Currently available human tumour cell line panels consist of a small number of lines in each lineage that generally fail to retain the phenotype of the original patient tumour. Here we develop a cell culture medium that enables us to routinely establish cell lines from diverse subtypes of human ovarian cancers with >95% efficiency. Importantly, the 25 new ovarian tumour cell lines described here retain the genomic landscape, histopathology and molecular features of the original tumours. Furthermore, the molecular profile and drug response of these cell lines correlate with distinct groups of primary tumours with different outcomes. Thus, tumour cell lines derived using this methodology represent a significantly improved platform to study human tumour pathophysiology and response to therapy. Despite methodological improvements, it remains extremely difficult to routinely establish cell lines from human primary tumours. Here, the authors develop a culture medium that allows efficient cell line establishment and use this to generate 25 ovarian cancer cell lines. More than 60 years have passed since the establishment of the first human cancer cell line, HeLa, in 1951 , not otherwise specified (n=4) or mixed mullerian (n=1) histology. Attempts to establish cell lines from clear-cell and endometrioid cancers, or from moderately differentiated carcinomas were not successful. Importantly, it took >5 months for the first passage, and the cell lines could be propagated only up to 15 passages6The challenges associated with establishing ovarian carcinoma cell lines is illustrated by a comprehensive study, which reported that only 11 out of 90 tumour samples could be established as cell lineset al.et al.et al.et al.The difficulty of establishing human tumour cell lines extends to many other tumours types as well. For example, since the first breast cancer cell line was establishedThese experiences provide clear indication that the great diversity of tumour subtypes and patient groups are not adequately represented in the existing tumour cell line collections. Consistent with this view, comparison of copy-number changes, mutations and messenger RNA (mRNA) expression profiles revealed pronounced differences between commonly used ovarian cancer cell lines and ovarian cancer tumour samplesIn general, the standard cell culture media such as DMEM, Ham's F12, Roswell Park Memorial Institute medium (RPMI), McCoy's and MCDB105 are composed of 40–50 ingredients. Because they are unable to support living cells on their own, these media are supplemented with tissue extracts, feeder layers, anti-apoptotic drugs and serum. While these supplements have improved culture results14in vitro models and by the encouraging results with a chemically defined culture medium that we described previously21-fold expansion of cell numbers, in contrast with the 10–15 doublings in the standard mediumHence, we set out to develop a highly efficient culture system that preserves the phenotype of the original tumour as much as possible for common human cancers, driven both by the clear need for improved Here, we report the phenotypic properties of 25 new ovarian cancer cell lines (OCI) derived using cell culture media optimized for human ovarian cancer subtypes . This panel of cell lines will be available to the research community and nearly doubles the number of existing 26 cell lines available from public cell lines repositories, such as ATCC and ECACC.In standard culture media we were able to establish tumour cell lines with a success rate of <1%, consistent with prior reports. In the single successful case, the ovarian tumour line OCI-U1a is derived in RPMI medium , in whicImportantly, the DNA copy-number variations (CNVs) identified in the cell line that is established in RPMI differ significantly from those found in the uncultured tumour tissue, consistent with clonal outgrowth of select subpopulations or the acquisition of additional genetic aberrations during tissue culture . ConsistTo derive a representative set of human ovarian cancer cell lines from multiple types of ovarian tumours, we tested many culture conditions over 10 years and developed a medium (OCMI) that allowed establishment of 25 new ovarian cancer cell lines in 26 attempts. Through a combination of theoretical deduction of metabolic pathways and trial and error, we found a mixture of variables with a large synergistic effect on culture efficiencyIn many cases, the individual components had incremental effects that emerged over many passages, varied between different histotypes and patients and produced combined effects that were non-obvious and synergistic, which made optimization difficult . Also, ein vitro clonal selection or a need to acquire additional genomic or epigenetic aberrations  lines . In OCMIrrations . Furtherrrations .The capital letter after the ovarian carcinoma designation ‘OCI' refers to the histological subtype of the original tumour: papillary serous, clear cell, endometrioid and mucinous cancers, and rare types such as carcinosarcoma and dysgerminoma. Together, the papillary serous, clear cell, endometrioid and mucinous subtypes account for >90% of ovarian adenocarcinomas; accordingly this panel of cell lines is broadly representative of ovarian cancer. The number after the histotype designation refers to the unique case number.The lower case letter at the end of each cell line name refers to tissue source; 14 of the cell lines were established from primary solid tumours, 7 from ascites fluid and 4 from primary mouse xenografts derived from direct implantation of human tumours into immunocompromised mice. All 25 OCI lines formed soft agar colonies consistent with retention of a transformed phenotype in culture . In two Adenocarcinoma of the ovary is a heterogeneous disease that is comprised of many histopathological subtypes with distinct features. In many cases, the original subtype of tumour that was used to establish the ‘SOC' cell lines is unknownWe observed that none of the OCI lines we tested can be efficiently cultured in existing standard media . In contThe SOC lines are cultured in a variety of different standard media, and no single standard medium appears to support culture of all existing SOC lines, making it difficult to compare a large panel of SOC lines with one another because they require different media. Our results indicate that OCMI medium has the potential to serve as a universal culture medium for SOC lines, facilitating comparisons across cell lines.et al., who used an apoptosis inhibitor (Y-27632) that inactivates Rho-associated kinase (ROCK) to help culture human embryonic stem cells192122We also observed that OCI cell lines cannot be efficiently cultured in the medium recently described by Watanabe Major genetic alterations may accumulate during cell culture in standard media2526Next, we compared CNV patterns of OCI cell lines with the ovarian tumours analysed in the TCGA data setA persistent problem in the cell culture field has been cross-contamination and misidentification of up to 15–20% of the cell linesUnsupervised hierarchical clustering of the mRNA expression of 25 OCI and 6 SOC lines reveal two major clusters. There are 558 upregulated mRNAs in cluster 1 and 265 mRNAs in cluster 2  . In cont30313031These results suggest that some of the tumours that are histologically classified as endometrioid or clear-cell cancers may have a papillary serous-like gene expression signature. In summary, mRNA cluster 