The facts that LPA is present at high concentration in ovarian cancer patients’ ascites and it may serve as a stimulator to cell migration, implicate the role of LPA in the ovarian cancer metastasis. Since LPA mediates various biological functions through its interaction with LPA receptors, we aim to investigate the correlation between the expression of LPA receptors and the metastasis of ovarian cancer.To test whether the LPA responsiveness correlated with the metastatic capability of ovarian cancer cells, we performed LPA induced invasion assay and peritoneal metastatic colonization assay with a panel of established human ovarian cancer cell lines. The expression of LPAR1-3 in different ovarian cancer lines was examined by qRT-PCR. We also tested the effects of LPAR1 inhibition or overexpression on ovarian cancer cell's invasiveness. To confirm our laboratory results, we detected LPARs expression in specimens from 52 ovarian cancer patients by qRT-PCR and immunohistochemistry.T-test showed that LPAR1, not LPAR2 or LPAR3, expression was significantly higher in the metastatic cell lines than in the non-metastatic cell lines (P = 0.003). Furthermore, silencing LPAR1 alone could significantly reduce LPA-induced invasion (P < 0.001). Finally, we analyzed the correlation between the LPARs expression and clinicopathological features of the clinical cases. It indicated that LPAR1 expression rate increased significantly along with the more advanced stages . Besides that, LPAR1 expression was detected in all the 13 cases with abdominal metastasis more than 2 cm, 10 cases with retroperitoneal lymph node metastasis and 6 cases with hepatic metastasis. Moreover, the expression rate of LPAR2 significantly increased in ovarian cancer than in normal specimens (P = 0.039). LPAR3 expression showed the same trend as LPAR2, though the difference is not statistically significant (P = 0.275). Besides that LPAR2 and LPAR3 expression increased along with poorer differentiation .Thirteen ovarian cancer cells were enrolled in the invasion assay. Ovarian cancer cell lines which responded well to LPA-induced invasion, also displayed good capability for metastatic colonization. On the contrary, cell lines with poor LPA responsiveness showed inferior metastatic potential in peritoneal colonization assay. High expression level of LPAR1 was detected in all of the metastatic ovarian cancer cell lines. Metastatic capability of ovarian cancer cells correlated well with their responsiveness to LPA for cell invasion. LPAR1 acts as the main mediator responsible for LPA-stimulated ovarian cancer cell invasion. LPAR2 and LPAR3 might play an role in carcinogenesis of ovarian cancer. Ovarian cancer is the most lethal disease among all the gynecological cancers. The high mortality rate of ovarian cancer is mainly due to the complications of metastasis. Once the epithelial cells covering the ovaries undergo neoplastic transformation, they exfoliate from the primary tumor and disseminate to the peritoneal cavity through implantation pattern. It is widely recognized that the accumulation of malignant ascites is one of the most typical behaviors of ovarian cancers and may help the cancer cells to seed the abdominal cavity organs with tumor implants .Lysophosphatic acid (LPA) is a growth factor-like phospholipid that elicits multiple cellular events, including cell migration, proliferation, and survival , 3. LPA It has been long recognized that the ability of cancer cells to invade the surrounding tissues is essential for metastasis. The facts that the levels of LPA are elevated in the ascites in ovarian cancer patients, and that LPA may serve as a stimulator to cell migration as well as protease production/activation, prompted us to hypothesize that LPA-stimulated cancer cell invasion may play a critical role in ovarian cancer metastasis. Here, we presented that the peritoneal metastatic colonization of ovarian cancer cells is associated with their ability to respond to LPA for cell invasion. Besides, we demonstrated that LPAR1 acts as the main mediator responsible for LPA-stimulated ovarian cancer cell invasion. LPAR2 and LPAR3 might play an role in carcinogenesis of ovarian cancer.2. LPA was purchased from Avantis Lipid . DMEM, serum, and other cell culture supplies were purchased from Maixin Biotechnology .The human ovarian cancer cell lines, ES2, OVCAR429, HEY, OVCAR433, OVCAR5, SK-OV3, OCC1, OVCAR3, TOV21G, HEC1A, IGROV1, A2780, and OVCAR4 were provided by the Department of Biochemistry and Molecular Biology, Georgia Regents University  as a kind gift. Cells were cultured in Dulbecco's modified Eagle’s medium (DMEM) supplied with 10 % (w/v) fetal bovine serum (FBS) at 37 °C in a humidified incubator containing 5 % COClinical specimens were obtained from 52 primary epithelial ovarian cancer patients and 15 non-tumor patients, who underwent ovariectomy due to other diseases at the Department of Obstetrics and Gynecology, Zhongnan Hospital of Wuhan University, Wuhan, China, from December, 2011 to December, 2015. All experiments were approved by the Ethics Committee of Wuhan University. The fresh specimens were frozen in liquid nitrogen and stored at −80 °C. The formalin-fixed/paraffin-embedded samples were also collected. Surgical pathological stages were assessed according to the International Federation of Gynecology and Obstetrics (FIGO) criteria. The range of carcinoma invasion and metastasis were confirmed by surgical exploration and postoperative pathological examination.5/well, in log phase) were detached and plated into the upper Matrigel-coated invasion chambers. The cells were then allowed to invade for 48 h. The remaining cells in the chambers were removed by cotton swabs and the invaded cells on the lower surface of the chambers were fixed and stained with crystal violet. Subsequently, the crystal violet-stained cells were solubilized with 10 % acetic acid and quantitated on a microplate reader at 600 nm. Fold increase in cell invasion was calculated to evaluate the effects of LPAR1-3 silence on cells’ responsiveness for LPA(OD600 LPA-induced cell invasion /OD600 base cell invasion).The effect of LPA on cell invasion ability was measured by Matrigel invasion assay . LPA dissolved in serum-free medium (20 μM) was added into the underwells of invasion plates as a chemoattractant to induce cell invasion, while the serum-free medium without LPA was used as control. Serum-starved ovarian cancer cells (107 cells/mice), and monitored for five weeks. The mice were then sacrificed and autopsied. Visible metastatic implants were observed and photographed. All animal experiment procedures were approved by the Animal Center of Wuhan University.Six-week-old athymic female homozygous nu/nu mice were purchased from and cultured in the Animal Biosafety Level-3 Laboratory of Wuhan University under sterile environment. Ovarian cancer cells in log-phase were trypsinized, washed, and resuspended in PBS. The mice were intraperitoneally injected with different cell lines (10−3 were designed and introduced into HEY and SK-OV3 cells. The shRNAs for each target genes were designed with the aid of web-based Invitrogen Block-It program and inserted into pLV-shRNA vector (Biosettia). The target sequences were as follows: sh-LPAR1: 5’-GGATACCATGATGAGTCTTCT-3’, sh-LPAR2: 5’-GCCTGGTCAAGACTGTTGTCA -3’, sh-LPAR3: 5’-GCCAAGGTGCAGTCTGCAATA-3’. Matrigel invasion assay was performed in LPAR1-3 knockdown cell lines, respectively. Vector containing coding sequence of LPAR1 were got from Biochemistry and Molecular Biology Department, Georgia Regents University, as a gift. Lentiviral vector encoding LPAR1 were prepared by subcloning the coding sequence into pCDH-CMV-MCS-EF1-Puro vector. TOV21G and OVCAR3 cells were respectively infected with lentiviral vectors encoding LPAR1 for 2 days and chosed by puromycin. The efficiency of LPAR1 over-expression was tested by RT-PCT. After 2 days starvation, cells were stimulated with 20 μM LPA and followed by the Matrigel invasion assay.To test the role of LPARs in LPA-induced cell invasion, specific shRNAs to LPAR1@Universal PCR Master Mix Kits . The reaction was performed using ABI 9500Fast Real-time machine and the conditions were as follows: 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The expression levels of the target gene were standardized by comparing the Ct value of target genes to the GAPDH, and presented as 2 [Ct(GAPDH) - Ct(target gene)]  .Paraffin-embedded sections (4 μm-thick) were deparaffinized and rehydrated. Hydrogen peroxide treatment was used to block endogenous peroxidase activity. Sections were blocked with goat serum and incubated with polyclonal antibodies against LPAR1(Cat#: PAB10126), LPAR2(Cat#: A-ALS10695) or LPAR3 (Cat#: A-ALS10242). Primary antibodies were purchased from Amyjet Scientific Inc. Antigens were visualized by streptavidin-biotin-peroxidase complex method. Immunostaining was evaluated by two pathologists without knowledge of patients’ clinical information. All three antigens were found to be localized in cytoplasm. Extent of immunostaining was graded based on the percentage of cells displaying staining. “-” is considered as negative staining (<10); “+”, “++” and “+++” were considered as positive staining .t test. Chi-square test and Fisher's exact test were used to compare covariates between LPARs expression and clinicopathological parameters. All of the statistical tests were two-sided and P-values of less than 0.05 were indicated as statistically significant.Statistical analyses of the invasion assay were performed by ANOVA and independent To test whether the LPA response correlated with the metastatic capability of ovarian cancer cells, we performed LPA-induced invasion assay and peritoneal metastatic colonization assay with a panel of established human ovarian cancer cell lines. Invasion assay revealed that not all the cell lines responded well to LPA. A significant increase in cell invasion of ES2, OVCAR429, HEY, OVCAR433, OVCAR5, SK-OV3, and OCC1 lines was observed with LPA stimulation. However, the OVCAR3, TOV21G, HEC1A, IGROV1, A2780, and OVCAR4 cell lines showed poor or no response to LPA with regard to cell invasion. Subsequently, the metastatic potential of these cell lines was assessed by analyzing metastatic colonization with a well established peritoneal seeding model , 23. Anit-test established that LPAR1 expression was significantly higher in metastatic cell lines than in non-metastatic cell lines (P = 0.003). However, we also noticed that not all of the non-metastatic cell lines were low level LPAR1 expressing ones. OVCAR3, IGROV1 and TOV21G also expressed a moderate level of LPAR1. On the contrary, we did not detect any statistically significant difference in LPAR2 and LPAR3 transcript levels between metastatic lines and non-metastatic lines. These results indicate the possibility of LPAR1 as the key factor for ovarian cancer cell