumor initiation versus progression, it seems more likely that it is a secondary (somatic) event involved in tumor progression. After the metastatic cancer cell has reached its secondary site, it may restore d to RMS . We reporoglycan , 15. AltLARGE1 expression. This defect was reverted by ectopic expression of LARGE1 cDNA in all of the cell lines that were tested, which then showed normal levels of matriglycan modifications and restored the ability of α-dystroglycan to bind to laminin. Thus, ectopic expression of LARGE1 may be worthy of further investigation as a potential therapeutic approach for RMS.We have demonstrated that the modification of α-dystroglycan with matriglycan is dramatically reduced in a panel of RMS samples, and that this result is reproduced in RMS-derived cell lines. In some of the cell lines studied, the loss of matriglycan modifications could be attributed to the silencing of


Purpose: The PI3K/Akt signaling pathway regulates cell growth, proliferation and viability inhematopoietic cells. This pathway always dysregulates in acute lymphoblastic leukemia (ALL).PTEN and P53 are tumor suppressor genes correlated with PI3K/Akt signaling pathway, and bothhave a tight link in regulation of cell proliferation and cell death. In this study, we investigatedthe effects of dual targeting of PI3K/Akt pathway by combined inhibition with nvp-BKM-120(PI3K inhibitor) and MK-2206 (Akt inhibitor) in relation with PTEN and P53 on apoptosis andproliferation of leukemia cells.Methods: Both T and B ALL cell lines were treated with both inhibitors alone or in combinationwith each other, and induction of apoptosis and inhibition of proliferation were evaluated byflow cytometry. Expression levels of PTEN as well as p53 mRNA and protein were measured byreal-time qRT-PCR and western blot, respectively.Results: We indicated that both inhibitors (BKM-120 and MK-2206) decreased cell viability andincreased cytotoxicity in leukemia cells. Reduction in Akt phosphorylation increased PTEN andp53 mRNA and p53 protein level (in PTEN positive versus PTEN negative cell lines). Additionally,both inhibitors, particularly in combination with each other, increased apoptosis  and reduced proliferation (Ki67 expression) in leukemia cells.However, administration of IL7 downregulated PTEN and P53 mRNA expression and rescuedcancer cells following inhibition of BKM-120 and MK-2206.Conclusion: This investigation suggested that inhibition of Akt and PI3K could be helpful inleukemia treatment. Both of them regulate cell proliferation and cell death. Their activity is dependent on each other.23 Conventional cancer therapies, including chemotherapy and radiotherapy, could not completely prevail overexpression and activation of the PI3K/Akt pathway in cancer cells.25 To overcome drug resistance and side effects of traditional cancer therapies, application of various small molecule inhibitors could increase the specificity and efficacy of cancer targeting. Numerous preclinical studies have been focused mainly on PI3K/Akt signaling as an attractive object for targeted therapy in leukemia.26Acute lymphoblastic leukemia (ALL) is the most common and aggressive form of cancer in children.27 In addition, MK-2206 is an orally active, selective and potent allosteric inhibitor of Akt; preclinical studies have demonstrated its efficacy in the treatment of various cancers.28 Here, we evaluated whether inhibition of PI3K/Akt signaling by BKM-120 and MK-2206 would affect the phosphorylation and activation of Akt in correlation with PTEN and P53. Finally, we used interleukin-7 (IL-7), as a growth factor for the development of T and pre B-ALL, to understand whether IL-7 would rescue PI3K/AKT signaling inhibition.BKM120 (NVP-BKM120 or Buparlisib) is an orally bioavailable, highly potent and selective pan-class I PI3K inhibitor preventing the binding of ATP to PI3K active sites. It has exhibited strong antiproliferative activities, antitumor effects and has induced apoptosis in various cancers.29 and T-ALL (Dnd-4130 and Molt-431 ) cell lines were purchased from the National Cell Bank of Iran . Nalm-6, Reh-6 and Molt-4 cell lines are PTEN positive whereas Dnd-41 cell line is PTEN negative. Four ALL cell lines were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Gibco), 100 mg/mL streptomycin and 100 units/mL penicillin G  in a 37°C incubator with a humidified atmosphere of 5% CO2.The B-ALL 4cells per well) with a different dose of BKM-120 and MK-2206 inhibitors as incubation at 37°C with 5% CO2. Untreated cells served as control. Following treatment for 24, 48 and 72 hours with BKM-120 and MK-2206 inhibitors, colorimetric methylthiazol tetrazolium bromide (MTT) solution was added to a final concentration of 5 mg/mL in each well and incubation was prolonged for 4 hours at 37°C. After, acidified detergent (10% SDS in 0.01 M HCl) was added to each well and incubated overnight at 37°C with 5% CO2 due to solubilization of the formazan. Complete solubilization of the purple formazan crystals by SDS was measured by ELISA reader. We used GraphPad Prism software to determine IC50 value of inhibitors, using regression analysis to fit a dose- response curve.A total of 200 µL cell suspensions were seeded and treated in triplicate in flat bottomed 96-well plates  according to the silica based method. Then First-strand cDNA was synthesized via reverse transcription of 1 μg of total RNA by Prime ScriptTM RT reagent kit  according to the manufacturer’s instructions. The cDNA was amplified using SYBR Green . Real-time PCR was performed using the Corbett RotorGene™ 6000 HRM  thermocycler. PCR primers were designed using Oligo7 software.32 Primer pairs sequence (5′3′) used for PCR, including Akt (forward: ATTGTGAAGGAGGGTTGGCTG and reverse: CTTGAGGAGGAAGTAGCGTGG), PTEN (forward: AGGAAGTGAATCTGTATTGGG and reverse: TTGCTGTGTTTCTTACCTATG), P53 (forward: TCAGTCTACCTCCCGCCATAA and reverse: AGTGGGGAACAAGAAGTGGAG), GAPDH (forward: CAAGATCATCACCAATGCCT and reverse: CCCATCACGCCACAGTTTCC).The total RNA was extracted from 1×10/Wash buffer and incubated with anti-Ki-67 or anti-caspase 3 antibody at room temperature for 20 minutes and analyzed by BD FACS Calibur with FlowJo (7.6.1) software for acquisition and analysis.To assess cell proliferation, flow cytometric analysis was performed using PE-conjugated monoclonal anti-Ki-67 antibody  and apoptosis performed by Caspase 3 kit . For intracellular staining, cells were collected and washed twice with ice-cold PBS, resuspended with BD cytofix/cytoperm solution and incubated at 4°C for 20 minutes, cells were washed twice using PermAlso, flow cytometric analysis was performed using Annexin V-FITC/PI staining. Following 48h treatment, cells were collected and rinsed twice with ice-cold PBS. Then cells washed and incubated with Annexin V-FITC/PI (eBiosciences) for 15 min at room temperature. Cells were analyzed by BD FACS Calibur with FlowJo (7.6.1) software for acquisition and analysis.Cell lines were treated with inhibitors for 48 hours and lysed in RIPA buffer  supplemented with the protease and phosphatase inhibitor cocktail. Protein concentration was determined using the BCA Protein Assay kit . The lysates were diluted with sample loading buffer and equal amounts of protein extracts were separated on 12% SDS–PAGE gels and transferred to polyvinylidene difluoride membranes . The membranes were blocked for 1 hour at room temperature with 5% low-fat dry milk or BSA. The membrane was incubated with primary monoclonal antibodies β-actin (1:1000), Akt, p-Akt, PTEN, p-PTEN and p53  for overnight at 4˚C. After washing for three 10-minute, the membrane was incubated with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody for 1 hour. Then, the blots were visualized using a chemiluminescent detection Kit  and X-ray film .The anti-proliferative effects of BKM120 and MK-2206 in leukemia cell lines were examined in T-ALL (Molt-4 and Dnd-41) and B-ALL  cell lines with distinct concentrations of BKM120 and MK-2206 (between 0.25 and 10 μM) for 24, 48 and 72 hours.