1 generally correlates with papillary serous histology and OCI cell lines, and mRNA cluster 2 correlates with non-papillary serous histology and SOC cell lines. To exclude medium-based differences, both OCI and SOC lines were cultured in OCMI medium for these experiments.In addition, SOC lines were cultured in standard versus OCMI media in a control experiment. We observed that SOC lines cluster next to each other regardless of whether they are cultured in OCMI or in standard media .These results indicate that the mRNA expression differences observed between OCI versus SOC cell lines and between cluster 1 versus 2 are unlikely to be due to culture medium differences.To uncover the pathways that are differentially expressed between cluster 1 versus -2 cell lines, we used Ingenuity Pathway Analysis . This anWe next used reverse-phase protein array (RPPA) to examine the expression levels of 226 proteins in OCI and SOC cell lines. These proteins were selected because they represent major signalling pathways in cancer cellsIt is worth noting that in the above experiments the mRNA and protein extracts were prepared in triplicates from three different passages in two separate experiments. Reassuringly, we observed that replicates from different experiments cluster together .Moreover, we confirmed that OCI lines maintain their phenotype during long-term culture by comparing the RPPA profile of 10 pairs of cell lines that were analysed at two different times separated by 10–48 passages . Unsuperin vivo tumour phenotype of the OCI lines, we injected each cell line into immunocompromised mouse hosts.To examine the Ovarian carcinomas are characterized by very distinct morphological features31While it is difficult to entirely phenocopy a primary tumour in a xenograft, we hoped to recapitulate some of the cardinal features of different histotypes. For papillary serous carcinoma, these morphologic features would include finger-like structures (papillae) that consist of central stromal cores giving rise to smaller branches lined by a malignant epithelium with minimal cytoplasm and very large, high-grade, round nucleiIn general, SOC lines produce poorly differentiated xenograft tumours in mice, without any of the distinctive histopathologic features of specific ovarian tumour subtypes .In contrast, the OCI lines produce tumour xenografts with a histopathology strongly resembling the original human tumour . OCI-P5xIn summary, quite remarkably, the OCI lines form tumours that are morphologic phenocopies of corresponding human ovarian carcinomas at the histopathologic level, unlike SOC lines, which generally lack this characteristic.Several molecular features help distinguish the histological subtypes of human ovarian carcinomas3637363738in vivo histotype-specific molecular features described above are preserved to a great extent in OCI cell lines. On the basis of an algorithm described previously3637in vivo tumour histotypes.The Two of the SOC cell lines we examined—SKOV3 and A2780—account for nearly 60% of ovarian carcinoma-related publications according to a recent reportThe putative cells of origin of ovarian cancer include the normal ovarian surface, ovarian inclusion cysts and fallopian tube epithelia that are almost uniformly cytokeratin-7 (CK7) positive184240In contrast, some SOC lines such as OV90 and TOOV-112 do not express any of the ovarian-specific markers  and otheNext, we compared the mRNA expression profile of the cell lines with primary human tumours. Clustering analysis of the OCI and SOC cell lines with 285 primary human ovarian tumour specimens reveal two distinct patient clusters. Patient cluster 1 that includes only OCI lines and patient cluster 2 that includes all of the SOC lines . Completin vitro cell line mRNA Clusters 1 and 2, with the exception of a single cell line (OCI-C4p)  have a worse progression-free and overall survival compared with patients with SOC-like tumours (patient cluster 2)  . This reuster 2) .The striking difference between the outcome of OCI-like versus SOC-like patient clusters prompted us to test whether the drug response of the corresponding cell lines are different as well. To test this, we treated OCI and SOC cell lines with taxol and cisplatin, which are the two most commonly used first-line drugs for the treatment of ovarian cancer.In these experiments, we tested a subset of OCI and SOC lines from mRNA/RPPA cluster 1 and cluster 2 that are representative of a spectrum of different tumour subtypes , and tissue sources  . FinallyConsistent with the worse outcome of patients in the OCI-like cluster, we observe that the OCI lines in mRNA/RPPA cluster 1 are more resistant to taxol, cisplatin and vincristine compared with SOC/OCI lines in mRNA/RPPA cluster 2 .It is worth noting that there was at least 5–36 passages between the RPPA analysis and taxol/cisplatin-sensitivity experiments . NeverthTo explore the possible basis for the relative drug resistance of OCI cells, we compared the protein profiles of cluster 1/OCI lines with cluster 2/SOC lines , and fou454647484950The drug-sensitive SOC lines in cluster 2 express higher levels of several pro-apoptotic proteins, for example, Bim, SMAC-DIABLO, cleaved caspase 7 and lower levels of inactive phosphorylated BAD (pS112), a BH3-only pro-apoptotic protein. High Bim levels have been inversely correlated with activated Erk (MAPKpT202), which phosphorylates Bim and targets it for ubiquitination and degradation525354n=734) in the US (TCGA) and Australia (AOCS) classified ovarian cancers based on their molecular profiles5656We also examined the correlation of the OCI cell lines with molecular subtypes of ovarian cancer. Two large studies (R=0.93)   and was R=0.93) . These c.93) 5859.in vitro culture and culture media that poorly recapitulates the conditions in living tissue. The resulting panels of cancer cell lines have hampered many attempts at preclinical drug development, in that the behaviour of the limited set of available cell lines, both in vitro and in vivo, are frequently unpredictive of clinical responses.Over the past six decades, the great majority of human cancer subtypes have proven to be difficult to propagate in cell culture. This problem derives from the suboptimal conditions of in vitro. Use of this method, in the form of a novel medium and alterations in oxygen and serum levels, has yielded to date a panel of 25 new ovarian cancer cell lines that are extensively characterized, with histopathological and molecular analyses that includes whole-genome profiles of DNA and RNA data and protein arrays. In this report, we provide detailed description of the medium and methods in the Here, we present a method for propagating a diverse array of ovarian carcinoma cell types The molecular profile of OCI cell lines we describe here demonstrate a remarkable stability over time and consistency across multiple platforms including their DNA, mRNA and protein profilesin vitro drug responses of these OCI lines may indeed correlate closely with in vivo patient responses to drug treatments. Such a correlation between in vitro cell line data and in vivo patient data is especially encouraging since it has been recognized that such correlations are rare with standard tumour cell lines, which tend to be more drug sensitive than human tumours, leading to false-positive hits in cell culture-based drug screens. The closer correlation between OCI lines and human ovarian tumours is perhaps not surprising, since we observed