metastasis.LPA-induced cellular events can be potentially mediated by multiple LPA receptor subtypes , 18. In P < 0.001). On the contrary, LPAR2-shRNA displayed slight and LPAR3-shRNA exhibited no effect on LPA-induced cell invasion . These results indicated that LPA-stimulated ovarian cancer cell invasion was mediated mainly through LPAR1. Our data above also showed some cell lines with moderate LPAR1 expression were non-metastatic ovarian cells. To rule out the possibility that LPA non-responsive lines contained potential function-impairing mutation in LPAR1 sequence, we lentivirally transduced LPAR1 into IGROV1, TOV21G and OVCAR3 lines. However, these lines with LPAR1 overexpression remained non-responsive to LPA for cell invasion. These results indicate that the inability of LPA to stimulate cell invasion in LPA non-responsive line is not at the step of LPA receptors.Specific shRNAs targeting LPAR1-3 were designed and introduced into HEY and SK-OV3 cells. The efficiency of target gene knockdown was confirmed by qRT-PCR Fig. . SilenciP = 0.039). LPAR3 expression rate is also increased in cancer than in normal specimens, though the difference is not statistically significant (P = 0.275). On the contrary, no significant difference in LPAR1 expression between normal or cancer specimens were observed (P = 0.658). To further evaluate the role of LPARs in ovarian cancer metastasis, we analyzed the relationships between the expression of LPARs and clinicopathological features. As presented in Table P = 0.003). Besides, LPAR1 expression was detected in all the 13 cases with abdominal metastasis, more than 2 cm; 16 cases with retroperitoneal lymph node metastasis; and 6 cases with hepatic metastasis. We also found that LPAR2 and LPAR3 expression rate increased along with the more advanced pathologic grades . The immunohistochemistry also demonstrated that LPAR1 positive percentage increased along with the clinical stages , while LPAR2 and LPAR3 positive percentage increased along with the pathologic grades  . The qRT-PCR results showed that LPAR1, LPAR2, and LPAR3 were positive in 75.00 %, 12.50 %, and 6.25 % in the 15 of the normal ovarian specimens, respectively; and 69.23 %, 42.31 %, 17.31 % in the 52 of the ovarian cancer specimens, respectively. The expression rate of LPAR2 was much higher in ovarian cancer specimens than in normal ones , and then stimulate multiple downstream signaling pathways including Ras-MAPK, Rho GTPase, and KT/PKB. Eventually, it can trigger a series of biological events [2 to be less efficacious [2 and LPA3 mRNAs were higher in most ovarian cancer cell lines as compared with normal ovarian epithelial cells. However, in our study, we demonstrated that LPAR1 expression in invasive ovarian cancer cells was significantly higher than in non-invasive ones; while the expression of LPAR2 and LPAR3 had no statistical correlation with the metastatic potential of ovarian cancer cells. This difference may be attributed to differences in the cell lineage. Moreover, we observed that LPAR1 was highly expressed in all invasive ovarian cancer cells and all the three low LPAR1 expressing cells are non-invasive ones through there are still three of non-invasive lines expressing a moderate level of LPAR1. Besides that, silencing LPAR1 alone could significantly reduce LPA-induced invasion. Our in vitro data suggested that LPAR1 is the major receptor of LPA-induced ovarian cancer metastasis. As multiple signaling pathways are involved in tumor cell migration, there may also be other key factors in signal transmission of ovarian cancer besides LPAR1. Lack of certain downstream factors may lead to the depression of tumor cell invasion. This may explain why LPAR1 overexpression could not render IGROV1, TOV21G and OVCAR3 lines capable of responding to LPA for invasion in our study.LPA mediates various biological responses through its interaction with LPA receptors, namely LPAR1, LPAR2, and LPAR3, which belong to the G protein-coupled receptor (GPCR) superfamily. By binding to LPARs, LPA can activate three distinct G-protein subfamilies (Gl events . Most ofl events . Mayumi l events . Chen eticacious . In a si2/LPA1 in cancer tissues contributes to pathogenesis in cancer biology [LPA receptors are widely distributed in embryos, tissues, and cell lines, and each subtype has a distinct specificity. Recent studies have evaluated the expression of LPA receptors in clinical specimens. For instance, Shida et al. observed a reduced expression of LPAR1 and increased expression of LPAR2 in colorectal cancers as compared with normal mucosa. The ratio of LPA biology . The exp biology . In our In summary, our study demonstrated that LPA response might be a prerequisite for peritoneal metastasis of ovarian cancer cells, and that LPAR1 is the major mediator for LPA-induced ovarian cancer invasion as well as metastasis. Although our results were supported with established ovarian cancer cell lines, which may not completely simulate the clinical settings, the consistency seen in multiple cell lines, the convergence of loss- and gain-of-function findings, and especially, the significant correlation observed between LPAR1 expression and advanced disease stage/wider spreading range strongly argue against any confounding influence derived from our experimental studies. As LPA receptors are located on the cell surface and easily influenced by drugs, there lies immense potential in developing a therapeutic approach by targeting LPAR1 and its downstream factors.


Chordomas are rare, slowly growing tumors with high medical need, arising in the axial skeleton from notochord remnants. The transcription factor “brachyury” represents a distinctive molecular marker and a key oncogenic driver of chordomas. Tyrosine kinase receptors are also expressed, but so far kinase inhibitors have not shown clear clinical efficacy in chordoma patients. The need for effective therapies is extremely high, but the paucity of established chordoma cell lines has limited preclinical research. Here we describe the isolation of the new Chor-IN-1 cell line from a recurrent sacral chordoma and its characterization as compared to other chordoma cell lines. Chor-IN-1 displays genomic identity to the tumor of origin and has morphological features, growth characteristics and chromosomal abnormalities typical of chordoma, with expression of brachyury and other relevant biomarkers. Chor-IN-1 gene variants, copy number alterations and kinome gene expression were analyzed in comparison to other four chordoma cell lines, generating large scale DNA and mRNA genomic data that can be exploited for the identification of novel pharmacological targets and candidate predictive biomarkers of drug sensitivity in chordoma. The establishment of this new, well characterized chordoma cell line provides a useful tool for the identification of drugs active in chordoma. They are rare tumors, accounting for 1–4% of bone cancers, that occur more frequently in males than females and have a peak of incidence at around 60 years of age, although adolescents and children can also be affected3. Despite being slow progressing, chordomas are locally aggressive and invasive, and can also spread distally generating metastases in soft tissues, liver, lung, lymph-nodes and skin2. No therapeutic options have so far revealed to be efficacious for this indication, which is highly resistant to conventional chemotherapy, and consequently no approved standard of care exists. Extensive surgery and/or high dose radiotherapy is currently used to treat the disease, but tumor location, frequently close to cranial or lombo-sacral nerves and blood vessels, makes the achievement of negative surgical margins very challenging5. In most cases local relapses occur and represent the main cause of death for patients7. Chordomas therefore retain a strong unmet medical need, and new efficacious therapies are urgently needed.Chordomas are malignant bone tumors that arise along any region of the axial skeleton, more frequently at sacro-coccygeal or skull-base ends8. Brachyury re-expression in notochord remnants is believed to play a major role in chordoma onset and maintenance and its expression is considered the main distinctive molecular marker of chordomas9. Brachyury expression is therefore considered a mandatory feature for the validation of chordoma cell lines10. Chordomas consistently also express other molecular markers, such as epithelial membrane antigen (EMA), vimentin, cytokeratin 19, CD24v and CAM5.211. Moreover, variable expression and activation of several receptor tyrosine kinases (RTKs) and downstream signaling molecules have been reported. Among these, EGFR, PDGFR and MET represent the most frequently expressed and activated RTKs in chordoma14.Chordomas are thought to originate from remnants of the notochord, a transient structure required for patterning the surrounding tissues in all chordate embryos, that disappears later in development and is absent in adults. Among other developmental signals, notochordal cells express the transcription factor brachyury, a key molecule for mesoderm specification that is silenced in adult tissueswww.chordomafoundation.org) include the prototype cell lines U-CH1 and U-CH2 and a few other more recently established cell lines17.Very few bona fide chordoma cell lines have been available until recently, limiting the identification of relevant targets and the development of effective drugs. Validated chordoma cell lines available from the Chordoma Foundation , in order to compare genomic profiles and to evaluate the expression of kinases which might represent potential new therapeutic targets.Here we describe the generation and characterization of the new Chor-IN-1 chordoma cell line, established from a surgical sample of a sacral chordoma. The Chor-IN-1 cell line was shown to display the morphological and growth features of chordoma and to express brachyury, as well as other key relevant markers associated with chordoma diagnosis. This newly established cell line was characterized in parallel with U-CH1The original surgical sample was obtained from a 55 year old man diagnosed with a locally advanced sacral chordoma. A sacral nodule of 2 cm of diameter, macroscopically invading the surrounding soft tissues, was surgically excised. Histological diagnosis of chordoma was made according to WHO classification (2013). Morphologically, the tumor recapitulated the features of conventional chordoma, exhibiting lobulated growth of round epithelioid cells separated by fibrous septa. The cells, arranged in ribbons and nests, showed eosinophilic and/or vacuolated cytoplasms  and were embedded into abundant extracellular matrix. Immuno-phenotyping revealed expression of vimentin, S100, brachyury and EMA Fig. .Figure 118. The cell line was confirmed to express brachyury and EMA by immunocytochemistry (p13), −2, add(3)(q21), +7, del(9)(p21), −13, −22, +mar[cp10], a