-dependent manner, whereas Dnd-41 cell line showed a much lower sensitivity. After 24, 48 and 72 hours of treatment with inhibitors, the IC50 of each cell line was measured. The best IC50 was achieved after 48 hours of treatment. Thus, BKM-120 IC50 of Nalm-6 (9.15 μM), Reh-6 (10.15 μM) Molt-4 (8.61 μM) and Dnd-41 (37.01 μM) cell lines and MK-2206 IC50 of Nalm-6 (0.79 μM), Reh-6 (0.89 μM) Molt-4 (2.22 μM) and Dnd-41 (24.25 μM) cell lines was defined.Both BKM120 and MK-2206 reduced cell viability on Nalm-6, Reh-6 and Molt-4 cell lines in a concentrationDnd-41 as a PTEN positive T cell line showed a much lower sensitivity to inhibitors in comparison to Molt-4 as a PTEN negative T cell line, but PTEN positive pre-B cell lines, Nalm-6 and Reh-6, similar to Molt-4 displayed the cytotoxic effect in response to inhibitors .Nalm-6, Reh-6 and Molt-4 cell lines were treated with BKM-120 and MK-2206 inhibitors for 48 hours and apoptosis were evaluated by Annexin V/PI staining. Both inhibitors significantly increased the percentage of apoptosis in all cell lines. BKM-120 and MK-2206 had somewhat the same effect on apoptosis in Reh-6 and Molt-4 cell lines. BKM-120 had an intensive effect on apoptosis rather than MK-2206 in Nalm-6 cell line. Interestingly, the percentage of apoptotic positive cells strongly increased when we used both drugs together in all cell lines .We also evaluated caspase3 expression to more strengthen this observation, which has a critical role in apoptotic cell death. The apoptotic effect due to activation of caspases-3 significantly increased following treatment with each drug alone in comparison with the control group in all cell lines, although BKM-120 had a greater apoptotic effect relative to MK-2206. Using inhibitors together had a higher rate of apoptosis rather than using alone in all cell lines .To understand whether inhibitors affect proliferation of cell lines, we evaluated Ki67 expression following using these inhibitors. The proliferation of treated groups significantly reduced in comparison with the control group in all cell lines, especially BKM-120 treated groups shown more increased KI67 expression in comparison with MK-2206 inhibitor treated groups in all cell lines. KI67 expression did not notable change when inhibitors administrated together in comparison with BKM-120 alone in all cell lines .To inspect the impact of PI3K and Akt inhibition on Akt gene and protein expression and phosphorylation, we performed quantitative RT-PCR and western blot analysis after using BKM-120 and MK-2206 inhibitors and a combination of them for 48 hours. As displayed in 33 We evaluated the effect of PI3K and Akt inhibition on the PTEN mRNA and protein expression and phosphorylation. Nalm-6, Reh-6 and Molt-4 cells were treated with IC50 of BKM-120 and MK-2206 inhibitors and a combination of them for 48 hours to measure changes in PTEN gene and protein expression and phosphorylation. We used Real Time PCR assay to assess the effect of PI3K and Akt inhibition on the PTEN mRNA. As shown in . For this purpose, because BKM-120 had an intensive effect on cell lines proliferation and apoptosis rather than MK-2206, cell lines treated with IC50 of BKM-120 in the presence of IL-7 for 48 hours. Simultaneous treatment of cells with BKM-120 and IL-7 or with MK-2206, BKM-120 and IL-7 showed that inhibition of PI3K by BKM-120 in the presence of IL-7 caused downregulation of mRNA expression level of PTEN and P53 in comparison with using PI3K inhibitor alone .35 In this regard, development of new therapeutic strategies against ALL cells for negative modulation of PI3K/Akt signaling is extremely important to achieve better clinical outcomes and restore the functional balance between tumor suppressors and growth signaling pathways.The PI3K/Akt signaling pathway dysregulation is effectively involved in cancer onset and progression, making this pathway an interesting target for therapeutic intervention.38 Earlier studies indicated that multiple negative and positive feedback regulatory loops were involved in signal transduction of the PI3K/Akt network.39 Therefore, to overcome feedback loops limitation against antitumor effects of PI3K and Akt inhibitors given in monotherapy, simultaneously targeting PI3K/Akt signaling pathway at different levels with different inhibitors might be a promising therapeutic strategy to treat leukemia.Vertical inhibition of different targets of the PI3K/Akt signaling leads to better outcomes than use of using single or dual inhibitors in solid tumor treatment.41 It seems that combination of a PI3K inhibitor with an Akt inhibitor have greater antitumor activity in cancer treatment.In some previous studies, the usual dual approach in targeting the PI3K/Akt pathway was combining a PI3K inhibitor with a mTOR inhibitor. Interestingly, some rapalogs amplified mTORC2-dependent Akt phosphorylation on Ser 473 and increased cell survival via inhibition of a negative feed-back loop based on mTORC1/p70S6K/IRS1/PI3K.Hence, the present study aims to investigate the therapeutic potential of BKM-120 and MK-2206 for inhibition of PI3K/Akt signaling activity by regarding PTEN and P53 as two main tumor suppressors associated with this pathway. We showed that both inhibitors decreased cell viability, proliferation, and induced apoptosis in PTEN positive Pre B-ALL cell lines  and PTEN deleted T-cell line (Molt-4), whereas PTEN positive T-cell line (Dnd-41) was not sensitive to inhibitors. Furthermore, inhibition of PI3K pathway exerted a greater anti-cancer effect than Akt signaling inhibition. BKM-120, the pan-class I PI3K inhibitor, was more powerful than the allosteric Akt inhibitor MK-2206, in both T and Pre B-ALL cell lines. Inhibition with BKM-120 and MK-2206 showed additive efficiency, improved antitumor effectiveness on leukemic cells, enhanced apoptosis, and decreased proliferation in comparison to using each drug alone. Both BKM-120 and MK-2206 inhibited Akt phosphorylation on Ser473, whereas there were no significant changes in total Akt protein