that cytological, morphological and molecular features of the OCI lines and their xenograft tumours resembled specific subtypes of human ovarian cancer, which has not been the case for most SOC lines.Consistent with these results, we found that OCI lines in cluster 1 lines are more resistant to taxol and cisplatin, both first-line treatments for ovarian cancer, compared with SOC/OCI cell lines in cluster 2. This result provides the beginning of evidence that the While the overall correlation between cell line taxol/cisplatin response and patient outcome is encouraging, long-term prospective follow-up studies will be required to determine whether OCI lines will be predictive of patient response.Currently, the number of cells in our OCI panel is too small to carry out robust statistical analysis to determine whether there are >2 significant clusters. It is possible that with a larger panel of cell lines we will discover additional molecular subtypes. Furthermore, more diverse data sets from human tumours will be needed for histotype-specific studies, because over 95% of the nearly 1,500 samples in 12 publically available gene expression data sets with outcome data are from stage 3–4, high-grade (3) serous ovarian cancers5818in vivo proliferation rates of tumours can be markedly different, such as the c5 subtype, the successful culture of tumours involves expansion of proliferating cells. Hence, the in vitro proliferation rate differences are likely to be less pronounced compared with in vivo differences.Regarding mRNA expression subtypes of ovarian cancer, it is worth pointing out that several of the AOCS/TCGA subcategories are characterized by immune (c2), stromal (c4) or proliferation (c5) signatures. As we described, the immune and stromal cells have to be eliminated from the culture for successful establishment of tumours as cell lines. Otherwise fibroblasts will typically outgrow the tumour cells. In OCMI medium, the non-tumour cells are naturally eliminated during the first several passages. Thus, the OCI cell lines are free of stromal and other cells, and as a result we did not expect them to replicate tumour subtypes that are defined by non-tumour signatures such as the c2 and c4 subtypes. While the In conclusion, we describe here the initial instalment of our efforts as a proof of concept towards establishing a comprehensive ovarian carcinoma cell line panel. Even at this early stage the results are encouraging, and we are in the process of establishing additional lines representing each subtype that will be needed to explore the histotype-specific differences and intra-tumoral heterogeneity of ovarian tumours.While we have successfully cultured a number of colon and lung cancer cells in OCMI medium, we do not recommend this medium as a universal culture environment for all tumours at this time. Our experience with other tumour types indicates that to achieve high success rates and maintain the phenotypic properties of the original tissue, each tumour type will require tissue-specific modifications in medium formulation for optimum results.in vitro drug-sensitivity profile of each patient's tumour can be assessed real time and this information can be used to guide treatment decisions.In the future, a robust and efficient culture system yielding cancer cell populations that predict patient responses to various drugs should improve development of new drugs for personalized treatment of cancer patients. It should also improve the ability to identify mechanisms of resistance and to develop rational drug combinations. Our preliminary results suggest that the methodology we describe here can be modified with tissue-specific changes to culture other tumour types such as leukaemias, as well as normal breast, ovarian and fallopian tube cells1618http://sylvester.org/shared-resources/Live-Tumor-Culture-Core. To establish the cell lines, fresh tumour tissue fragments are minced and plated on Primaria (BD Biosciences) plates before and after digestion with 1 mg ml−1 collagenase (Roche). The tumour cells are cultured in nutrient medium described previously2 and regular O2 at 37 °C as monolayers attached to Primaria culture plates. The endometrioid and mucinous tumours are initially cultured in 5% CO2 and low O2 (5%) at 37 °C as monolayers attached to Primaria culture plates. Adjustments in O2 and plates are made for individual cell lines as necessary  the plates are treated with diluted trypsin first, to deplete stromal cells. The remaining cells that are still attached to the culture plate are treated with 0.25% trypsin for subculturing. In general, tumour cultures are free of stromal and normal cell types within 4–6 passages. The SOC cell lines were short tandem repeat validated and cultured as per the instructions of the vendor. OCI lines will be available from the Ince laboratory upon publication. All study procedures were approved by the Institutional Review Boards of the University of Miami, Brigham & Women's and Massachusetts General Hospitals to collect discarded tissues with written consent from all patients. See The OCI cell lines and OCMI medium are available from the Live Tumor Culture Core at Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami ecessary . The tumProtein expression was analysed by RPPA, as described previously65The relative sensitivities of OCI and SOC cell lines to chemotherapy drugs was measured by seeding an equal number of cells in six replicates in 96-well black-walled clear-bottom Corning plates at 3,000 cells per well, and allowing attachment in OCMI for 12 h. Both OCI and SOC cell lines were exposed to drugs in OCMI medium. The cell lines were cultured in the presence of drug or vehicle control for 96 h. The fraction of metabolically active cells after drug treatment was measured by incubation with 2:10 (v/v) CellTiter-Blue reagent (Promega Cat# G8081) in media for 2 h, and the reaction was stopped by addition of 3% SDS. Fluorescence was measured in a SpectraMax M5 plate reader  using SoftMax software (555EX/585EM). See Single-cell suspensions were prepared in a Matrigel: medium mixture (1:1) and 1–5 million cells per 100 μl volume were injected in one intraperitoneal and two subcutaneous sites per mouse. Tumour cell injections were performed on 6–8-week-old female immunodeficient nude (Nu/Nu) mice . Tumours were harvested 5–9 weeks after implantation. Tumour histopathology was assessed with haematoxylin and eosin staining of formalin-fixed paraffin-embedded sections. All mouse studies were approved by the University of Miami, the BWH or MGH Institutional Animal Care and Use Committee.Mutation detection was carried out at the MD Anderson Cancer Center Sequencing and Microarray Facility by amplifying purified DNA with primers designed to amplify coding regions of the p53 gene (exons 5–9). Primers were designed using a variety of software applications including, but not limited to, Primer Express v3.0 (Applied Biosystems). The PCR products were purified with ExoSAP-IT (USB) and sequenced in both directions using BigDye Terminator chemistry (Applied Biosystems) and run on a 3730 DNA Analyzer (Applied Biosystems). The sequence data files were aligned and compared with a reference sequence (from Ensembl TP53: Gene ID ENSG0000014150 Transcript TP_002 ENST00000445888) in SeqScape v2.5 Software (Applied Biosystems) and mutations analysed.Analysis of mRNA and protein expression profiling data, LOH and DNA copy-number changes are described in the Accession codes: the mRNA (GSE40785), MIP (GSE40786) and SNP (GSE40787) data are available in Gene Expression Omnibus under accession code for the study GSE40788.How to cite this article: Ince, T. A. et al. Characterization of twenty-five ovarian tumour cell lines that phenocopy primary tumours. Nat. Commun. 