pseudo-diploid karyotype with chromosomal numerical and structural abnormalities typically found in this tumor  Fig. 9.The Chor-IN-1 cell line was then characterized for the expression of key RTKs and downstream signaling molecules, in comparison to the other chordoma cell lines U-CH1, U-CH2, MUG-Chor1 and JHC7. Western Blot analysis revealed that Chor-IN-1 expresses significant levels of EGFR, PDGFRβ and c-Met proteins, with activation of STAT3 and a very weak P-AKT Fig. .Figure 2Brachyury protein was expressed at comparable levels in all cell lines Fig. . InteresThe Chor-IN-1 cell line was authenticated by Short Tandem Repeat (STR) analysis in parallel with the parental tumor sample. The identity of the other chordoma cell lines was also confirmed by comparison with the published STR profiles , which allows the complete sequencing of the exonic regions of a subset of 4,813 genes harbouring disease-causing mutations, covering genes reported in the Human Gene Mutation Database (HGMD), in the Online Mendelian Inheritance in Man (OMIM) catalog and most of the genes currently reviewed in clinical research. Variants (SNVs and short InDels) detected in the Chor-IN-1 cell line and in the original tumor were highly consistent (>80%). All but four of these variants were also present in the DNA extracted from the non-tumor tissue component, therefore indicating that they represent germline variants. Tumor unique variants were identified in the MUC1, KEL, TECTA and SART3 genes  in the Chor-IN-1 and in all the other chordoma cell lines, which was also present in the original tumor tissue and in its normal component, as confirmed by Sanger sequencing  to investigate the kinome gene expression in chordoma (~ 500 kinases). Gene expression values for the whole kinome are provided as Supplementary Table https://cran.r-project.org/web/packages/kohonen/index.html). This analysis showed a very consistent kinase expression profile among chordoma cell lines, which differs from that of control placenta tissue . ULK4, NPR1 and CDKL4 were the top most expressed kinases in the Chor-IN-1 cell line, while FGFR3, KDR and WNK2 were less expressed or absent in Chor-IN-1 cells as compared to the other chordoma lines. The differential expression of these kinases in Chor-IN-1 was confirmed by RT-qPCR .In keeping with the indolent nature of this tumor, chordoma cell lines display very long doubling times in culture and require several months to reach stabilization. The Chor-IN-1 cell line has been maintained We decided to characterize Chor-IN-1 in parallel with four other cell lines of sacral origin, including the most widely used U-CH1 and U-CH2, as well as the more recently established MUG-Chor1 and JHC7 cell lines.19. Also, the availability of patient DNA from the non tumoral tissue highlighted that the SNVs identified in the tumor tissue and Chor-IN-1 cell line were mainly germline, with four exceptions involving TECTA, SART3, KEL and MUC1 genes. Interestingly, MUC1  is the gene encoding EMA, a transmembrane glycoprotein, commonly used as a biomarker for chordoma diagnosis23. This variant lies within an insertion region of unknown significance, located within the signal peptide present in a subset of MUC1 isoforms. The biological significance of the mutations identified in these genes will require further studies.In general, the TruSight One analysis did not identify gene variants common to the different cell lines, in agreement with literature data24.Next we investigated gross chromosomal rearrangements by mean of aCGH and NGS analysis. Several chromosomal alterations shared among the chordoma cell lines were identified. In particular, all chordoma cell lines apart from JHC7 share a biallelic loss of 9p21. This region includes the loci for CDKN2A and CDKN2B tumor suppressor genes and is reported to be frequently altered in chordoma tumor samples (~59%)26. Interestingly, the EGFR gene is located in the short arm of chr7 and is therefore not affected by the partial trisomy in the JHC7 cell line. In general, the major chromosomal abnormalities, common to the chordoma cell lines reported here, well recapitulate the most common alterations reported in Progenetix database for chordoma clinical samples.Similarly, trisomy of chr7, clearly evidenced also by karyotype analysis of the Chor-IN-1, is a feature shared by all the chordoma cell lines with the exception of JHC7, which bears a partial chr7 trisomy involving only the long arm. This trisomy was previously reported in a significant fraction of chordoma clinical samples27; LIG4, whose mutations are responsible for LIG4 syndrome, a disease characterized by dysmorphic features and microcephaly28 and WWP1, an E3 ubiquitin ligase involved in the regulation of osteoblast functions29. Of interest, an evident amplification of the MALAT1 locus, associated with the proliferation and metastasis of tumor cells, was observed.Besides these features, typical of chordoma, the Chor-IN-1 genome harbors peculiar chromosomal alterations in regions containing bone dysmorphism-associated genes, including DACH1, a gene involved in regulation of gene expression and cell fate determination during development, reported to downregulate EGFR and cyclin D1 and associated with osteosarcoma development30. It is tempting to speculate that the newly identified “chromosome A” might have been assembled from portions of different chromosomes following a catastrophic chromothriptic event that occurred in the development of this tumor.Chor-IN-1 major alterations were also detected by karyotype analysis. In particular, this analysis revealed the presence of an anomalous submetacentric C-like chromosome (chromosome A), possibly originated from the rearrangement of different portions of other chromosomes. This might account for the presence of the short arm of chr2 and of partial long arm of chr13 observed in aCGH analysis and not in the karyotype. Interestingly, chordomas were described as being among tumors that undergo “chromothripsis”, a peculiar event involving massive genomic rearrangements of one or few chromosomes31. We reasoned that detection of abundantly/differentially expressed kinases in chordoma cell lines might represent a convenient strategy for the identification of potential new pharmacological targets in this disease.We next focused on the characterization of protein kinase gene expression in chordoma cell lines. Kinases constitute one of the largest known families of enzymes that control different cellular functions and whose deregulation plays a causal role in cancer. A number of receptor tyrosine kinases have been reported to be implicated in chordoma pathogenesis, including EGFR, PDGFRβ, and c-METet al., manuscript submitted). The current analysis highlighted AXL, DDR1, DDR2, EPHA4, EPHB4, EPHA2, FGFR1, FGFR2, MET and ERBB2 as other interesting kinases that deserve further investigation as potential biologically relevant targets in chordoma.We provide here data on kinome gene expression in the different chordoma cell lines, which can be exploited to investigate the molecular basis for the sensitivity of each cell line to the different kinase inhibitors. Several among the most widely expressed kinases are inhibited by drugs currently undergoing clinical development, which may represent a new therapeutic option also in chordoma. We recently investigated in depth the role of EGFR inhibitors, providing a rationale to start a clinical trial with afatinib in this setting  family. Although little is known about ULK4 function, its role in chordoma deserves further investigation since the other members of the family have been implicated in autophagic pathways. CDKL4 (Cyclin Dependent Kinase Like 4), a member of the CDK family which includes CDK4 and CDK6, is also highly expressed. Interestingly, the use of CDK4/6-specific inhibitor palbociclib was reported to efficiently inhibit tumor cell growth In conclusion, we generated and extensively characterized Chor-IN-1, a new chordoma cell line that represents a valuable contribution to the preclinical research in the field, in view of the paucity of current cell models and of the heterogeneity of chordoma tumors. The Chor-IN-1 cell line will be made available to be used for preclinical studies aimed at further understanding the pathogenesis of chordoma and its sensitivity to available drugs. Moreover, we have generated genomic data at the DNA and RNA level that can be exploited for the identification of biomarkers of drug sensitivity, and for the identification of novel pharmacological targets in chordoma.The surgical sample was obtained from a patient initially diagnosed with sacral chordoma. The patient refused surgery, and received imatinib and radiotherapy. After three years, the patient experienced local subcutaneous progression at lombo-sacral level, received again imatinib and, upon further progression, also metformin. A tumor biopsy was performed when the patient finally underwent surgery. A sacral nodule of 2 cm of diameter, invading macroscopically the surrounding soft tissues, was surgically excised. All methods were performed in accordance with the relevant guidelines and regulations.The patient gave his informed consent to study his tumor, followed by the approval of the Fondazione IRCCS Istituto Nazionale dei Tumori  Ethical Committee for the genotypic and phenotypic characterization of the cell line to confirm the identity with original tumor.2. Cells were monitored daily to evaluate growth and morphology, and were carefully detached and re-seeded upon reaching confluence. After three months in culture the resulting cell line, named Chor-IN-1, displayed stable morphological and growth features and was further maintained in culture up to 50 population doublings, which required fourteen months. For doubling time calculation, Chor-IN-1 cells were seeded into 12 well plates  into 1 ml of culture medium. Cells were carefully detached from two wells every day and counted using a Multisize 3 (Beckman Coulter). Doubling time was calculated on the exponential part of the growth curve, using the formula DT = (Log2 Cell number[Tx] − Log2 Cell number[T0])/Tx (hr).Fresh aseptic surgical chordoma sample was minced and incubated with collagenase  for three hours and the obtained cell suspension was filtered through a 45 µL nylon mesh, washed with PBS/FCS 0.5% and seeded on collagen coated plates using 70% DMEM 30% IMDM2 supplemented with 10% of fetal bovine serum. When confluence was reached, cells were carefully detached and passed in new flasks at 20,000 cells/cm2. Cultures were all routinely checked for mycoplasma contamination using MycoAlert (Lonza– USA).The other chordoma cell lines were obtained from the Chordoma Foundation  or purchased from ATCC (MUG-Chor1). Chordoma cell lines were maintained using collagen-coated flasks (Cellcoat Cat 658950 Collagen Type I – Greiner Bio-one) in an appropriate culture medium (IMDM/RPMI 1640 4:1 ratio), supplemented with 10% (v/v) heat-inactivated fetal bovine serum  at 37 °C in a humidified atmosphere containing 5% COThe following antibodies were used: Anti-EGFR ; anti-PDGFRβ ; anti-MET ; anti-P-AKT ; anti- AKT ; anti-P-STAT3 ; anti-STAT3 ; anti-brachyury , anti- GAPDH .HRP-conjugated secondary antibodies were used 1:10000 (Immunopure goat anti-mouse and Immunopure Goat Anti-rabbit from Thermo-Scientific). Detection was performed using SuperSignal west Pico Chemiluminescent substrate from Thermo ScientificRepresentative sections obtained from the formalin fixed paraffin embedded (FFPE) tumoral sample of the case were selected and phenotyped. IHC was done with 2-µm FFPE sections. Anti-Brachyury  and Anti-vimentin , were diluted 1:400, while Anti-EMA  was diluted 1:250. Antigen retrieval and development was carried out using the fully automated (DAKO) instrument in accordance with manufacturers’ instructions.Chor-IN-1 cells were detached from plates using 1% trypsin, washed twice with PBS and then fixed using 10% PBS-buffered formalin. After fixation, cells were included in 300 µl of pre-heated (60 °C) 2% agar solution . After agar solidification, cells were included in paraffin according to standard procedures. IHC was done with 3-µm cell block sections. Brachyury and EMA IHC were performed as described above.IHC images have been scanned with Aperio Scan Scope XT (Aperio Technology). Length indicators are reported below. Magnification is 40× for all images. Digital zoom and scale bar are as follows: H&E ; brachyury ; EMA ; physaliferous morphology ; S-100 ; vimentin ; brachyury in cell line ; EMA in cell line .Karyotype was obtained according to standard procedures: chromosome preparations were obtained from semi-confluent petri at the seventh passage by ipotonic treatment  followed by Cornoys fixative (Methanol:Acetic Acid = 3:1). Slides were G banded with Wright staining and analyzed at 100 X magnification with a Leica DM6000D microscope.2 for 30 minutes, then washed with PBS containing 1% FCS (staining buffer) and counted. 0.5 × 106 cells of each cell line were resuspended in 100 µl staining buffer and stained with 10 µl PE-conjugated mouse anti-human CD24 or its correspondent PE-conjugated isotype control  for 20 minutes at room temperature. Samples were washed with staining buffer, fixed with 1% formaldehyde for 10 minutes at 37 °C and permeabilized with 90% methanol for 30 minutes on ice. After washing with staining buffer, 10 µl of FITC-conjugated mouse anti-cytokeratin (CAM5.2) or its correspondent FITC-conjugated isotype control  were added and incubated for 20 minutes at room temperature. Samples were washed with staining buffer, then acquired and analyzed with a FACSCalibur cytometer and CellQuest software . Analysis was performed on 10,000 events, gating out debris and doublets.U-CH2 and Chor-IN-1 cells were collected by trypsinization and recovered in their culture medium at 37 °C and 5% COGenomic DNA was isolated from all chordoma cell lines and from frozen primary parental tumor tissue using NucleoSpin Tissue kit , according to manufacturer’s instructions.DNA from patient-derived FFPE clinical samples for the Chor-IN-1 parental tumor and adjacent normal tissues was extracted from 7 µm slices using the RecoverAll Total Nucleic Acid Isolation kit , after tumor area selection and deparaffinization with xylene and absolute ethanol, according to manufacturer’s instructions. DNA yield and purity were determined by absorption measurement at 260 nm and by evaluation of the 260/280 ratio value using a Nanodrop 1000 Spectrophotometer .RNA was extracted from the cell lines using the RNeasy Mini Kit , as per manufacturer’s instructions. RNA yield and purity were evaluated by measurement of UV absorption at 260 nm and analysis of the 260/280 ratio using a Nanodrop 1000 Spectrophotometer .The STR identity profiles of all the chordoma cell line samples and of the Chor-IN-1 original tumor tissue were verified using the PowerPlex 16 HS System kit  which allows the simultaneous amplification of 15 STR loci, together with the amelogenin gender marker.Briefly, 0.5 ng of purified genomic DNA was amplified with the recommended PCR protocol and capillary electrophoresis was performed using the 3100 Genetic Analyzer  according to the PowerPlex 16 HS System kit manufacturer’s guide.33.Resulting data were processed using the GeneMarker HID v 2.4.0 software  and comparative analysis was performed with the CLIFF (Cell Line Identity Finding by Fingerprinting) softwareThe DNA polymorphism  in the “T” gene (Brachyury) was characterized by PCR amplification using the following primer pairs: FW 5′-TCACCAACAAGCTCAACGGA-3′; REV 5′-AAGCAGTCACCGCTATGAAC-3′  in a total reaction volume of 50 µl, containing 1× PCR buffer, 0.2 mM each dNTP mix, 0.8 µM of each primer, 1.25 units of FideliTaq DNA Polymerase  and 100 ng of genomic DNA as template.Primers for PCR amplification and sequencing were designed using the freely available Primer3 software (http://primer3.ut.ee/) and synthesized using an Applied Biosystems 3900 Synthesizer .For direct sequencing, the obtained PCR product was electrophoresed in agarose gel, then purified using NucleoSpin Gel and PCR clean-up , according to the manufacturer’s protocol, and subjected to Sanger sequencing with a ABI 3100 Genetic Analyzer instrument  with the same primers used for PCR amplification.Chordoma clinical samples and cell lines were profiled using TruSight One (TSO) sequencing panel kit , following manufacturer’s instructions. The protocol allows the detection of single nucleotide variants and short InDels over the entire coding sequence of 4,813 genes (http://www.illumina.com/products/trusight-one-sequencing-panel.html). Briefly, 50 ng of input DNA were used for each sample for library preparation. Pooled libraries were quantified with the Qubit 2.0 Fluorometer System using the Qubit dsDNA HS assay kit . The libraries were sequenced in paired-end on the MiSeq platform  using the v3 reagent kit (2 × 150 bp). A minimum mean coverage depth of 100× was achieved for all the profiled samples.The FASTQ files obtained from the instrument were analyzed following GATK gold standard procedures. Briefly, the sequences were mapped against the human reference genome (hg19) using BWA (v. 0.7.7) and the gene variants were identified using the Unified Genotyper (GATK v1.6) variant caller. Synonymous variants, variants with minor allele frequency >2% (reported in dbSNP) and low coverage variants  were excluded from further analysis. Somatic variants characterizing the Chor-IN-1 cell line were also excluded by identifying variants in the normal tissue counterpart and subtracting them from the intersection of Chor-IN-1 cell line variants with the original tumor variants. A pairwise similarity matrix was computed comparing the variants identified in each chordoma cell line.www.progenetix.org.In each genomic interval, coverage values obtained from TSO analysis were used to calculate the log2 ratio of the local versus sample mean coverage, imposing a cutoff of log2 > 0.2 for the regions of gain and of log2 < −0.2 for the regions of loss. Using TSO coverage data from all the analyzed chordoma cell lines, a consensus profile was created. For each genomic interval, we counted how many cell lines show a gain or loss in their profile and then plotted the percentage of cell lines showing that chromosomal aberration on the y-axis. The profile of the 50 chordoma clinical samples was obtained from the Progenetix database22; The Array Comparative Genomic Hybridization (aCGH) characterization was performed using the oligonucleotide-based microarray CytoChip Cancer 8 × 60 K v2.0 for Chor-IN-1, U-CH1 and U-CH2 samples and CytoChip ISCA 8 × 60 K v2.0 for MUG-Chor1 and JHC7 samples . Both arrays consist of 60,000 60-mer oligonucleotides spaced at about 50 kb density over the full genome. Labeling and hybridization were performed following the manufacturer’s protocol. 500 ng of the DNA extracted from each sample was co-hybridized with an equal amount of reference DNA. Reference sample was chosen according to the gender of the analyzed sample . The analysis was performed using InnoScan 710 Microarray scanner  and Mapix software . Genomic profiles were obtained using Blufuse Multi v4.3 software .34. Raw count quantification was performed using Bedtools Coverage tool (v. 2.22.0)35, starting from the bam files. Normalization was performed using DESeq. 2 (v. 1.12.4)36 with default parameters and data were log2 transformed. Due to the similar length of the produced fragments, a length normalization step was not performed. R was used to calculate the distance matrix (distance method = maximum) between the kinases in the chordoma cell lines and “complete linkage” clustering was applied (hclust function) to generate 3 groups: high, low and medium expressed kinases. These clusters were then used to generate a plot of kinase expression pattern in chordoma cell lines by means of Kohonen maps, used for visualization.A custom panel interrogating 487 human kinases was selected from the available pre-designed assays for TruSeq Targeted RNA expression kit  using the Design Studio software . Custom TREx is based on amplicon technology generating PCR products with an average insert size between 70 and 80 bp. Library preparation was performed starting from 200 ng RNA input. To assess RNA quality, the RIN parameter (RNA Integrity Number) was evaluated using 2100 Bioanalyzer instrument, RNA 6000 nano kit . All the analyzed samples had a RIN value >8. The libraries were sequenced in single-end on MiSeq platform  with sequencing reagent kit v3 (1 × 50 bp). Fastq files were aligned to the human reference genome (hg19) using STAR (v. 2.5.1b)Real Time quantitative PCR (RT-qPCR) was carried out using SYBR green technology; specific primers were designed for the genes of interest using the freely available Primer3 software (http://primer3.ut.ee/) and synthesized using an Applied Biosystems 3900 Synthesizer.37.RT-qPCR data were obtained using TaqMan Reverse Transcription Reagents  and random hexamer priming to reverse transcribe RNA to complementary DNA (cDNA), according to manufacturer’s instructions. Real Time quantitative PCR (qPCR) was carried out on a ABI Prism 7900HT Applied Biosystems Sequence Detector , with reagents and materials from Applied Biosystems/Thermo Fisher Scientific (Power SYBR® Green PCR Master Mix), according to manufacturer’s instructions, in a volume of 12.5 μl per reaction, each containing approximately 10–12 ng cDNA (diluted in TE buffer 1×), 300 nM primers for the different targets and for 3 endogenous reference controls (glucuronidase beta (GUSB), peptidylprolylisomerase A (PPIA), and 18S ribosomal RNA), as detailed in Suppl. Table The NGS data reported in the submitted manuscript have been deposited in the BioProject database with the following ID: PRJNA382303, and will be released upon paper acceptance.Supplementary Information