levels, confirming the efficacy of the drugs in inhibition of PI3K/Akt signaling activity. However, Akt mRNA increased in all cell lines, unlike the protein levels, which did not significantly change, indicating that Akt was inactivated at the posttranscriptional level.43Similar results were observed in several cancer models. For example, it was previously reported that LY294002 and rapamycin had a much greater degree antiproliferative effect in PTEN-negative breast cancer cells, indicating that PTEN-negative breast cancer cells were more sensitive to PI3K inhibition.44 Silva et al demonstrated that LY294002 treatment was not affective against T-ALL patient samples with normal levels of PTEN expression, PTEN phosphorylation, and PI3K/Akt pathway activation, whereas other PTEN-expressing samples showing PI3K/Akt pathway hyperactivation were sensitive to LY294002 treatment. Normal levels of PTEN protein expression in cells were not necessarily synonymous with normal function of PTEN. PI3K/Akt pathway hyperactivation occurs as a result of posttranslational modification of PTEN by CK2-mediated phosphorylation and ROS-dependent oxidation.45Also previous research showed that wortmannin, a PI3K inhibitor, by mimicking PTEN function and enhancing of P53 stability, could induce sensitization to treatment with p53-inducing drugs in refractory tumors with wt p53.46We showed that Dnd-41 cell line with PTEN positive status did not show sensitivity to the inhibitors, but Molt-4 was sensitive to PI3K and Akt inhibitors. On the other hand, Nalm-6 and Reh-6 cell lines with PTEN positive status were sensitive to PI3K and Akt inhibitors that may be implies to posttranslational modification of PTEN and loss of normal function of PTEN in these cell line. Furthermore, Earlier studies indicated that Akt was emphatically activated in PTEN-negative cell lines. Activation of Akt results in Mdm2 phosphorylation and promotes translocation of Mdm2 from the cytoplasm to the nucleus. Moreover, inhibition of PI3k/Akt by PTEN results in accumulation of MDM2 in the cytoplasm. In PTEN-positive cells, MDM2 is localized mainly in the cytoplasm, whereas MDM2 is expressed mostly in the nucleus in PTEN-negative cells. However, it showed that PTEN-expressing cell lines did not display significant reduction of PI3k and Akt-p. In addition, transfection of PTEN did not significantly inhibit Akt-p expression in PTEN negative cell lines. These results indicate that PTEN-mediated cell survival and apoptosis are partially independent of the PI3k/Akt pathway.However, assessment of PI3K/Akt pathway activity in patient samples at diagnosis could be helpful to determine treatment programs.33 Various genetic and epigenetic alterations, as well as translational and post-translational modifications of PTEN imply decreased or complete loss of PTEN protein expression and activity in various cancers.47 Another important tumor suppressor is p53 that acts as a transcription factor and is involved in activation of the gene network responsible for a cell cycle arrest and apoptosis in response to DNA damages and cell stresses.49 Maintenance of low levels of p53 is required for cell viability in normal cells that is provided by its rapid ubiquitination. Stabilization of p53 is essential for its tumor suppressor function.50 Activation of PI3K/Akt signaling negatively controls p53 levels through promotion of Mdm2 movement in the nucleus.51 Mdm2 is an oncoprotein regulating the p53 protein levels and transcriptional activation through ubiquitin ligase activity.22 PTEN inhibits activation of PI3K/Akt signaling, prevents translocation of Mdm2 in the nucleus and positively regulates p53. Thus, it is clear that PTEN can control P53 function via inhibition of PI3K/Akt signaling with decrease of Mdm2 and p53 interaction and degradation.51 There are multiple layers of crosstalk between PTEN and P53,52 including transcription and protein levels affecting the cell death and survival.53 Mutation and inactivation of these genes were the most common elements in human cancers.54 Moreover, lack of PTEN tumor suppressor function cooperates with p53 loss for tumor development.23 Expression of PTEN in PTEN-null glioblastoma cells increases the expression of p53 target genes involved in cell cycle arrest.55 In fact, p53 upregulates PTEN, and PTEN protects p53 from survival signals by antagonizing PI3K/Akt signaling. PTEN–p53 associations demonstrated their dependence on each other in tumor suppression activity.56 We illustrated inhibition of PI3K/Akt signaling with BKM-120 and MK-2206 inhibitors upregulated PTEN gene and increased PTEN protein phosphorylation, whereas total PTEN protein levels did not change significantly. In addition, we found that inhibition of PI3K and Akt by BKM-120, and MK-2206 inhibitors caused upregulation of P53 mRNA and protein expression levels in all cell lines. It is likely that enhanced levels of PTEN phosphorylation upon PI3K/AKT inhibition affect p53 stability and increase P53 protein levels in all cell lines.PTEN as an important tumor suppressor that negatively controls PI3K/Akt pathway through dephosphorylation of PIP3.These findings are consistent with previous observations that mutation and loss of PTEN lead to deficiency or absence of p53 protein. Our results showed that p53 protein in PTEN negative cell line (Molt-4) displayed lower levels than PTEN positive cell lines.58IL-7 cytokine, as one of the important growth factors produced by bone marrow and thymic stroma, functions as a powerful proliferative stimulus for leukemia cells.59 IL-7 as an antiapoptotic factor plays a crucial role in normal T cell development through down-regulating the expression of the cyclin dependent kinase (cdk) inhibitor p27kip1, resulting in up-regulation of Bcl-2 expression, and cell cycle progression.60 Severe T cell deficiency was observed in defective IL-7R expression.61 IL-7 stimulates proliferation of pro–B cells and development of early B-cell stages.62 IL-7-mediated signaling contributes to survival and cell cycle progression63 and it has been demonstrated that IL-7 can active PI3K/AKT signaling in T-ALL.18 A previous study indicated that LY294002, a PI3K inhibitor, could prevent induction of proliferation by IL-7 in T ALL cells.64 Another study also showed that inhibition effect of rapamycin in B-precursor acute lymphoblastic leukemia lines could be reversed by IL-7. Our finding showed that inhibitory effects of BKM-120 and MK-2206 in the presence of IL-7 could be reversed, and IL-7 was capable of reversing the inhibitory effect of inhibitors by downregulation of PTEN and p53 mRNA levels.65IL-7 regulates lymphoid development and may stimulate proliferation and survival of ALL cells.This study was approved by ethical committee of Tabriz University of Medical Sciences with ethical number: TBZMED.REC.1394.752.Authors declare no conflict of interest in this study.This work was supported by Tabriz University of Medical sciences under Grant (5/104/996). We thank the Stem Cell Research Center for technical support.