6:7419 doi: 10.1038/ncomms8419 (2015).Supplementary Figures 1-22, Supplementary Tables 1-16, Supplementary Note 1, Supplementary Methods and Supplementary ReferencesDNA from 12 primary uncultured tumors, 12 matched OCI cell lines, and nine SOC lines are analyzed using 250K Sty Affymetrix SNP arrays. Unsupervised hierarchical clustering of mRNA expression levels of OCI and SOC ovarian cancer cell lines reveal two clusters. Ingenuity Pathway Analysis (IPA) of the 823 genes that are differentially expressed between Cluster 1 vs. in Cluster 2 (p < 0.05) . The 558 transcripts up-regulated in Cluster-1 are associated with 37 core pathways in IPA (p < 0.05). The 265 transcripts up-regulated in Cluster-2 are associated with 37 core pathways in IPA (p < 0.05). The unsupervised clustering of protein expression (measured by RPPA) in OCI cell lines together with SOC ovarian cancer cell lines reveal two distinct clusters. List of Antibodies used for RPPA analysisThe heatmap profiles derived from unsupervised clustering of data from RPPA analysis of OCI cell lines. Each column depicts a different antibody and each row depicts an individual replicate from each cell lineAnalysis of RPPA data from OCI and SOC lines reveal a subset of proteins and phosphor-proteins that are differentially-expressed in the Taxol resistant OCI lines in cluster 1 vs. SOC lines in cluster 2


We had previously shown that the bLZip domain-containing transcription factor, Zhangfei/CREBZF inhibits the growth and the unfolded protein response (UPR) in cells of the D–17 canine osteosarcoma (OS) line and that the effects of Zhangfei are mediated by it stabilizing the tumour suppressor protein p53. To determine if our observations with D-17 cells applied more universally to canine OS, we examined three other independently isolated canine OS cell lines—Abrams, McKinley and Gracie.Like D–17, the three cell lines expressed p53 proteins that were capable of activating promoters with p53 response elements on their own, and synergistically with Zhangfei. Furthermore, as with D–17 cells, Zhangfei suppressed the growth and UPR-related transcripts in the OS cell lines. Zhangfei also induced the activation of osteocalcin expression, a marker of osteoblast differentiation and triggered programmed cell death.Osteosarcomas are common malignancies in large breeds of dogs. Although there has been dramatic progress in their treatment, these therapies often fail, leading to recurrence of the tumour and metastatic spread. Our results indicate that induction of the expression of Zhangfei in OS, where p53 is functional, may be an effective modality for the treatment of OS. Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents, although its incidence in dogs is ten times greater than in humans . SpontanWe had previously shown that the basic leucine zipper (bLZip) domain-containing transcription factor, Zhangfei/CREBZF/SMILE inhibits the growth and the unfolded protein response (UPR) in the D–17 canine osteosarcoma (OS) cell line  and thatZhangfei was initially identified through its interaction with the host cell factor (HCF1) a protein required for the initiation of herpes simplex virus gene expression . Unlike To confirm the effects of Zhangfei we had observed in D–17 OS cells we examined three other canine OS cell lines. We have shown that Zhangfei exerts its effect on cell growth and the UPR by stabilizing p53  and it tTo determine if the p53 proteins in the cell lines were functionally active, we transfected the cells with a plasmid that expressed the reporter protein chloramphenicol acetyl transferase (CAT) regulated by a promoter with two copies of a p53 response element (pCATp53RE). As a negative control, cells were transfected with a plasmid (pCAT3B) without the response elements. Parallel cultures were transfected with a plasmid expressing Zhangfei. Figure We next compared the effect of Zhangfei on the growth characteristics of Abrams, McKinley, and Gracie cells with its effect on D–17 cells. The cells were infected with adenovirus expressing either Zhangfei (Adeno-ZF) or the control protein β-galactosidase (Adeno-LacZ). Cell growth was monitored by the WST-1 Cell Proliferation Assay. In agreement with previous results, all four Adeno-ZF-infected cells failed to divide as early as day 1 after infection as determined by their ability to convert WST-1 Cell Proliferation reagent and absorb light at 405 nm. Mock infected cells continued to grow for three days and the growth of Adeno-LacZ-infected cells was indistinguishable from mock-infected cells Figure . Apoptos—osteocalcin .”All experiments were done following biosafety procedures and precautions approved by the University of Saskatchewan Biosafety committee and under Biosafety Permit VMB-03. All experiment were done on established cell lines and no animals were used.


B-cell receptor (BCR) and JAK/STAT pathways play critical roles in diffuse large B-cell lymphoma (DLBCL). Herein, we investigated the anti-lymphoma activity of cerdulatinib, a novel compound that dually targets SYK and JAK/STAT pathways. On a tissue microarray of 62 primary DLBCL tumors, 58% expressed either phosphorylated SYK or STAT3 or both. SYK and STAT3 are also phosphorylated in a panel of eleven DLBCL cell lines although ABC and GCB subtypes exhibited different JAK/STAT and BCR signaling profiles. In both ABC and GCB cell lines, cerdulatinib induced apoptosis that was associated with caspase-3 and PARP cleavage. The compound also blocked G1/S transition and caused cell cycle arrest, accompanied by inhibition of RB phosphorylation and down-regulation of cyclin E. Phosphorylation of BCR components and STAT3 was sensitive to cerdulatinib in both ABC and GCB cell lines under stimulated conditions. Importantly, JAK/STAT and BCR signaling can be blocked by cerdulatinib in primary GCB and non-GCB DLBCL tumor cells that were accompanied by cell death. Our work provides mechanistic insights into the actions of cerdulatinib, suggesting that the drug has a broad anti-tumor activity in both ABC and GCB DLBCL, at least in part by inhibiting SYK and JAK pathways. Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL) and accounts for approximately 40% of all NHL cases. The tumor progresses rapidly and treatment is normally initiated immediately after a patient is diagnosed with the disease. The standard chemoimmunoregimen, rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), is effective in ~60% of patients, but nearly 50% of patients treated with R-CHOP will eventually progress and relapse. The death rate of DLBCL remains at approximately 30%. Thus, there is an urgent demand for the development of more effective therapies based on the understanding of molecular pathogenesis.3) and diacylglycerol (DAG). This event is followed by activation of multiple distal signaling pathways for B-cell activation, such as RAS-MAPK pathway, PKC activation and formation of CARD11/BCL10/MALT1 complex, and subsequent NFκB activation [Aberrant B-cell receptor (BCR) signaling is implicated in B-cell malignancies including DLBCL. The BCR complex consists of surface immunoglobulins (sIg) that bind antigen in association with disulfide-linked heterodimer CD79A and CD79B proteins . Upon anThe first evidence that dysregulated BCR activation is a major contributor to DLBCL pathogenesis came from gene expression profiling analysis. Based on relatedness of gene expression profiles to normal B-cell subsets, DLBCL were classified into three cell-of-origin subtypes: germinal center B-cell (GCB) subtype, activated B-cell (ABC) subtype and primary mediastinal B-cell lymphoma (PMBL) –5.CD79A/B, CARD11, TNFAIP3 (A20) and MYD88. For PMBL DLBCL, key molecular abnormalities include CIITA translocations, amplification of REL, amplification of chromosome region 9p24 containing PD-L1, PD-L2 and JAK2 loci, and activation of NF-κB pathways [Subsequent studies revealed that different signaling pathways are involved in these distinct subtypes of DLBCL . The maipathways .Additional information regarding the molecular features of DLBCL was revealed by analysis of gene expression profiles with consensus clustering which groups lymphomas by functional relatedness of genes . This anPreviously, several groups including ours have explored the potential of inhibiting BCR pathways in DLBCL cell lines and primary tumor cells –14. We sFrom a clinical perspective, targeting the BCR pathway, however, has met with limited success in DLBCL patients compared to patients with other types of NHL and chronic lymphocytic leukemia (CLL). Fostamatinib, a SYK inhibitor, produced an objective response in 5 of 23 (22%) relapsed/refractory (R/R) DLBCL patients in a phase I/II study . EnzastaIn vitro inhibition of STAT3 activity with either JAK inhibitors or STAT3 knockdown results in decreased cell proliferation and increased apoptosis in ABC tumor cell lines [The Janus kinase and Signal Transducer and Activator of Transcription (JAK-STAT) pathway represents another important signaling pathway in the pathogenesis of DLBCL. One mechanism of STAT3 activation in ABC DLBCL (or non-GCB) has been defined. STAT3 is both overexpressed and activated primarily as a function of autocrine secretion of IL-6/IL-10 by tumor cells , a survill lines , 23. Morll lines , STAT3 kll lines  may be bC481S mutation, cerdulatinib is able to overcome ibrutinib resistance by completely blocking the proliferation of the resistant cells [Thus, literature evidence provides a strong rationale to target both BCR and JAK-STAT pathway in DLBCL. Cerdulatinib (previously known as PRT062070) is a novel orally available small-molecule ATP-competitive inhibitor that demonstrates inhibition of SYK, JAK1, JAK2, JAK3, and TYK2 in a biochemical assay   and p-SYK (Y525/526) on a tissue microarray of 62 DLBCL primary tumors, including 41 germinal center-like (GCB) and 21 non-germinal-center-like (non-GCB) tumors classified using Han's algorithm  Figure . PatternWe then determined the basal expression levels of BCR signaling and JAK/STAT signaling molecules and their phosphorylation status in a panel of GCB and ABC DLBCL cell lines using immunoblotting analysis. The cell lines studied include six GCB cell lines: OCI-LY1 (LY1), OCI-LY4 (LY4), OCI-LY8 (LY8), OCI-LY18 (LY18), SUDHL6 (DHL6) and VAL, and 5 ABC cell lines: OCI-LY3 (LY3), SUDHL2 (DHL2), HBL1, U2932 and OCI-LY10 (LY10). As shown in Figure 50's < 200 nM . Early clinical results  suggest that high level of SYK and JAK inhibition is achieved in treated patients, with an acceptable safety profile. In biochemical assays, cerdulatinib demonstrated inhibitory activity against 24 kinases with IC994382. E50 at or below ~2 μM . Overall, the results demonstrate potent and broad activity of cerdulatinib in DLBCL cell lines. Sensitivity to cerdulatinib was confirmed in an independent cell growth assay  can result from either cell death or cell cycle inhibition or both. We therefore analyzed the effects of cerdulatinib on DLBCL cell viability. Using annexin V and 7-AAD double staining, we demonstrated that with the exception of LY18, viability of all cell lines was reduced by cerdulatinib treatment in a concentration- and time-dependent manner Figure . ApoptosAfter establishing apoptosis induction as one of the drug's actions, we then studied the effect of cerdulatinib on cell cycle progression using BrdU incorporation. As shown in Figure Since the BCR pathway may be chronically active in many DLBCL, we next examined the capability of cerdulatinib to inhibit cell cycle and induce apoptosis under the condition of BCR stimulation. Figure To determine whether cerdulatinib inhibits BCR and JAK-STAT signaling pathways, we first measured the phosphorylation of SYK, PLCγ2, AKT and ERK in BCR-stimulated DLBCL cells treated with or without cerdulatinib Figure . ImmunobWe also determined whether cerdulatinib inhibits the JAK-STAT signaling pathways under the condition of cytokine stimulation. Cells stimulated with IL-6 and IL-10 were treated with cerdulatinib or vehicle, phosphorylation of STAT3 at Y705 was measured. As shown in Figure To confirm whether data obtained with the cell lines are reproducible with primary patient tumors, we tested several human DLBCL samples, including 3 GCB, 2 non-GCB and one unclassified (patient information shown in p = 0.0117). Meanwhile, no significant linear correlation was identified between the extent of p-AKT inhibition and the cell death response . The results suggest that indirect inhibition of downstream ERK phosphorylation by cerdulatinib may play a role in the final cellular outcome, and ERK inhibition may mediate the therapeutic effects of cerdulatinib.We next studied the inhibition of p-STAT3, p-ERK and p-AKT in response to cerdulatinib treatment in these primary patient cells stimulated with anti-BCR. Phospho-flow assay demonstrated a marked and simultaneous down-regulation of p-STAT3, p-AKT and p-ERK in response to increasing concentrations of cerdulatinib in all six primary DLBCL cells regardless of their subtype designation Figures , but inhAvailable clinical data indicate that BCR-directed inhibitors such as SYK (fostmatinib), BTK (ibrutinib) and PKCβ (enzastaurin) are active in ≤ 20% of DLBCL patients. This highlights the need to continue exploring novel agents with expanded activities in this disease. In this study, we first demonstrated the presence of active SYK and JAK in their phosphorylated forms in a significant fraction of DLBCL primary tumor tissues and cell lines. We then showed that cerdulatinib, a potent inhibitor of SYK and JAK, has broad anti-tumor activity as demonstrated by inhibition of 1) cellular metabolic function, 2) cell viability, 3) cell cycling, 4) signal transduction through SYK-PLCγ2-AKT or ERK, and 5) signal transduction through JAK-STAT. We further demonstrated that cerdulatinib induced cell death in primary DLBCL cells and the degree of cell death significantly correlated with decreased p-ERK.While the broad activity of cerdulatinib in DLBCL cell lines may be ascribed to mechanisms beyond SYK and JAK inhibition Table , the datCerdulatinib demonstrated broader anti-tumor activity relative to several BCR-specific inhibitors we have evaluated. Previously, we have shown that dasatinib (targets mainly SRC family kinases at low concentrations) and PRT318 (highly specific for SYK) exert anti-tumor activity primarily by affecting cell cycle with minimal impact on cell viability , 13. HowTNFAIP3 (A20), which negatively regulates NFκB activity. Loss of A20 function results in constitutive activation of NFκB that promotes tumor growth and survival. ABC cell lines, HBL1, LY3, LY10 and SUDHL2, along with GCB cell line SUDHL6 