Cytokines and chemokines are widely involved in cancer cell progression and thus represent promising candidate factors for new biomarkers. 2 Profiler PCR Array; Qiagen, Hilden, Germany), and expression profiles were compared.  Four renal cell cancer (RCC) cell lines  and a nonmalignant renal cell line (RC-124) were examined with respect to their proliferation. The cytokine and chemokine expression pattern was examined by a DNA array (Human Cytokines & Chemokines RT Caki-1 and 786-O cells exhibited significantly increased proliferation rates, whereas RCC4 and A498 cells demonstrated attenuated proliferation, compared to nonmalignant RC-124 cells. Expression analysis revealed 52 cytokines and chemokines primarily involved in proliferation and inflammation and differentially expressed not only in malignant and nonmalignant renal cells but also in the four RCC cell lines.  This is the first study examining the expression of 84 cytokines and chemokines in four RCC cell lines compared to that in a nonmalignant renal cell line. VEGFA, NODAL, and BMP6 correlated with RCC cell line proliferation and, thus, may represent putative clinical biomarkers for RCC progression as well as for RCC diagnosis and prognosis. Renal cell cancer (RCC) represents the deadliest neoplasm of the urinary tract, due to the fact that a majority of patients are diagnosed at a very advanced stage , 2. TumoAmong other mechanisms, cytokines and chemokines are suspected to play a crucial role in proliferation and progression of various malignancies. For instance, CCL11 controls cancer cell growth and invasion in ovarian cancer and CCL21 as well as CCR7 represent pivotal regulators of bladder cancer progression , 7. MoreIn the study presented here, we used an RCC model system consisting of four malignant RCC cell lines compared to a nonmalignant renal cell line. After classification of the RCC cells in cell lines of high and low cell growth rates, a transcriptional profiling specific for 84 cytokines and chemokines  was carr2).RCC cell lines Caki-1, 786-O, A498 , and RCC4  and the nonmalignant renal cell line RC-124 (Cell Lines Service) were used. Caki-1 and A498 cells were cultivated in a minimum essential medium (MEM) supplemented with 79.6 mg/l nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% penicillin/streptomycin (P/S), and 10% FBS . 786-O cells were propagated in RPMI 1640 medium containing 2 mM L-glutamine, 1% P/S, and 10% FBS (PAN Biotech); RCC4 cells were cultivated in Dulbecco's Modified Eagle Medium (DMEM) with 1 mM sodium pyruvate, 1% P/S, and 10% FBS (PAN Biotech); and RC-124 cells were cultivated in McCoy's 5a medium supplemented with 2 mM L-glutamine, 1% P/S, and 10% FBS (PAN Biotech). All cells were propagated in a humidified atmosphere .Proliferation of cells was examined by cell counting . Adherent cells, detached by trypsin/ethylenediaminetetraacetic acid (EDTA) treatment, were suspended in CASYton (Roche Applied Science) as 1 : 100 dilution. Measurement was performed using a capillary of 150 2 Profiler PCR Array  according to the supplier's instructions (http://www.sabiosciences.com). Briefly, 0.5 μg of total RNA was reversely transcribed and polymerase chain reaction (PCR) was done on a CFX96 Real-Time System  and analyzed with the CFX Manager software (Bio-Rad).RCC cells and nonmalignant RC-124 cells were grown on a 6-well cell culture plate to 80% confluency and total RNA was prepared . Analysis of 84 human cytokines and chemokines  was perfIL6, IL8, IL11, IL15, and VEGF concentrations in cell culture supernatant of RCC cells and nonmalignant RC-124 cells were determined by use of target-specific DuoSet ELISA kits with a Substrate Reagent Pack containing stabilized hydrogen peroxide and tetramethylbenzidine  according to the supplier's instructions. Cells were sedimented , the supernatant was incubated with capture antibodies , and soluble antigens were determined applying the BMG FLUOstar OPTIMA Microplate Reader with OPTIMA software 2.10 . Each sample was analyzed in duplicates.For data evaluation, the graphics and statistics software Graph Pad Prism V5.01  was used. Principal component analysis (PCA) was performed using Multibase 2015 software .In contrast to the elevated proliferation rates of Caki-1 and 786-O cells compared to those of nonmalignant RC-124 cells, both, RCC4 and A498 cells, showed a markedly reduced cell growth . These d2 Profiler PCR Array Human Cytokines & Chemokines, the expression of 84 target mRNAs  and the lowly proliferative  RCC cells. This observation has been confirmed by several studies. CSF-1 and VEGFA expression is associated with RCC progression and bad prognosis for patient survival , 18, 19.genicity –23. ExpeVice versa, the expression of BMP6 was significantly increased in the lowly proliferative cell lines RCC4 and A498, whereas an expression in highly proliferative Caki-1 and 786-O cells was not detectable. Since BMP6 is considered to be a tumor suppressor in breast cancer , upregulTaken together, there are a number of cytokines and chemokines which are differentially expressed not only in malignant and nonmalignant renal cells but also in the four RCC cell lines. These results may reflect heterogeneity among RCC patients as well as among RCC subtypes. In addition, this may suggest a functional substitution of members of the cytokine/chemokine system due to an activation of alternative factors involved in cytokine/chemokine signalling. Notably, two proliferative factors  and a tumor suppressive factor (BMP6) have been found which may significantly contribute to the malignant phenotype of RCC and thus may be suitable as novel marker proteins in RCC diagnosis and therapy.


We found that all EBV-positive T and NK cell lines and patient samples tested displayed activation of the JAK3/STAT5 pathway. Treatment of these cell lines with tofacitinib reduced the levels of phospho-STAT5, suppressed proliferation, induced G1 cell-cycle arrest and decreased EBV LMP1 and EBNA1 expression. An EBV-negative NK cell line was also sensitive to tofacitinib, whereas an EBV-infected NK cell line was more sensitive to tofacitinib than its parental line. Tofacitinib significantly inhibited the growth of established tumors in NOG mice. These findings suggest that tofacitinib may represent a useful therapeutic agent for patients with EBV-associated T and NK cell lymphoma.Epstein-Barr virus (EBV) infects not only B cells, but also T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoma. These lymphoid malignancies are refractory to conventional chemotherapy. We examined the activation of the JAK3/STAT5 pathway in EBV-positive and -negative B, T and NK cell lines and in cell samples from patients with EBV-associated T cell lymphoma. We then evaluated the antitumor effects of the selective JAK3 inhibitor, tofacitinib, against these cell lines  The Epstein-Barr virus (EBV) is a ubiquitous virus that has infected up to 95% of the adult population worldwide. After primary infection, EBV establishes a latent infection for the entire lifetime of the host. Although EBV predominantly infects B cells, it also infects T or NK cells, especially in association with lymphoid malignancies including NK/T-cell lymphoma, hydroa vacciniforme-like lymphoma, and chronic active EBV disease (CAEBV) –4. As soThe Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway is one of the critical intracellular cascades in transduction of extracellular signals from cytokines and growth factors. Various physiological functions such as cellular growth, differentiation and immune regulation are controlled by the JAK/STAT pathway , 11. JAKThe JAK family member JAK3 is specifically related to T cell development and proliferation. Recent data have also indicated that JAK3 activating mutations are found in human hematological malignancies including NK/T-cell lymphoma –16. UnliTofacitinib, a pyrrolo-pyrimidine JAK3-selective inhibitor, was originally developed as an orally active immunosuppressant, and has already received FDA approval for the treatment of rheumatoid arthritis , 19. ConIn this study, we investigated the activity of the JAK3/STAT5 pathway in EBV-associated B, T and NK cell lines as well as in lymphocytes from patients with EBV-associated T cell lymphoma, and evaluated the antitumor effects of tofacitinib on those cell lines in culture and in a murine xenograft model.To determine the presence and activation status of the JAK3/STAT5 pathway in EBV negative or positive B, T and NK cell lines, we examined the expression of JAK3, and the expression and phosphorylation status of STAT5, in an EBV-negative B cell line (BJAB), an EBV-transformed lymphoblastoid cell line (LCL), EBV-negative T cell lines (Jurkat and MOLT4), EBV-positive T cell lines , an EBV-negative NK cell line (KHYG1), and EBV-positive NK cell lines (KAI3 and SNK6). The characteristics of each cell line are summarized in Table To determine whether the growth of B, T and NK cell lines was sensitive to tofacitinib, the cells were exposed to 0.1 to 5 μM of tofacitinib, and cell counts were determined after 48 and 72 h. Neither fresh medium nor additional drugs were added during the observation period. As shown in Figure Additionally, to examine the effect of IL-2 on JAK3/STAT5 pathway activation, we compared the expression of phospho-STAT5 in IL-2-independent and -dependent SNT16 cell lines. As shown in Figure To directly compare the effects of tofacitinib on EBV-positive and -negative cells, we administered tofacitinib to the EBV-positive NK cell line, TL1, and to the EBV-negative parental cell line, NKL. As shown Figure To evaluate whether tofacitinib induces apoptosis, apoptosis of various tofacitinib-treated cells was analyzed by flow cytometry after Annexin V staining. Tofacitinib treatment of KHYG1, an EBV-negative NK cell line, induced a significant increase in apoptotic cells, as shown by an increase in the Annexin V-positive and 7-AAD-negative fraction, when compared with dimethyl sulfoxide (DMSO)-treated cells Figure  and 4B. We next investigated whether growth inhibition of T and NK cells by tofacitinib was a result of cell-cycle arrest. T and NK cells were treated with 5 μM of tofacitinib for 24 h, were stained with propidium iodide, and were then analyzed using flow cytometry. Both an increase in cells in the G1 phase and a decrease in cells in the G2 phase were observed after treatment of SNT15, SNT16, KHYG1, SNK6, and KAI3 cells with tofacitinib Figure . ConsistWe next analyzed the effect of tofacitinib on the expression of EBV-encoded genes in T and NK cell lines. Expression of the following four viral genes was analyzed using real-time RT-PCR: lytic genes encoding BZLF1 and gp350/220 and latent genes encoding EBV nuclear antigen (EBNA) 1, and latent membrane protein (LMP) 1. The expression of LMP1 and EBNA1, which are known oncogenes or