Canine and human osteosarcomas (OS) are notably similar and have a high rate of metastasis. There is a poor understanding of the tumor development process, predisposing causes, and varying levels of aggression among different cell lines. By characterizing newly developed canine osteosarcoma cell lines, treatments for people and pets can be developed. Of the seven subtypes of OS, three are represented in this group: osteoblastic (the most common), fibroblastic, and giant cell variant. To our knowledge, there are no other giant cell variant canine OS cell lines in the published literature and only one canine fibroblastic osteosarcoma cell line. Understanding the differences between the histologic subtypes in dogs will help to guide comparative research.Alkaline phosphatase expression was ubiquitous in all cell lines tested and invasiveness was variable between the cell lines tested. Invasiveness and oxidative damage were not correlated with in vivo growth rates, where TOT grew the fastest and had the higher percentage of mice with metastatic lesions. TOL was determined to be the most chemo-resistant during cisplatin chemotherapy while TOM was the most chemo-sensitive.Further comparisons and studies using these cell lines may identify a variety of characteristics valuable for understanding the disease process and developing treatments for osteosarcoma in both species.Some of this data was presented as a poster by KMF at the August 5th, 2017 National Veterinary Scholars Program in Bethesda, MA.Characterization of 5 newly generated canine osteosarcoma cell lines.Kelli Franks, Tasha Miller, Heather Wilson-Robles. Immortalized cell lines are an invaluable resource and imperative to cancer research. However, the artificial conditions required to keep these cells alive in culture induce a variety of changes that likely would not occur in vivo –3. The lOsteosarcoma is the most common type of primary bone cancer that develops from osteoblasts and cells that form the osteoid matrix –9. ThereTreatment for both species typically involves surgical resection and chemotherapy, with 60% of human patients reaching a 5 year survival time . With meThe purpose of this manuscript is to describe five newly generated canine OS cell lines in an effort to better understand this devastating disease and allow for additional translational research to be performed in the future.A 72-h proliferation assay was performed and yielded doubling times for each cell line. TOL, TOM, TOT and TOK were similar with doubling times of 35.7 h, 34.85 h, 33.78 h, and 32.07 h respectively. TOB had the fasted doubling time at 29.3 h. The previously immortalized and published canine OS cells lines, Abrams and BWKOS, had doubling times of 18.72 h and 17.02 h, respectively .ALP staining performed on each cell line determined the percent of ALP positive cells present in each cell line. ALP expression was consistent between all cell lines as well as the normal osteoblastic cell line (TNO) and the two previously established OS cell lines, Abrams and BWKOS. All cell lines demonstrated 100% positive expression of alkaline phosphatase, though the degree of staining was variable between the cell lines  and TOT (p = 0.046) cell lines had significantly higher invasive ability when compared to the normal osteoblast cell line TNO, as did Abrams (p = 0.02) and BWKOS (p = 0.002)  of cells invading through the membrane compared to controls. This was the closest cell line to the invasiveness found in both the previously established cell lines, Abrams  and BWKOS . TOT had a mean of 50.42% (SD 4.58%) invasion followed by TOB with 45.86% (SD 2.13%) invasion through the basement membrane. TOK and TOM had the lowest mean percent invasion at 38.54% (SD 3.93%) and 9.26% (SD 4.3%), respectively. TNO was not invasive. Both the TOL (p = 0.01). TOT had similar levels with a mean of 14.47 nM . TOL had a mean of 9.83 nM  and TOM had the lowest degree of oxidative damage with a mean of 7.61 nM . Both TOB and TOT had a significantly higher degree of oxidative DNA damage when compared to the TNO cell line . The previously established cell lines, Abrams and BWKOS, had low levels of inherent oxidative damage present. Abrams was very similar to TNO with a mean of 1.07 nM (SD 0.25) and BWKOS had a mean concentration level of 3.8 nM (SD 0.03)  has been implicated in cancer development and progression . When exCisplatin is a platinum chemotherapy drug frequently used to treat osteosarcoma in both dogs and humans. Sensitivity to the drug was measured by determining cell viability after exposure to the drug at a variety of concentrations over several time points. Determination of IC50 revealed TOK to be the most chemo-resistant, with higher concentrations of cisplatin needed to inhibit growth and initiate cell death at 2 of the 3 time points measured. TOM and TOB are the two most chemo-sensitive with lower concentrations of cisplatin needed to induce cell death at all three time points. Indeed, an IC50 could not be determined for TOM at 72 h due to excessive cell death. The TOT cell line demonstrated initial resistance to cisplatin at the 24 h timepoint, however, the IC50 dropped significantly after 48 and 72 h of exposure to the drug. The previously established OS cell lines, Abrams and BWKOS, demonstrated similar sensitivities to cisplatin as the TOB and TOM cell lines. Table p < 0.0001, SED 6.2). The TOL cell line had 44.64% positive cells in the control samples compared to 86.42% cells in the ADM treated samples  and the TOK cell line had 17.2% positive cells in the control samples compared to 50% positive cells in the ADM treated cells . The TOB and TOM cell lines had 70.2 and 87.13% positive cells in the controls versus 77.2%  and 96.4%  in the ADM treated samples, respectively. Abrams was incapable of adipocyte differentiation even with ADM media stimulation with a mean of 1.2% positive cells in the control and 0.8% in the ADM media . The BWKOS cell line was capable of some differentiation with 5% positive cells in the control versus 22% in the ADM media , however, this was minimal compared to the newly derived cell lines even though it was significant . In the TOT cell line, the control contained a mean of 24.11% cells staining positive with ORO (oil red O) compared to a mean of 92.5% positive cells on the ADM treated slides . For the TOM cell line there was a mean of 2.2 osteoid islands per 10x field compared to 28.8 in the ODM sample , and for TOB there was a mean of 1.2 osteoid islands per 10x fields whereas the ODM sample had a mean of 33.8 osteoid islands per 10x field . The TOT cell line was surprisingly resistant to ODM with a significantly larger number of osteoid islands in the control (mean = 7.2) compared to the ODM sample . The two previously established canine OS cell lines, Abrams and BWKOS, were highly capable of creating osteoid islands. Abrams generated very large osteoid islands with nearly every cell staining positive for cytoplasmic osteoid. For the Abrams cell line there was a mean of 1 osteoid islands per 10x field compared to 56.2 in the ODM treated cells . BWKOS has very similar osteoid island production to that seen with TOL, TOM and TOB .TOL, TOM and TOB were capable of creating a significantly higher number of osteoid islands in vitro when exposed to osteogenic differentiation media (ODM). The control sample for TOL had a mean of 5 osteoid islands per 10x field compared to 28.6 osteoid islands in the ODM sample . The TOB