all carry activating MYD88 mutations, which promote IRAK1/4 and TRAF6 dimerization and subsequent NFκB activation.It is also noteworthy that many cell lines resistant to BCR-targeted inhibitors were sensitive to cerdulatinib. Among the cell lines studied, LY3 carries an activating mutation in CARD11  Supplem; LY3, LYC481S mutation [The ultimate activation of NFκB, resulting from these BCR-associated or MYD88 mutations, leads to increased IL6 and IL10 autocrine secretion that subsequently activates JAK-STAT3 pathway to enhance cell survival and proliferation. This perhaps represents one of several mechanisms rendering tumor cells resistant to inhibitors of early BCR components. As a matter of fact, high levels of baseline phosphorylated STAT3 exhibited in the ABC cell lines Figure  may be amutation , 29. The2 incubator. Frozen primary DLBCL cells were obtained from the tumor bank of the Department of Pathology at Weill Cornell Medical College after Institutional Review Board review and approval. All primary cells were thawed as previously described [GCB cell lines OCI-LY1, OCI-LY4, OCI-LY8, OCI-LY18, SUDHL6 and VAL were cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 20% Fetal Bovine Serum (FBS) and 100 μg/mL penicillin/streptomycin. ABC cell lines SUDHL2 and HBL1 were cultured in RPMI1640 supplemented with 20% FBS and 100 μg/mL penicillin/streptomycin. OCI-LY3 cells were grown in RPMI1640 supplemented with 20% FBS, 100 μg/ml penicillin/streptomycin and HEPES. ABC cell lines OCI-LY10 was cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 20% human serum and 100 μg/mL penicillin/streptomycin. ABC cell line U2932 was cultured in high glucose RPMI1640 supplemented with 20% FBS, 100 μg/ml penicillin/streptomycin. All cells were maintained in a humidified 37°C/5% COescribed  was kindThe study included 62 DLBCL samples in two tissue microarrays from 2000–2011. Twenty-five cases were from nodal sites and 37 from extranodal sites. Only tumors with enough tissues were included. Immunohistochemical stains of p-STAT3  and p-SYK  were performed and the results were evaluated by proportion of lymphoma cells that were stained. The antibody reactivity in ≥ 30% of the lymphoma cells was considered positive. Paraffin-embedded normal tonsil tissue was included as a baseline expression level for p-STAT3 and p-SYK.Immunoblotting were performed as previously described . Antibod50 was calculated using the Sigma Plot generated with GraphPad Prism 6 software . Cell growth was measured at every 24 h counting live cells with flow cytometry as previously described [DLBCL cell lines were treated with various concentrations of cerdulatinib for up to 72 h. The metabolic activities of cells were determined with MTT assay according to manufacturer's instruction  at 72 h time point. ICescribed . Cells wDLBCL cells were treated with various concentrations of cerdulatinib for 48 h. Cells were incubated with 10 μM BrdU  at 37°C for 2 h, and stained with PE conjugated anti-BrdU antibody (BD Biosciences) according to the supplier's manual. The percentage of cell cycle distribution was analyzed with FlowJo .DLBCL cells were treated with cerdulatinib for 6 h followed by stimulation with 5 μg/mL of goat F (ab')2 anti-human IgM and IgG antibodies  at 37°C for 15 min. Cells were fixed in 4% formaldehyde at room temperature for 10 min, and permeabilized with 100% methanol on ice for 20 min before flow cytometric analyses.A One-way ANOVA test was performed to compare the percentage of S phase distribution or the MFI fold changes between the treated and untreated DLBCL cell lines/primary cells. The relationships between the percentage of cell death response and inhibition of ERK1/2 or AKT phosphorylation were analyzed by Spearman correlation


Prostate cancer (PCa) is the most common type of cancer among males. Human epidermal growth factor receptor type 2 (HER2) expression in PCa has been reported by several studies and its involvement in the progression towards androgen-independent PCa has been discussed. External irradiation is one of the existing therapies, which has been demonstrated to be efficient in combination with androgen deprivation therapy for the treatment of advanced PCa. However, 20–40% of patients develop recurrent and more aggressive PCa within 10 years. The current study investigates the involvement of HER2 in survival and radioresistance in PCa cells and we hypothesized that, by monitoring HER2 expression, treatment may be personalized. The PCa cell lines, LNCap, PC3 and DU-145, received a 6 Gy single dose of external irradiation. The number of PC3 cells was not affected by a single dose of radiation, whereas a 5-fold decrease in cell number was detected in LNCap (P<0.00001) and DU-145 (P<0.0001) cells. The HER2 expression in PC3 exhibited a significant increase post irradiation, however, the expression was stable in the remaining cell lines. The administration of trastuzumab post-irradiation resulted in a 2-fold decrease in the PC3 cell number, while the drug did not demonstrate additional effects in LNCap and DU-145 cells, when compared with that of irradiation treatment alone. The results of the present study demonstrated that an increase in membranous HER2 expression in response to external irradiation may indicate cell radioresistance. Furthermore, imaging of HER2 expression prior to and following external irradiation may present a step towards personalized therapy in PCa. Prostate cancer (PCa) is the most common type of cancer in males. This cancer type has a heterogeneous nature and the characteristics vary during development. Initially, PCa develops in the prostate gland and is dependent on the androgen, testosterone for proliferation, growing slowly. At present, no optimal treatment has been identified due to the difficulties of predicting the disease progression ,2. Howevin vivo molecular imaging of HER2 expression in PCa may contribute to an improved patient selection, as well as improved therapy outcomes.External irradiation is a common treatment for PCa, and novel treatment regimens have been developed in order to increase the doses received by the tumor, whilst sparing the surrounding tissues. By using image-guided radiation therapy or intensity modulated therapy, doses of 78–81 Gy may be administered while the healthy tissue surrounding the tumor is spared. Combinations with selected radiation boost regimens such as brachytherapy, may achieve doses of >116 Gy . HoweverThe specific aims of this study were to analyze and evaluate PCa cell survival, as well as the HER2-expression as an acute response to external irradiation and anti-HER2 drug treatment, such as trastuzumab. In total, three cell lines, LNCap , PC3  and DU-145  were selected for this study. Together, these cell lines may represent the tumor heterogeneity due to differences in androgen sensitivity and aggressiveness. The cell panel was treated with external irradiation, modeling one of the current therapy modalities for a localized disease, alone or in combination with a HER2-targeting drug. Cell survival, as well as membranous expression of HER2 in response to therapy, was investigated. Trastuzumab, a clinically approved therapeutic monoclonal antibody (Herceptin), which binds to the extracellular domain of HER2 and downregulates its expression , was sel2 at 37°C.The cell lines LNCap, PC3 and DU-145 originally from the American Type Culture Collection  were provided by LGC Standards . The HER2-receptor expression of the