anti-apoptotic genes, was significantly decreased in SNT13, SNT15, KAI3, and SNK6 cell lines by treatment with tofacitinib Figure . In contin vivo xenograft model to validate the significance of the in vitro findings. We subcutaneously inoculated 2 × 106 SNT15 cells into mice. On the day of tumor inoculation, mini-osmotic pumps were implanted and vehicle alone or tofacitinib (30 mg/kg/day) was delivered for 28 days. Subcutaneous inoculation of SNT15 cells into NOD/Shi-scid/IL-2Rγnull (NOG) mice resulted in tumor formation at the site of injection in all mice except for one treated mouse. The mice generally tolerated tofacitinib with no apparent toxicity such as death, weight loss or not doing well throughout the experiment. An antitumor effect of tofacitinib on tumor growth was evident on day 13, and tumor growth was significantly suppressed in the tofacitinib-treated group versus the control group at the end of the experiment  of two patients (Patient 1 and Patient 2) and a healthy donor (control) using magnetic sorting, and confirmed activation of the JAK3/STAT5 pathway in these cells. Phospho-STAT5 was detected in the patient samples and treatment of these cells with tofacitinib inhibited STAT5 phosphorylation  into NOG mice resulted in tumor formation, and that tofacitinib significantly suppressed the growth and invasion of the tumor without remarkable side effects. Furthermore, we found that EBV-infected γδ T cells of patients with EBV-associated T cell lymphoma showed activation of the JAK3/STAT5 pathway. Patients with EBV-associated T cell lymphoma are occasionally treated with stem cell transplantation because most EBV-associated T cell lymphoma cases are refractory and resistant to conventional chemotherapies. Our results indicate that inhibition of the JAK3/STAT5 pathway might be a promising strategy for treatment of EBV-associated T cell lymphoma.Previous studies have reported that inhibition of the JAK3/STAT5 pathway induced apoptosis of tumor cells; however, its effect on the cell cycle has not been fully investigated , 15. In In conclusion, we demonstrated that tofacitinib has the potential to be a new therapeutic agent against EBV-associated T and NK lymphoma. Although there is still concern regarding side effects of tofacitinib treatment, very strong adverse side effects have not been reported and clinical trials of tofacitinib for the treatment of many diseases are currently being conducted , 30. FurThe cell lines used were as follows: LCL cells were established by infection of B cell with EBV, strain B95-8. SNT13, SNT15 and SNT16 are EBV-positive T cell lines, SNK6 and KAI3 are EBV-positive NK cell lines, Jurkat and MOLT4 are EBV-negative T cell lines, and BJAB and KHYG1 are EBV-negative B- and NK-cell lines, respectively. SNT13, SNT15, SNT16, SNK6 and KAI3 cells were derived from patients with CAEBV or EBV-associated NK/T cell lymphoma –33. The Cells were lysed directly in sample buffer. Equal amounts of protein were subjected to SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and incubated with antibody. Antibodies against JAK3, STAT5, phospho-STAT5 (Tyr694), p27Kip1 and caspase-3 ; β-actin and PARP (Sigma-Aldrich); CDK2, cyclin D3 (BD Biosciences); LMP1 and EBNA1 ; and BZLF1  were use5/ml, and cell numbers were assayed by trypan blue exclusion using a Countess automated cell counter (Invitrogen). Experiments were performed at least in triplicate.Cells were seeded in 24-well plates at a density of 2 × 10Apoptosis was measured using an Annexin V-PE/7-AAD apoptosis assay kit (BD Pharmingen Biosciences) in accordance with the manufacturer's instructions. Cells were analyzed using flow cytometry. Viable cells were defined as negative for Annexin V-phycoerythin (PE) and 7-aminoctinomycin (7-AAD) staining, whereas apoptotic cells were defied as positive for Annexin V-PE and negative for 7-AAD staining.Cells were treated with 5 μM of tofacitinib for 24 h, fixed with 70% ethanol, and then washed with ice-cold PBS. Fixed cells were treated with DNase-free RNase and stained with propidium iodide (Sigma-Aldrich). The stained cells were analyzed using flow cytometry and ModFit LT software (Verity Software House). Experiments were performed in triplicate.Viral mRNA expression was quantified using RT−PCR, as described previously, using β2-microglobulin as an endogenous control and a reference gene for relative quantification , 37. Eac6 cells per flank) were suspended in a volume of 100 μL and subcutaneously inoculated into the right flanks of mice. Mini-osmotic pumps (ALZET) were implanted into the left flanks on the same day. Tofacitinib was dissolved in a sterile solution of 50% DMSO, 10% PEG and 40% saline solution and was delivered at a flow rate of 0.5 μL/h for four weeks [NOG mice were obtained from the Central Institute for Experimental Animals  and were maintained under specific pathogen-free conditions in the animal facility of Nagoya University . SNT15 cells  and the VECTASTAIN Elite ABC kit (Vector Laboratories), according to the manufacturers' protocols.All mice were humanely killed and the body tissues including tumors, brain, heart, kidney, lung and spleen were removed. Formaldehyde-fixed, paraffin-embedded tissues were cut into serial sections about 3-μm thick. Sections were then stained with hematoxylin and eosin and were stained for EBER by in situ hybridization assay [+ T Cell Isolation kit (Miltenyi Biotec). Informed consent was obtained from all participants or their guardians according to the Declaration of Helsinki. This study was approved by the Institutional Review Board of Nagoya University Hospital.PBMCs were obtained from 3 patients with hydroa vacciniforme-like lymphoma and from 2 healthy donors. Hydroa vacciniforme-like lymphoma was diagnosed according to the World Health Organization (WHO) criteria . EBV-infon assay . γδ T ceU test. Probability values of < 0.05 were considered to be statistically significant.Statistical analyses of cell proliferation, cell cycle, and tumor volume were performed using the Mann-Whitney


Zanthoxylum armatum DC has been an important traditional plant known for its medicinal properties. It is well known for its antimicrobial, larvicidal and cytotoxic activities.Z. armatum on breast (MDA-MB-468 and MCF-7) and colorectal (Caco-2) cancer cell lines using MTT, neutral red uptake(NRU) and DAPI stain assays were evaluated.The potential anticancer effects of the methanol extract and the crude saponins from fruit, bark and leaves of Zanthoxylum armatum, showed significant and dose dependent growth inhibition of MCF-7, MDA MB-468 and Caco-2 cancer cell lines in a dose of 200 μg/ml and above. The saponins  showed significant activity against MDA MB-468  as compared to MCF-7  and Caco-2  respectively. The extracts were further tested in more sensitive NRU assay and its was found that Zf extract showed higher cytotoxic activity as compared to Zb and Zl extracts with 100 μg/ml concentration. The breast cancer cell lines showed more sensitivity toward the crude saponins from fruit and bark with maximum inhibition of up to 93.81(±2.32) % with respect to 71.19(± 2.76) of Actinomycin-D. DAPI staining experiment showed that saponins from fruit induced apoptosis mode of cell death in all three types of cell lines while saponins form leaves and bark showed similar results against MDA MB-468 indicated by nuclear fragmentation and chromatin condensation. The effect of saponins from fruit, bark and leaves  against Caco-2 cell lines inhibited the growth of Caco-2 by 53.16 (±3.31) %, 66.43 (± 3.24) and 45.96 (± 10.67) respectively with respect to Actinomycin-D (4 μM) which showed the growth inhibition of 65.40(±4.29) %.In MTT assay the methanol extract of fruit (Zf), bark (Zb) and leaves (Zl) of Zanthoxyllum armatum DC., has the potential to exert its cytotoxic effect on cancer cell lines isolated form human by a mechanism involving apoptosis. The overall finding demonstrate that this plant specially fruits, could be potential source of new anticancer compounds for possible drug development against cancer.The current study clearly demonstrates that the extract and crude saponins from fruit, bark and leaves of traditional medicinal plant  Podophyllum peltatum, Taxus brevifolia, and Cantharanthus roseus and many others provided bioactive compounds including podophyllotoxin, taxol, and vincristine etc. for the management of various cancers [Zanthoxylum armatum fruit, bark and leaves in cultured human breast cancer cells (MCF-7 and MDA MB-468) and colorectal cancer cell lines (Caco-2).Cancer has become one of the most annihilating diseases globally with more than 10 million new cases annually. The compounds isolated from natural sources have been the source of most of the active components of medicines. One of the studies revealed that since 1994 50 % of the drugs approved are originated from natural sources. The drugs approved cover a variety of therapeutic indications, most of these are related to cancer treatment [ cancers . There iZanthoxylum usambarense (Engl.). In this study the extracts of Z. usambarense showed significant activity against breast cancer cell lines MCF-7 (IC50 42.9 mg/mL) and revealed to have induced cell death through apoptosis [Zanthoxylum armatum DC. extract has the ability to induce clumped chromosomes at metaphase stage of cell division coupled with mitotic arrest, DNA degradation, and chromatin condensation [The genus Zanthoxylum have been explored for various activities based on traditional uses. The active metabolites isolated from these plants showed anti-cancer potential . A similF-7 IC50 2.9 mg/mLensation .Zanthoxylum armatum (DC) belongs to family Rutaceae. It is a common plant in Southeast Asia. In Pakistan it is found wild in Dir, Hazara, and Murree hills of Pakistan [Z. armatum are used as tooth brush (miswak) for washing the teeth while the powder of fruit is applied in toothache [Zanthoxylum genus possess antimicrobial, larvicidal and cytotoxic activity [Z. armatum include terpenes, sterols, flavonoids, alkaloids, saponins, and coumarins [Pakistan . The aerPakistan . The smaoothache . Many plactivity –11. The oumarins , 12–14.