cell line was surprisingly resistant to the CDM producing significantly fewer chondroid islands (mean- 0.6 per 10x field) compared to controls . The TOT line again produced fewer chondroid islands in the CDM media (mean-0.6) compared to controls . The TOL line produce slightly more chondroid islands in the CDM (mean- 6.3) compared to the controls  but this was not a significantly higher amount. Neither of the previously established canine OS cell lines was capable of any significant chondroid production with a mean number of 2.6 chondroid islands per 10x field in the control versus 3 chondriod islands in the CDM media  for the Abrams cell line and 0 chondroid islands for the control versus 0.4 chondroid islands in the CDM media   compared to controls . Several, though not all, of the TOM xenografts also demonstrated a rapid growth rate compared to the other cell lines with large tumors necessitating euthanasia in all 6 mice by day 56 . Five of the 6 mice developed tumors in each of the TOL  and TOB  groups and were euthanized due to ulcerations of the masses on day 84 after injection. None of the 6 mice injected with the TOK cell line were able to develop tumors after 12 weeks of monitoring. The Abrams cell line was also injected into 6 mice and growth rates recorded for 52 days . This cell line produced tumors in all 6 mice and had a similar growth rate to the TOM cell line  stained slides from 4 of the 5 cases were compared to H&E stained slides of the murine xenografts generated from each cell line. TOK did not produce tumors in mice so there was no tissue available for comparison. Additionally, slides from the primary tumor used to generate Abrams were not available to us for comparison. Histologic comparisons were made by an osteopathologist (RP). In general, the histologic characteristics for the tumors were preserved in vivo Fig. . For themor Fig.  a were pmor Fig.  e. A fusThe TOM xenografts Fig.  f retainThe tumor from which TOL was derived consisted of three major cellular patterns including a meshwork of hyperchromatic polygonal cells of moderate cell density containing small numbers of tiny spicules of tumor with angular profiles, a moderately dense filigree of small and larger haphazard spicules of tumor bone some of which is mineralized, and random sheets of highly cellular tumor containing a few spicules of tumor bone in which tumor cells were loosely packed but had tiny slit like spaces between the hyperchromatic tumor cells having angular profiles Fig.  c. The TThe TOB cell line was derived from a metastatic lesion Fig.  h collecWhole lung quantitative DNA analysis was performed on 4 of the 6 mice in each group. Species specific primers were used to identify foreign canine DNA in the lungs of the mice. This assay can predictably detect as little as 0.02% foreign DNA in samples. The TOB (2 of 4 mice), TOL (1 of 4 mice) and TOT (4 of 4) cell lines each had evidence of micrometastatic lesions present in lung samples. The amount of canine DNA was highest in the TOT xenograft mice. The mean percent of DNA in the TOM and TOK lung samples were 0 (SD 0) and 0.003 (SD 0.007), respectively. TOB had the highest mean percent canine DNA in the lung samples with 0.094% (SD 0.17). TOT has a mean percent of canine DNA of 0.082% (SD 0.068) and TOL had a mean percent of 0.015% (SD 0.01) with only one mouse registering above the 0.02% minimum reliable detection level of the assay. For the Abrams cell line the mean percent of canine DNA detected was 0.021% (SD 0.003) with 3 of the 4 mice having means above the 0.02% cut off  and only the second of a canine fibroblastic osteosarcoma (TOB) in the literature . These tSome of the more interesting findings in the characterization of these cell lines include the longer doubling times of the new cell lines compared to the existing cell lines, the difference in the degree oxidative in the newly generated cell lines compared to Abrams and BWKOS and the degree of plasticity in the newly generated cell lines compared to the more established cell lines.Newly generated cell lines often take time to immortalize and “learn” to grow on plastic in flasks. For this reason, it is not unexpected that the new cell lines require a little more time to divide and grow to confluence within the flask. It is expected that over time these cell lines, especially the more aggressive ones, will shorten their doubling times to be more in line with those of Abrams and BWKOS.The increased degree of oxidative damage present in the new cell lines as compared to the established cell lines was unexpected. Cancer cells and cell lines have been shown to have decreased levels of antioxidant enzymes such as manganese superoxide dismutase (MnSOD), leading to higher levels of ROS and increased DNA damage . This daThe previously established cell lines were only capable of differentiating when exposed to the ODM (Abrams and BWKOS) and ADM (BWKOS). This lack of plasticity was unexpected for these established cell lines. The newly generated cell lines demonstrated an increased ability to differentiate along adipocyte lines, however, there was a fair amount of lipid present within the cytoplasm of the cells even within the control cells. Altered metabolism is an emerging hallmark of cancer. Cancer cells are known for their ability to obtain energy from glycosis even with ample amount of oxygen present. This is referred to as the Warburg effect. This metabolic pathway causes cancer cells to increase the generation of cytoplasmic lipid droplets in the same way that cells with reduced access to nutrients and lipids would , 26. ThiThe TOT cell line consistently demonstrated an aggressive biologic behavior. This cell line had a high level of invasiveness, a high degree of oxidative damage, a high propensity for adipocyte plasticity, a rapid growth rate in a murine model with 100% xenograft formation and 100% metastatic rate. The cell line also had the highest MI in the xenografts examined. This aggressiveness mirrors the clinical picture associated with the primary tumor from which it was derived. This patient was treated aggressively with amputation of the affected limb and carboplatin chemotherapy. However, the progression free interval (PFI) was only 183 days when the patient presented with bone metastasis and the overall survival time (OST) was only 233 days., but a moderate metastatic rate. This was the only cell line derived from a metastatic lesion and this patient has the longest clinical course of the 5 dogs represented in this study. This patient, also treated with amputation and carboplatin chemotherapy, had a PFI of nearly 7 years (2383 days) with the presentation of lung and sternebra metastasis and an OST of 2402 days. Histologically, this tumor produced the most bone in the murine studies and had a moderately high MI. The characteristics of this cell line are not universally aggressive and the ability of this cell line to invade and spread may be sequelae of the fact that it was derived from a metastatic lesion rather than the true aggressive nature of the primary tumor.The TOB cell line had high invasive capabilities, a relatively quick doubling time, high oxidative damage, cisplatin sensitivity, very little plasticity, slow growth in vivoThe TOL cell line had many similarities to the TOT cell line in that it had the highest invasive ability, a moderate amount of oxidative DNA damage and plasticity, moderately high in vivo and in vitro growth rates and was fairly