cell lines was evaluated in a previous study . The celin vitro treatment. The drug was diluted in cell cultivation medium to 0.05 mg/ml. External irradiation was performed using a Gammacell 40 Exactor .Trastuzumab  was used for 2395 , was used. Radiolabeling of Z2395 with technetium-99m was performed as described by Ahlgren et al , treatment with trastuzumab (group III), or a combination of the two (group IV). One group of cells was used as a control (group I) and treated in the same manner as all other cells, without exposure to any drug or irradiation. All experiments were performed in triplicate.Cells were treated according to protocol A . Cells w2395 was added to half of the dishes and the cells were subsequently incubated for 1 h at room temperature. This was followed by the addition of 10 nM 99mTc-labeled Z2395 to all dishes and incubation for 1 h. The cells were subsequently trypsinized, resuspended, collected and counted according to protocols described previously . To evaluate the changes in receptor expression as response to external irradiation alone, cells were seeded as described, one day prior to the first irradiation exposure and treated according to protocol B . For theeviously ,21. The PCa cell lines treated with a 6 Gy dose of external irradiation exhibited a varied response with regards to cell survival and HER2 expression. Irradiation treatment alone was not observed to exert any effect on PC3 cells, whereas a 5-fold decrease in cell survival was detected in the LNCap (P<0.00001) and DU-145 (P<0.0001) cells . After 2The HER2-targeting drug, trastuzumab, was administered following irradiation treatment to evaluate its potential additive effect . The PC3The most common first line treatment for disseminated PCa is external irradiation, which is often combined with removal or blockage of androgen. However, 20–40% of patients relapse within 10 years following treatment. The occurrence of relapsing and more aggressive PCa with androgen independent clones is considered to be caused by an activation of androgen-independent pathways, required for cancer progression . HER2 isIn the current study, a panel of PCa cell lines was used to represent the heterogeneity of the disease. As the tumor is not homogenous, the use of one cell line would not adequately represent the various characteristics of PCa. Therefore, three PCa cell lines, LNCap, PC3 and DU-145, were selected, which exhibit different metastatic potentials and proliferation rates, as well as different degrees of androgen independence. Together the cell lines constitute a broad panel for preclinical PCa studies.The level of HER2 expression in the selected cell lines is considered to be 20,000–50,000 receptors/cell  which coIn this study, PCa cells were exposed to one dose of external radiation (6 Gy), which corresponds to one dose of fractionated therapy, and the short-term response in cell survival and HER2 expression was analyzed as a model for early therapy modeling. The rapid response to this treatment was observed as a 5-fold reduction in cell number in LNCap and DU-145 cells when compared with untreated controls , whereasIn order to investigate whether the HER2 targeting drug, trastuzumab, exhibits an effect post irradiation, PCa cells were exposed to trastuzumab treatment following external irradiation, or to trastuzumab treatment alone. Treatment with trastuzumab alone did not exhibit an inhibitory effect on cell survival, and notably demonstrated a pro-proliferative effect on PC3 cells . AlthougThis study preclinically evaluated a combination of clinically available therapies and drugs, and their effect on cell survival and HER2 expression. External irradiation therapy in combination with HER2-targeting drugs was demonstrated to alter the receptor expression profile in radioresistant PCa cell lines. The observed changes in HER2 expression as a response to this treatment regime may be used to monitor the response to therapy.In conclusion, measurement of HER2 expression prior to and following therapy may present a step towards patient stratification and more personalized treatment, as well as emphasizing the potential requirement for additional therapy. Additional studies investigating how analysis of HER2 in a xenograft model may serve as a marker to identify non-responders that may benefit from other treatments are required.


Dear Editor,BRAF and NRAS wild type remain limited  alone or in combination with BRAF inhibitor treatment has significantly improved the survival of patients with BRAF and 16 BRAF/NRAS wild-type cell lines (Table S1). The sensitivity of each cell line to trametinib was assessed using Syto60, a nucleic acid-based assay, after 6 days of exposure to 9 different escalating doses of trametinib (range 0.08–10 nM). This assay provides a robust estimate of cell viability  suggesting that utilisation of the MAPK pathway is an intrinsic feature of these melanomas. These results confirm and extend the validity of a recent study showing that BRAF/NRAS wild-type melanoma cell lines are sensitive to trametinib and suggest that they can be as sensitive to MEK inhibition as melanomas with BRAF mutations  and sequencing (Table S1). Nine of the 16 BRAF/NRAS wild-type cell lines analysed by Western blotting displayed undetectable NF1 protein levels while 7 expressed NF1 protein . Trametinib induced a significant decrease of ETV5 and PHLDA1 levels in 3/4 and 4/4 cell lines, respectively . Our collection comprised 9 cell lines carrying activating mutations of BRAF/NRAS wild-type melanomas are highly sensitive to the MEK inhibitor trametinib, and that loss of NF1 protein expression alone does not stratify sensitive cell lines. Overall, our findings mandate further investigation of the efficacy of trametinib in BRAF/NRAS wild-type melanoma. Given the limited therapeutic options for BRAF/NRAS wild-type melanoma, trametinib may represent a useful therapeutic tool for patients with this subclass of the disease.In summary, we show that


Targeted anti-HER2 therapy has greatly improved the prognosis for many breast cancer patients. However, treatment for HER2 negative disease is currently still selected from a multitude of untargeted chemotherapeutic treatment options. A predictive test was developed using patient-derived spheroids to identify the most effective therapy for patients with HER2 negative breast cancer of all stages, for clinically relevant subgroups, as well as individual patients.Tumor samples from 120 HER2 negative patients obtained through biopsy or surgical excision were tested in the breast cancer spheroid model using scaffold-free cell culture. Similarly, spheroids were also generated from established HER2 negative breast cancer cell lines T-47D, MCF7, HCC1143, and HCC1937 to compare treatment efficacy of heterogeneous cell populations from patient tumor tissue with homogeneous cell lines. Spheroids were treated in vitro with guideline-recommended compounds. Treatment mediated impact on cell survival was subsequently quantified using an ATP assay.Differences were observed in the metabolic activity of the untreated spheroids, whereby cell lines consistently achieved higher values compared to tissue spheroids (p < 0.001). A higher number of cells per spheroid correlated with a higher basal metabolic activity in tissue-derived spheroids (p < 0.01), while the opposite was observed for cell line spheroids (p < 0.01). Recurrent tumors showed a higher mean vitality (p < 0.01) compared to primary