(DMEM) and 4, 6-diamidineo-2-phenylinldole (DAPI), MTT (methyl-thiazolyl tertrazolium), DMSO (dimthylsulphoxide) were obtained from Sigma Chemical Co. . The drugs Gentamycin, Streptomycin, Glutamine, Actinomycin-D used in the experiments were also purchased from Sigma Chemical Co. .Neutral red solution, Fetal Bovine serum (FBS), Actinomycin-D, Dulbecco’s modified Eagle medium MCF-7 and MDA-MB-468 cells were purchased from ATCC  through an authorized distributor, LGC Standards, Teddington, UK. The Caco-2 (human colon adenocarcinoma) cell line was obtained from the ECCC (European Collection of Cell Cultures) through Health Protection Agency, Salisbury, UK .Z. armatum were collected from Mansehra, Tanawal area of KPK Pakistan in the month of August, 2013. After authentication from taxonomist, the plant and its voucher specimen (PB025.13) were deposited in the herbarium of the Post graduate college, Abbottabad. Each part of the plant was washed under running water and dried in shade at room temperature and was ground to coarse powder. The powder drug was stored in air tight and light resistant container before extraction.Five kg of each of leaves, bark and fruit of (DMEM) containing 10% v/v FBS, 2 mM L-glutamine, Gentamycin (40 μg/ml), Penicillin (100 units/ml) and Streptomycin (1040 μg/ml). The cells were seeded into 96-well cell culture plates at a density of 1 × 104 cells per well in 100 μl aliquots of the medium. The cells were permitted to attach for a period of 24 h, the temperature was maintained at 37 °C, and 5% CO2 in air, in a humidified atmosphere in an incubator.A complete growth medium was provided for the cell growth: Dulbecco’s modified Eagle medium The powder material (200 g each) of leaves, bark and fruit was extracted with methanol solvent using soxhlet extractor for 20 h. After filtering through Whatman Grade-I filter paper, the filtrate was evaporated under reduced pressure on a vacuum rotary evaporator at 40 °C. The separation of saponins from powdered materials (200 g) of leaves, bark and fruit was carried out first by defatting with petroleum ether. In the next step extraction was carried out with methanol in Soxhlet apparatus. Evaporation of the solvent was done under reduced pressure to obtain a semi solid extract. This extract of the plant was further fractionated with n-butanol and water in equal proportions. The n-butanol fraction was separated. Petroleum ether was added to the extract in small proportions to precipitate the crude saponins . ExtractZ. armatum were tested for cell cytotoxicity against breast and colorectal cancer cell lines. A series of eight dilutions  of crude extracts were prepared in DMEM (100 μl) containing DMSO . After the initial screening results, a test dose of 200 μg for MTT assay and 100 μg for NRU assay was set for extracts based on their apparent IC50 values.The crude extract of the leaf, bark and fruit of After a 24 h exposure of test period, the determinations of toxic endpoints were carried out by two colorimetric methods namely; methyl-thiazolyl tertrazolium (MTT) assay and neutral red uptake (NRU) assay.n = 3) to calculate the standard error of mean. The results were calculated as percentage growth inhibition, untreated (control) cells versus treated cells according to the following formula:The quantification of cancer cells growth was carried out as described by , 17. AftAbsorbance of the media was subtracted, both from control and treated cells.n = 3). The results were calculated by the same equation as already given for MTT assay.This assay was performed as described by . The med4 cells/well in 500 μl of medium on sterilized glass cover slips in 96 well plates for 24 h. The cells were treated with the culture medium (negative control) and Actinomycin-D . The plates were incubated at 37 °C, 5% CO2 in air in a humidified atmosphere for 24 h. After treatment, cells were briefly equilibrated with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilised with methanol for 5 min, and mounted in a DAPI-containing medium . The morphology of the nuclei was observed using a confocal fluorescence microscope, Leica SP2 AOBS confocal microscope  with excitation at 350 nm and emission 460 nm under a ×40 oil objective [In this study, DAPI  stain was used to evaluate the morphological changes in nuclei of control and treated cells. The cells were seeded at density of 1 × 10bjective .All data were compiled from a minimum of three experiments. Data for statistical analysis were expressed as ‘standard error of the mean’, n (number of experiments). The software used was GraphPad Prism version 6.00 for Windows, . The test applied was Dunnett’s test with one-way ANOVA.Zanthoxylum armatum were subjected to MTT assay using two breast cancer cell lines, MDA-MB-468 and MCF-7, and the colorectal cell line Caco-2 to assess potential cytotoxicity.The methanol extract and crude saponins from leaves, bark and fruit of Z. armatum, showed a concentration dependent growth inhibition from 10 to 500 μg/ml of MCF-7 cancer cell lines. The Zf showed more effective inhibition and highly significant activity was observed with extract concentration of 200 μg/ml as compared to Zb (400 μg/ml) and Zl (300 μg/ml).The methanol extract fruit (Zf), bark (Zb) and leaves (Zl) of Z. armatum showed a concentration depended inhibition of Caco-2 cancer cell lines growth in tested concentration range of 10–500 μg/ml. The very high significance inhibition of the growth was observed above the concentration of 200 μg/ml for Zf and 300 μg/ml for Zb. However, Zl extract proved least active and showed maximum inhibition over 500 μg/ml. , bark (Zb.Sa) and Leaves (Zl.Sa) against cell lines MCF-7 and MDA MB-468 tested at a concentration of 100 μg/ml in MTT assays. The saponins Zf.Sa showed growth inhibition of MCF-7 and MDA-MB-468 by 79.89 (±7.45) % and 95 (±2.64) % respectively, the saponins Zb.Sa inhibited the growth by 9.43 (± 3.82) and 94.59 (± 3.00) and saponins of Zl.Sa inhibited with growth by 49.08 (± 5.21) and 85.33 (± 3.41) respectively with respect to Actinomycin-D (4 μM) which showed the growth inhibition of 62.04 (±1.43) % and 62.87 (±5.28) % respectively. The effect of Zf.Sa, Zb.Sa and Zl.Sa against cancer cells Caco-2 were tested at dose of 200 μg/ml for MTT assays. The saponins showed maximum effect and inhibited the growth of Caco-2 by 75.88 (±8.41) %, 61.82 (± 4.07) and 68.62 (± 2.48) respectively with respect to Actinomycin-D (4 μM) which showed the growth inhibition of 65.40(±4.29) % , bark (Zb.Sa) and Leaves (Zl. Sa) against cancer cells MCF-7 and MDA MB-468 were tested at concentration of 100 μg/ml in NRU assays. The saponins Zf.Sa showed inhibition of MCF-7 and MDA MB-468 by 81.67 (±4.15) % and 93.81(±2.32) % respectively, the saponins Zb.Sa inhibited the growth by 7.77 (± 4.83) and 95.25 (± 4.35) and saponins of Zl. Sa inhibited with growth by 48.58 (±7.36) and 71.19(± 2.76) respectively with respect to Actinomycin-D (4 μM) which showed the growth inhibition of 62.04 (±1.43) % and 62.87 (±5.28) % respectively. The crude saponins Zf.Sa, Zb.Sa and Zl.Sa inhibited the growth of Caco-2 by 53.16 (±3.31) %, 66.43 (± 3.24) % and 45.96 (± 10.67) % respectively with respect to Actinomycin-D (4 μM) which showed the growth inhibition of 65.40(±4.29) %. . Microscopic examination revealed that the cells lost their shapes and were shrunken. It was a clear indication of nuclear fragmentation and chromatin condensation. It was very clear that the numbers of apoptotic cells were higher as compared to untreated cells (Control). The crude saponins isolated from leaves (Zl.Sa) and bark (Zb.Sa) were evaluated against MDA MB-468 breast cancer cells and were stained with DAPI. The treated cells showed similar results and revealed nuclear fragmentation and chromatin condensation. The saponins from bark and leaves showed no prominent activity against caco-2 cancer cell lines Fig. .Fig. 1CyZ. armatum, observed in the present study, corresponds well with the cytotoxic potential of this genus [Z. nitidum, which has been reported to inhibit breast cancer cell lines [Screening of herbal drugs may lead to discovery of new mutagenic agents which can be an alternative source to the costly anticancer chemotherapeutic agents. Due to low toxicity and less cost, some medicinal plants have attracted the attention as alternative cancer therapies . The plais genus . An examll lines .Z. armatum, the fruit extract showed significance inhibition of all the three cell lines in MTT assay. The bark and leaves saponins/extract of Z. armatum were significantly active against MDA MB-468 cells only while mild activity was observed against MCF-7 and Caco-2 cells. The neutral red uptake (NRU) assay also showed similar results for Z. armatum and inhibited the three cell lines in a manner observed in MTT assay.MTT assay has been used to determine number of viable cells in an anticancer investigation . MitochoFurther studies were carried out on precipitated saponins fractions. Over 100 and 50 saponins from natural sources have been tested and found to be effective against different types of cancers. Due to this diversity in structure the saponins exert anticancer effect through variety of mechanisms and pathways. One of example is of steroidal saponins which induce apoptosis and cell cycle arrest of tumors . AnotherZ. alatum leaves. One such study was carried out against the Ehrlich ascites carcinoma in which the isolated compound Zanthonitirle showed the cytotoxic effect in dose dependent manner [Z. alatum against the pancreatic and lung carcinoma cell lines [There are evidences available from the previous studies that indicates the cytotoxic potential of t manner . Anotherll lines .Z. armatum DC exert its cytotoxic potential by mechanisms involving apoptosis [Z. armatum fruit (Zf.Sa) induced morphological changes in apoptotic cells which were observed in DAPI staining. The confocal microscopy demonstrates that the treatment with saponins (Zf.Sa) resulted in apoptotic body formation, chromatin condensation and nuclear fragmentation. It clearly indicates the potential of saponins to induce apoptosis against cancer cell lines.Here, we have studied the cytotoxic and apoptotic potential of the Zf.Sa, Zb.Sa and Zl.Sa on human cancer cell lines. The evidence already exist that the extracts of poptosis . DAPI stpoptosis . In thisZanthoxylum armatum DC., has the potential to exert its cytotoxic effect on cancer cell lines isolated from human by a mechanism involving apoptosis. The overall finding demonstrate that this plant could be a potential source of new anticancer compounds for possible drug development against cancer.In conclusion, the current study clearly demonstrates that the extract and crude saponins from fruit, bark and leaves of traditional medicinal plant