chemotherapy resistant. The metastatic rate for this cell line was low with only 1 in 4 mice demonstrating measurable metastatic disease however, if the mice had been allowed to live longer with their tumors this may have increased. The MI in the primary tumor was low but the xenografts had a moderately high MI of 30. The clinical course of this patient was altered by a surgical complication in which the dog developed a multi-drug resistant bacterial infection at the amputation site and was only able to receive 1 dose of chemotherapy before a significant treatment delay led to the development of pulmonary metastatic disease. The PFI for this patient was 130 days with an OST of 232 days.Despite an aggressive clinical course for the TOM cell line and the relative sensitivity to cisplatin seen in vitro this patient developed lung metastases 139 days after diagnosis even though she was treated with amputation and carboplatin chemotherapy. TOM was the most plastic of the cell lines evaluated with the ability to form exaggerated levels of adipocytes, bone islands and chondroid when stimulated to do so. The OST for this patient was 215 days even with additional chemotherapy. The TOM cell line represented one of the least biologically aggressive cell lines of those studied here and perhaps this patient would have benefited from cisplatin therapy rather than carboplatin.TOK cell line was the least aggressive cell line studied. Unfortunately, this cell line developed contamination during the study that could not be eliminated, and the cell line died out before completion of the oxidative damage assay as well as the osteogenic and chondrogenic differentiation assays could be done. This cell line had moderate ALP staining, moderate invasive characteristics and moderate plasticity with ADM. This tumor did not form any tumors in mice and no metastatic lesions were detected. It was, however, the most drug resistant cell line studied. This tumor displayed a fairly typical clinical behavior as the patient developed metastatic lesions at 286 days and lived a total of 380 days after diagnosis. This patient was treated with amputation and adjuvant carboplatin chemotherapy.Each of the 5 OS cell lines studied here display a variety of characteristics. There is a wide variety of biologic behaviors seen between the different cell lines with varying levels of invasiveness, ALP expression, and proliferation rates. The tumors in vivo varied widely in metastatic potential and growth rates as well. The cell lines also maintained many of the original histologic characteristics in the xenografts that were seen in the primary tumors. The three histological subtypes studied will broaden therapeutic treatment testing and provide new perspectives for understanding the complex process of tumor development and metastasis.2.Canine osteosarcoma (OS) cell lines TOB, TOL, TOK, TOM, and TOT were generated with owner permission from patients seen at the Texas A&M Small Animal Clinic Oncology Department in College Station, Texas (AUP 2015–0350 CA) . One milliliter of MSC growth medium was added to each slide. Slides were incubated at 37 °C in 5% CO2 until 75–80% confluence was achieved. The media was removed, and the slides were fixed with acetone and washed for 5 min in tap water The ImmPRESS AP Polymer kit was used to stain the cells for alkaline phosphatase  according to the manufacturer’s instructions. Briefly the cells were washed in buffer for 5 min before they were incubated in 2.5% normal horse serum as a blocking agent. The cells were incubated with a primary mouse antibody diluted 1:50 in buffer. The cells were washed again for 5 min in buffer and incubated with ImmPRESS-AP polymer Reagent and washed 2x’s in buffer before the alkaline phosphatase substrate was added for 24 h. The cells were rinsed in tap water and evaluated under the microscope.Cells were grown in flasks as described above to 75% confluence. They were trypsonized, collected and washed in PBS. 6 × 106 cells in 3 mL PBS and was added to each tube. The solution was centrifuged for 3 min at 1300 RPM. After decanting the supernatant, 2 mL of serum free media was added to the cell pellet and mixed with a pipette. On the 24 well plate, 600 μL of serum free media was placed in wells marked 0. Six hundred microliters of media containing serum was placed in wells marked + 1 and + 2. Thinserts were placed in wells designated 0 and + 2, while the coated thinserts were placed in the + 1 wells. 200 μL of the cells in serum free media were added to the top of the thinserts. The 24 well plate was placed in the incubator for 20 h at 37 °C. With a new 24 well plate, 450 μL of serum free media with 8 μM calcein-AM was added to each well. The thinserts were moved to the new plate containing the calcein-AM solution. The plate was incubated for 45 min at 37 °C. Each thinsert was removed and placed in a new well of a 24 well plate. The thinsert was aspirated and material on top of the thinsert was discarded. On a new 24 well plate 500 μL of trypsin-edta was added to each well. The thinserts were placed into the trypsin-edta wells. This plate was incubated for 15 min at 37 °C. Thinserts were discarded. The cells and trypsin were mixed and 200 μL were pipetted into new wells of a black 96 well plate in duplicate. The plate was read by the fluorescent plate reader (excitation 485 nm/emission 520 nm) .Coating buffer was made from 0.7% NaCl and 0.1 M tris in distilled water. It was filtered using a 0.2 μm sterile syringe filter and stored at 4 °C. Matrigel Matrix  was stored at 4 °C. The coating solution was made for a final concentration of 250 μg/mL. Fifty-four μL of matrigel was mixed into 1946 μL of coating buffer. The experimental, positive control and negative control wells were completed in triplicate and designated + 1, + 2, and 0 respectively. Three thinserts  per cell line were placed in a 24 well plate . These were coated in 100 μL of coating solution. The thinserts were placed in the incubator at 37 °C for 2 h. A cell suspension was prepared with 2 × 10Invasion index percentage was calculated using the formula:Invasion index % = /(positive control average – negative control average) × 100%.A white 96 well plate was labeled in triplicate with the cell line and number of cells plated. Initially, a variety of cell concentrations ranging from 500 to 3000 cells per well were plated to determine the best linear growth rate concentration. The appropriate amount of media was added to cell samples to reach a concentration of 100 μL needed per well. The plate was incubated for 2.5 h at 37 °C. Ten μL of the substrate and 10 μL of enzyme from the RealTime Glo Viability Assay  were mixed into 4980 μL of media to make a 2X solution. The solution was placed into a hot water bath for 5 min. Media was removed from the 96 well plate and 50 μL of new media was added to wells. Fifty microliters of substrate-enzyme solution was added to each well. The plate was incubated for 1 h at 37 °C. The Biotek Synergy 2 plate reader was prewarmed to 37 °C before the first reading. The plate was read on the prewarmed fluorescent plate reader  at 24, 48, and 72 h (488 nm) post plating. Results were graphed to determine linearity of concentrations. The following formula was used to determine the doubling time for each cell line:(t2 - t1)/3.32 x (log n2 - log n1) where t is time and n number of cells (27) {Hayflick, 1973 #8}.C. media was removed from the plate and the various serially diluted cisplatin concentrations in media were added to the wells in triplicates. 10 μL of MT Cell Viability substrate and NanoLuc Enzyme  were added to 4980 μL media and the solution was placed in the hot water bath for 5 min. 50 μL of the enzyme-substrate solution was added to all wells. The plate was incubated for 1 h at 37 °C and read on a prewarmed BioTek synergy 2 at excitation 485 nm/emission 520 nm. The plate was incubated at 37 °C before being read at 24, 48, and 72 h post plating.Cisplatin was received as a 1 mg/mL solution at 3.333 mM . From this a 400 μM solution was prepared. For the serial dilution of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM and 0 μM concentrations were generated. An acrid orange/ propidium iodide (AO/PI) stain was used to count live and dead cells per well for all cell lines. These surviving cell count numbers were determined to be linear. Media was added to cell volume to reach appropriate concentrations for 100 μL of cell solution per well. A white 96 well plate was used and all experiments were done in triplicate. The plate was incubated for 2.5 h at 37 °4 cells were counted and plated on chamber slides . One milliliter of MSC growth medium was added to each slide. For the chondrogenic assay, 1 × 105 cells were plated on chamber slides. One milliliter of MSC growth medium was added to each slide. Slides were incubated at 37 °C in 5% CO2. When the chondrogenic plate samples grew spheres at 60 h post plating, media was removed. Two slides received 1 mL of chondrogenic differentiation media and two slides received 1 mL of MSC growth medium. For the adipogenic assay cells reached 80–90% confluency before media was removed. Two slides received MSC growth media and two slides received adipogenic differentiation media. For the osteogenic assay cells reached 100% confluency before removing media and adding osteogenic differentiation media to two slides and MSC growth media to two slides. All differentiation slides were incubated at 37 °C. Every three days the media was changed. The chondrogenic and osteogenic slides were incubated for 21 days and the adipogenic slides was incubated for 14 days. Differentiation of cell lines was determined by doing a 500-cell count differential per slide of cells grown in the various differentiation media and cells grown in standard media as a control.Chondrogenic, osteogenic, and adipogenic differentiation media assays were performed. For the osteogenic and adipogenic assays, 6 × 106 cells/mL. DNA was extracted using the manufacturer’s protocol and quantitated spectrophotometrically (1OD260 = 50 μg/mL) to a final concentration between 200 and 1000 μg/mL. 100x cations, provided by the kit, were added to the DNA solution for final 1x concentration. The 8-OHdG standards and cell mixtures were added to a 96 well plate. Twenty-five microliters of the murine anti 8-OHdG monoclonal antibody solution (diluted 1:250) were added to each well (25 μL of diluent only were added to blank wells for negative controls) and thoroughly mixed. The plate was sealed and incubated at 25 °C for 1 h. Wells were washed 4 times with PBST and 50 μL of the goat anti-mouse IgG-HRP secondary antibody solution (diluted 1:500) was added to each well. The plate was again sealed and incubated at 25 °C for 1 h. The plate was washed again with PBST and 50 μL of pre-warmed TACS-Sapphire colorimetric substrate (provided by the kit) was added to all wells and the plate was incubated in the dark at 25 °C for 15 min. Fifty microliters of 0.2 M Hcl was added to each well to stop the reaction and absorbance was read immediately at 450 nm on a BioTek Synergy 2 fluorescent plate reader . The amount of oxidative damage present in each cell line was calculated using a standard curve generated with Graphpad Prism 7 software.The Trevigen HT 8-oxo-dG ELISA kit II  was purchased to assess the level of inherent oxidative DNA damage in each of the cell lines. The assay was completed according to the manufacturer’s instructions. Briefly, cells from each cell line were grown to confluence in T75 flasks (Corning), washed, trypsonized and collected. Cells were diluted in ice cold PBS at 1 × 106 cells in 170 μL Corning matrigel matrix was injected subcutaneously in the right flank. The animal’s weights were recorded, and the ears were notched for identification purposes. Every 2 to 3 days after injection, weight and tumor size were measured and recorded. When tumors reached 2 cm in diameter, became ulcerated, or when the mouse lost up to 20% of its body weight, they were humanely euthanized. Otherwise mice were followed for tumor growth for 10 weeks. Mice were euthanized in accordance with AVMA guidelines using CO2 inhalation provided by a compressed CO2 gas cylinder at a rate of 2–3 l per minute in a sealed 10 Liter volume chamber until they were unconscious (CO2 narcosis) followed by cervical dislocation. Tumors as well as whole lung tissue was collected from each mouse after euthanasia. For each cell line, tissues were collected and stored in Invitrogen RNAlater, formalin or flash frozen in liquid nitrogen. After 2 days in formalin all tissues were removed, rinsed and placed in 70% ethanol.A total of 30 female athymic nude mice were purchased from Envigo for the purposes of this study. All animal experiments were approved by the Texas A&M University Intuitional Animal Care and Use Committee (IACUC 2017–0127). Mice were housed in sterile cages with sterile bedding and 2–4 mice per cage. Mice were fed a sterile irradiated mouse chow. Mice were handled using sterile PPE under a sterile hood. Six mice were assigned per group in 5 groups by cell line. Media was removed from cell cultures and the T175 flask was rinsed with 5 mL PBS. The flasks were trypsonized and incubated for 12 min at 37 °C. Tuberculin syringes with 27-gauge needles were chilled in ice. A cell suspension of 1 × 10DNA was isolated from lung tissue using DNeasy® Blood & Tissue kit  in accordance with the manufacturer’s protocols. Quantitative PCR was performed using equal amounts of DNA with biological and technical triplicates . The primers are listed in Table p value of 0.05.Statistical analyses were performed using Graph Pad Prism 7 software. Alkaline phosphatase levels in each cell line were recorded and a Kruskal-Wallis test with a Dunn’s multiple comparison test was used to compare tumor cell lines to the TNO cell line. Cell line sensitivity to cisplatin was interpolated by calculating the fold change in viability based on a standard curve generated by exposure to cells of varying concentrations. Student’s T tests were used to determine the difference between cells grown in differentiation media and controls. For whole lung DNA analysis known canine and human DNA samples were used to generate sigmoidal standard curve from which all unknown values were determined. Once the unknowns were determined an unpaired t test was used to determine significant differences between treatment and control groups. Invasiveness, degree of oxidative damage, tumor growth rates and the number of mice with reported metastatic lesions were recorded and graphed. Significance was set at a Additional file 1: Figure S1. Differentiation Media in all cell lines. Five and 10X images of the control and differentiating media cells for the various cell lines. This file includes an image of the various cell lines (5 newly characterized ones) as well as 2 previously immortalized cell lines in differentiation medias .