In addition, in postmenopausal women with endocrine responsive breast cancer ZA prolongs DFS. The exact mechanism is still unclear. We have therefore investigated the effect of increasing concentrations of ZA in breast cancer cell lines in the absence or presence of estradiol to mimic the hormonal environment Using assays for cell proliferation  and cell death (Annexin/PI), we have analyzed the dose-dependent antiproliferative and pro-apoptotic effects of ZA in two hormone sensitive cell lines (MCF-7 and T47D) and a hormone insensitive, triple negative cell line (MDA-MB-231) in the presence of 0, 1 and 10 nM estradiol.In the absence of estradiol, ZA exerts dose-dependent antiproliferative and pro-apoptotic antitumor effects in both, hormone sensitive  and -insensitive (MDA-MB-231) breast cancer cell lines (p<0.0001). In the presence of estradiol, the antitumoral effect of ZA was significantly decreased only in the hormone sensitive MCF-7 and T47D cell lines .We have demonstrated that estradiol impairs the antiproliferative and proapoptotic effect of ZA in hormone sensitive, but not in hormone insensitive breast cancer cell lines. Our findings provide a possible explanation for the differential effect of ZA on DFS in pre- and postmenopausal patients with hormone sensitive early breast cancer, which has been demonstrated clinically. We further hypothesize that endocrine insensitive tumors such as triple negative breast cancer (TNBC) should benefit from ZA irrespective of their menopausal status. More than 50% of breast cancer patients present with early-stage endocrine-sensitive disease. In this subgroup, breast cancer outcomes have improved significantly over time partly because of the increasing availability of highly efficacious adjuvant therapies. Aromatase inhibitors and Tamoxifen have been shown to increase DFS in premenopausal breast cancer patients but these therapies are burdened with bone loss . FavorabMethodological heterogeneity has complicated the interpretation of trials examining adjuvant ZA in early breast cancer. Many of the completed trials investigated adjuvant bisphosphonate therapy for its effect on bone health –12. UnfoNevertheless, it has not yet been clarified which biological subtype of breast cancer will maximally benefit from ZA, nor has the relationship between efficacy of ZA to menopausal status in estrogen sensitive and–insensitive subtypes been investigated in detail.in vitro analysis on possible differences of the antitumor effects of ZA in hormone sensitive (MCF-7 and T47D) and hormone insensitive, triple negative (MDA-MB-231) breast cancer cells in the presence or absence of estradiol.This prompted us to conduct an Mycoplasma . ZA was purchased from Sigma-Aldrich .We analyzed the antitumor effect of ZA in the presence or absence of exogenous estradiol on the hormone sensitive human breast cancer cell lines MCF-7 and T47D and on the hormone insensitive, triple negative human breast cancer cell line MDA-MB-231. All cell lines were purchased from the American Type Culture Collection  and tested for the absence of 6 cells in T75 flasks and cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin and 1% L-Glutamine to 80% confluence for use in all assays. The cells were maintained at 37°C in a humidified atmosphere with 5% CO2 and split weekly. Medium and supplements were purchased from GibcoTM . To achieve an estrogen-free environment cells were cultured in RPMI 1640 complete medium supplemented with 10% Charcoal/Dextran Treated Fetal Bovine Serum , 1% penicillin-streptomycin and 1% L-glutamine for 5 days before treatment in all assays. Cells were harvested on day 6 and day 8 after estradiol and ZA treatment.Breast cancer cell lines were seeded at a density of 2x10Proliferation assays were performed over a period of 15 days using culture medium consisting of RPMI 1640 with 10% charcoal-treated fetal calf serum , 1% penicillin-streptomycin and 1% L-Glutamine without and with ß-Estradiol  at 1 nM or 10 nM. Cell numbers were examined using a Casy cell counter  and hemocytometer improved Neubauer  as well. After 6 and 8 days of culture a significant rise of proliferation was observed in estrogen sensitive MCF-7 and T47D, but not in estrogen insensitive MDA-MB-231 cells (data not shown).Cell viability given as optical density was determined using a formazan dye assay  as previously described .DNA replication was measured by BrdU. Following the manufacturers protocol, cycling cells were incubated for 48 hours with 5-bromo-2´deoxyuridine (BrdU), a synthetic analogue of thymidine, which incorporates into newly synthesized genomic DNA during the S-phase of mitosis .To examine the effect of ZA on apoptosis, we determined the content of cellular DNA using a fluorescent DNA-binding dye followed by flow cytometry . During apoptosis, genomic DNA is cleaved into smaller fragments. This is a specific marker of apoptosis and can be used to quantitate apoptosis. Using flow cytometry, PI (propidium iodide) stained cells will stain less intensely and show a peak below the G1 peak- the Sub-G1 peak. We performed the DNA flow cytometry according to the manufacturers protocol .6/ml and added 5 μl of fluorochrome-conjugated Annexin V to 100 μl of the cell suspension. After incubation for 15 minutes at room temperature, cells were washed in 1X Binding Buffer and resuspended in 200 μl of 1X Binding Buffer. We then added 5 μl of PI staining solution and analyzed the samples by flow cytometry [Annexin V Apoptosis Detection eFluor 450  was performed according to the manufacturers protocol: After dilution of 10X Binding Buffer to 1X using distilled water (1 ml 10X Binding Buffer + 9 mL dH20), we washed the cells once in PBS and once in 1X Binding Buffer. We then resuspended the cells in 1X Binding Buffer at 1x10ytometry .The antiproliferative and pro-apoptotic effects of ZA depending on dose, cell line and presence/absence of estradiol were determined. Analysis of Variance (ANOVA) was used to compare the effects of dose, cell line and estradiol on cell death and cell proliferation. The interaction between cell line and estradiol was assessed and reported if statistically significant. The significance level was 5%. The calculations were done in R 3.3.3.As expected, in the absence of ZA we found a significant proliferative stimulus by adding different doses of estradiol  after 5 days of estrogen depletion only in the two hormone sensitive cell lines MCF-7 and T47D , while there was no significant increase in proliferation in the triple negative cell line MDA-MB-231. There was no significant difference in the proliferative effect between 1nM and 10nM estradiol in the hormone sensitive cell lines MCF-7 and T47D (p = n.s.) .We have therefore chosen ZA concentrations of 0, 6.25 and 12.5 μM to investigate the effect of ZA on the three cell lines in more detail.Remarkably, in the absence of estradiol the antiproliferative effect of increasing concentrations of ZA was stronger in hormone sensitive MCF-7 and T47D cells when compared to hormone insensitive MDA-MB-231 cells (p<0.0001). The antiproliferative effect of ZA in triple negative MDA-MB-231 cells was significantly stronger when administered at a higher dosage (p = <0.0001). Using BrdU staining we analyzed the effect of ZA on DNA synthesis. As depicted in We then performed a cell cycle analysis by DNA content and found a dose-dependent pro-apoptotic effect of ZA in all three cell lines shown by the sub-G1-peak in This effect was stronger in the two hormone sensitive cell lines than in the triple negative cell line (p = 0.0019) without a significant influence of the addition of estradiol in all three cell lines (p = 0.104).To investigate if the pro-apoptotic effect of ZA is dependent on dosage, the addition of estradiol and the tumor subtype, we performed an Annexin V apoptosis detection test as shown in Bisphosphonates improve bone mineral density (BMD) in osteoporotic women and have also been shown to ameliorate anticancer treatment-induced decrease of BMD and bone fracture –25. ImpoThe Early Breast Cancer Trialists´ Collaborative Group (EBCTCG) performed a meta-analysis of 26 clinical trials including over 18,000 women with early breast cancer randomly assigned to receive bisphosphonates versus no treatment or placebo. The reduction in recurrence, distant recurrence, and breast cancer mortality were of borderline statistical significance, although a reduction in bone recurrence was found . A subset analysis of the EBCTCG meta-analysis revealed that use of bisphosphonates was associated with a statistically significant reduction in the risk of recurrence, bone recurrence, and breast cancer mortality in women with non-functioning ovaries . Women with functioning ovaries had no apparent effect on any outcome .Moreover, there is some preclinical evidence demonstrating that reproductive hormones can reduce the efficacy of bisphosphonates against cancer cells disseminated to the bone. The effects of ZA on the growth of disseminated MDA-MB-231 breast cancer cells in bone were compared in ovariectomised mice or sham-operated mice. ZA decreased the number of detectable tumors in bone only in the ovariectomised animals .Experimental work addressing the possible influence of tumor biology on the direct anticancer efficacy of ZA is still lacking. Most previous studies evaluated the effects of ZA in hormone sensitive breast cancer patients ,36,37. IPreclinical evidence presented by Verdijk et al demonstrated that sensitivity of ZA is most pronounced in estrogen receptor negative MDA-MB-231 cells . SimilarIn a meta-analysis of randomized trials looking at the effects of neoadjuvant chemotherapy with or without ZA on pathological response, significant higher pathologic complete response rates (pCR) were observed only in the subgroup of postmenopausal patients, whereas only a trend towards higher pCR rates was shown in triple negative breast cancer patients .Here we have demonstrated that ZA exerts direct antitumoral effects in breast cancer cells, irrespective of hormone receptor status and hormone-sensitivity or -resistance. ZA reduced cell multiplication and DNA synthesis, and caused cell cycle arrest and programmed cell death in all cell lines tested. Obviously, these ZA-mediated antitumor responses were not modulated by the presence or absence of exogenous estradiol in the triple negative hormone resistant MDA-MB-231 cells, whereas in hormone receptor positive MCF 7 and T47-D cells, administration of estradiol significantly reduced the antiproliferative and pro-apoptotic effects of ZA. In general, ZA was found to cause accumulation of the cells in the S-phase of the cell cycle, which is consistent with the findings of Neville-Webbe HL et al .in vitro. Evidently, a deteriorating effect of exogenous estradiol on the anticancer efficacy of ZA was detected only in hormone sensitive, but not in hormone insensitive breast cancer cells. Consequently, our findings underscore previous clinical evidence showing that postmenopausal patients with hormone sensitive breast cancer benefit most from adjuvant ZA, whereas patients with hormone insensitive breast cancer may benefit from adjuvant ZA independent of menopausal status. Based on these data specific clinical studies in various patient subgroups characterized by tumor biology and menopausal status are warranted in order to identify those patients that in fact would or would not benefit from (neo-) adjuvant ZA.Taken together, herein we present first experimental evidence demonstrating that ZA exerts direct dose-dependent anti-proliferative and pro-apoptotic effects in both estrogen sensitive and–insensitive breast cancer cells S1 FigCell proliferation of the three cell lines MCF-7, T47D, MDA-MB231 with and without estradiol and tamoxifen.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.