Mycoplasma contamination is a major problem in cell culturing, potentially altering the results of cell line-based experiments in largely uncharacterized ways. To define the consequences of mycoplasma infection at the level of protein expression we utilized the reverse phase protein array technology to analyze the expression of 235 proteins in mycoplasma infected, uninfected post treatment, and never-infected leukemic cell lines. Overall, protein profiles of cultured cells remained relatively stable after mycoplasma infection. However, paired comparisons for individual proteins identified that 18.7% of the proteins significantly changed between the infected and the never-infected cell line samples, and that 14.0% of the proteins significantly altered between the infected and the post treatment samples. Six percent of the proteins were affected in the post treatment samples compared to the never-infected samples, and 7.2% compared to treated cells that had never had mycoplasma infection before. Proteins that were significantly altered in the infected cells were enriched for apoptotic signaling processes and auto-phosphorylation, suggesting an increased cellular stress and a decreased growth rate. In conclusion, this study shows that mycoplasma infection of leukemic cell lines alters the proteins expression levels, potentially confounding experimental results. This reinforces the need for regular testing of mycoplasma.The online version of this article (10.1007/s10616-018-0244-2) contains supplementary material, which is available to authorized users. Mycoplasma contamination of cell lines is one of the major problems of cell culturing, with infection present in 5–30% of the cell lines, despite significant improvements in diagnostic and therapeutic possibilities to detect and eliminate mycoplasma contamination  technology.As leukemic cell line experiments are an essential tool to gain biological knowledge, it is important to know the consequences of mycoplasma contamination and to establish stable and reproducible cell cultures. A study by Miller et al.  already Leukemic cell line samples (n = 52) derived from either pediatric or adult AML , ALL , CML (e.g. KBM5) and lymphoma (e.g. Raji) including various molecular modifications were obtained from different laboratories. Cell lines underwent Short Tandem Repeat DNA fingerprinting for cell line identity confirmation and were cultured according to the ATCC cell culture methods. All cell lines were tested for mycoplasma using the mycoplasma Polymerase Chain Reaction (PCR) detection kit . Of these, 16 were positive for mycoplasma infection and from these a sample was collected for protein isolation for the ‘‘infected’’ samples. Infected cell lines were then treated with 25 µg/mL Plasmocin™ Treatment  for 2 weeks, and PCR was repeated immediately post treatment to check their mycoplasma status. Cell cultures that were successfully treated , were then cultured for another 2–4 weeks and again tested by PCR to verify if cell lines remained negative during culturing. From each cell line culture cells were collected for the production of protein for the “post treatment” sample from this ~ 4-week sample. As it is conceivable that mycoplasma infection could have permanent effects on cell line protein expression patterns, we attempted to assess this by obtaining another sample of the same cell lines, that had never had a mycoplasma infection, as best as we could ascertain, for comparison. Those cell lines were cultured similarly and checked by PCR on mycoplasma before preparation of protein sample from the ‘‘never-infected’’ sample. For three infected cell lines, no never-infected counterpart could be found. In addition, to assess the effect of Plasmocin™ treatment on protein expression, four never-infected cell lines were treated and added to the array. In summary, a total of 52 samples were available for comparison of protein expression between the different mycoplasma states; twelve cell lines had a complete set of an infected, an uninfected post treatment and a never-infected sample, one cell line had an infected and never-infected sample, three cell lines had an infected and post treatment sample, and four cell lines that had a never-infected and post treatment sample that had never been infected before. More information regarding the cell lines, including type, mycoplasma status, disease and culture method are provided in Supplemental Table S1.4 cell/µL and printed in five serial (1:2) dilutions onto slides along with normalization and expression controls. Slides were probed with 235 strictly validated antibodies and a secondary antibody to amplify the signal  to produce quantified data.RPPA was used to determine the protein expression of 52 leukemic cell line samples and ten normal CD34+ bone marrow samples. Methods and antibody validation techniques are fully described in previous publications .The unsupervised hierarchical clustering procedure was performed using the Ward-linkage rule. Principal component analysis was employed to further visualize the distribution of each cell line in relation to their mycoplasma status. Paired Wilcoxon signed-rank test was applied to compare protein expression of individual proteins between the three sample conditions . To compare changes in protein expression between the never-infected versus samples post treatment that were never infected before (n = 4), we applied the Student’s Protein names were uploaded in the Database for Annotation, Visualization and Integrated Discovery version 6.8 and analyzed for gene ontology of biological processes. Biological processes that were significantly enriched  were selected.p value. These observations are in line with our expectations based on the mRNA literature  biological processes Fig. a–c. ProtTaken together, in this study we demonstrated that mycoplasma infection of leukemic cell lines alters the protein expression levels of individual proteins, whereas the overall protein profiles remained relatively stable. Subsequent pathway analysis of the significantly changed proteins in the infected cells compared to the post treatment or the never-infected samples showed significant enrichment for apoptotic signaling processes in particular, but also showed changes in proteins involved in protein auto-phosphorylation and cell proliferation. This may suggest that mycoplasma infection leads to an increased cellular stress response and a reduced cell growth. Also, as infected cells expressed lower protein levels for the majority of the significantly altered proteins compared to their never-infected and uninfected post treatment counterparts, this might imply that the infected cell lacks sufficient energy to synthesize their normal protein levels, as they have to share their metabolism with the mycoplasmas, but that once the mycoplasma is eliminated their metabolism is at least partially restored. However, a larger study should be performed on mycoplasma infected cell cultures to assess the effect on cell biology, as well as to assess the additional effect of Plasmocin™ treatment on the protein expression. In addition, because the majority of the clean counterparts of the infected cell lines originated from different laboratories than the infected and post-treatment samples, and were thus previously cultured under different circumstances, a study that will artificially infect negative cell lines to keep culturing conditions between cell cultures stable, will likely result in more accurate comparisons between cell culture samples.In order to establish clean cell cultures to make cell line experiments more reliable and reproducible, we strongly encourage the regular testing of cell cultures for mycoplasma and to treat cell lines if cultures test positive to avoid having the consequences of altered expression of these proteins confounding the experimental results. Yet, as few alterations remained between the post treatment samples and the never-infected samples in our cohort, and as changes in protein expression were observed after treatment of never-infected cells, additional cell culturing for more than 2 weeks after completion of the antibiotic treatment, or taking a clean cell line aliquot, should also be considered.Supplemental Table S1. Cell line information for the 52 samples included in the analysis. Information is provided regarding mycoplasma status, disease, source, and the ATCC culture method (XLSX 175 kb)Supplemental Table S2. A “Rosetta Stone” of the antibody and protein nomenclature. This table contains information about the RPPA modified antibody name, the HUGO gene symbol and post-translational modification, and the MiMI and the full protein name. Secondary, RPPA staining details contain information about the manufacturer of each antibody, together with the antibody name, the antibody source, catalog number, and the primary and secondary antibody dilutions (XLSX 35 kb)Supplemental Table S3. Paired comparisons between the proteins expression levels of the infected, post treatment and non-infected cells. Paired comparison between the protein levels of infected vs. non-infected (n=13) samples, infected vs. post treatment (n=15) samples and post treatment vs. non-infected (n=12) using the Wilcoxon signed-rank test. The median expression is showed for the samples in each comparison (XLSX 52 kb)Below is the link to the electronic supplementary material.