tumors. Except for taxanes, treatment efficacy for most tested compounds differed significantly between breast cancer tissue spheroids and breast cancer cell lines. Overall a high variability in treatment response in vitro was seen in the tissue spheroids regardless of the tested substances. A greater response to anthracycline/docetaxel was observed for hormone receptor negative samples (p < 0.01). A higher response to 5-FU (p < 0.01) and anthracycline (p < 0.05) was seen in high grade tumors. Smaller tumor size and negative lymph node status were both associated with a higher treatment efficacy to anthracycline treatment combined with 5-FU .The tissue spheroid model reflects current guideline treatment recommendations for HER2 negative breast cancer, whereas tested cell lines did not. This model represents a unique diagnostic method to select the most effective therapy out of several equivalent treatment options. Although prognosis has vastly improved for HER2 positive breast cancer due to targeted anti-HER2 treatment options, the remaining majority of patients with HER2 negative disease lack directed therapy options. For these patients a number of guideline directed chemotherapy substances and regimen are available. Patients are stratified to a chemotherapy regimen according to routine clinicopathological criteria such as TNM-stage, hormone receptor status, grading and tumor histology, as well as prior medical history and existing comorbidities , 2. ThisPredictive tests or assays to direct cancer treatment decisions remain an unmet need in oncology diagnostics and in the field of personalized medicine –7. As ofBiomarkers currently being studied in clinical trials mostly focus on the molecular profiles which characterize the odds of recurrence –21. DepeOut of the newer techniques such as tumor infiltrating lymphocytes, Ki-67, circulating tumor cells, genetic profiles, and PIK3CA analysis, only the uPA/PAI1 analysis as well as the 21-Gene-Recurrence Score have been validated in prospective clinical trials, however, only for specific clinical breast cancer subtypes in primary disease –25.Laboratory models such as genetically engineered and patient-derived xenograft mouse models allow the study of deregulated pathways in the development of invasive tumors and the treatment efficacy in a living organism . HoweverBreast cancer cell lines are another frequently used model system and have previously been closely associated to the originating patient tumors regarding genomic aberrations, protein expression even after prolonged culture, and treatment efficacy –35. HoweOne main criticism of standard 2D cell culture is the absence of the surrounding cells and microenvironment which have an important influence on tumor development and progression  and drugThe aim of this study is to determine the treatment efficacy of the most frequently applied treatment options on tumor tissue-derived spheroids from HER2 negative breast cancer patients. These treatment results were compared to those obtained from established HER2 negative breast cancer cell lines, namely MCF7, T-47D, HCC1143, and HCC1937. Clinical subgroups were analyzed separately to determine if groups of patients or individual patients could be identified which deviate from the mean in regard to treatment efficacy in vitro, thus determining new treatment strategies for these patients.The tissue was provided by a representative patient cohort of 120 HER2 negative breast cancer patients between 2011 and 2015 out of a total of 200 collected. Samples were considered in this analysis if patients were histologically confirmed HER2 negative  and a valid assay readout was available , a total of 8 samples failed to fulfill both criteria. Tissue samples were consecutively collected either through an ultrasound-guided core biopsy procedure (n = 77) or through surgical excision (n = 43). Tissue samples derived from primary (n = 109) and recurrent tumors (n = 11). Tissue from the recurrent tumors was obtained from local recurrence (n = 6), lymph nodes (n = 2), or distant metastasis (n = 3). Samples from two different tumor sites were obtained from five patients, in two cases from the originating breast tumor and the simultaneous lymph node metastasis, one case of invasive lobular carcinoma obtained from the right and left breast, as well as tumor tissue from two different metastatic sites from two patients. Clinical and pathological data is reported from the time of tissue excision. A written and oral informed consent was obtained from all patients prior to tissue excision, and an approval by all applicable ethics committees was obtained prior to study start.All laboratory procedures were performed according to standardized, quality-controlled operating procedures. The mean cold ischemic time for the surgical samples was 22.13 min (range 0–60 min), the time from tissue excision to the beginning of the cell isolation procedure was mean 24.09 h (range 14.75–48 h). Biopsy samples were directly transferred to freshly prepared culture medium following excision. Breast cancer spheroids were directly generated from cancerous tissue as described recently . Briefly® . Cells were grown in appropriate culture medium and kept in culture for a maximum of 10 passages. For spheroid preparation, cells were detached using 1 mM EDTA and seeded 0.05 × 106 per well. All experiments were repeated a minimum of three times, and given values represent a mean of all experiments with exceptions specifically detailed. The last passage of all four cell lines used in the treatment experiments was sent for external STR-analysis for authentication . In addition cytospin samples of each cell line were prepared and stained using standard immunohistochemistry for confirmation of the epithelial origin, proliferation rate, and receptor status (data not shown).Cell line spheroids were generated using the representative hormone receptor positive breast cancer cell lines MCF7 (HTB-22) , T-47D (2) for a consecutive 48 h following seeding. The spheroids were then treated with guideline recommended cytostatic compounds or combination of compounds using the peak plasma concentrations as detailed in a previous publication [Both primary tissue and cell line spheroids were kept under standard culture conditions  years at sample excision. The majority of tumors were either ER and/or PR positive (66.8 %), stage cT1/2 (74.7 %), node negative (52.0 %), with a high-grade (50.2 %) invasive ductal/other histology (84.2 %). A mean Ki67 score of 39.22 % positive cells (range 0.01–100 %) was recorded for the collected study samples. Patient characteristics are summarized in Table –85 yearsMean cell viability of single cell suspensions following cell isolation from fresh tumor tissues was 87.0 % (range 26.2–100 %). No differences in cellular viability was found for cells isolated from biopsy or surgical tissue . Differences in viability were detected between primary and recurrent tumors, the measured viability was higher for the recurrent tumor samples .The number of isolated cells was highly dependent on the amount of tissue that was provided (p < 0.001). The surgical tissue weighed a mean of 1020.6 mg (range 58.6–7232.3 mg) and the biopsy samples a mean of 86.3 mg (range 10.5–513.3 mg). No significant difference in the number of cells per mg tissue was observed between surgical specimen 5201.1 cells/mg (range 284.5–58949.8 cells/mg) and biopsy samples 6061.0 cells/mg  or between primary  and recurrent tumors . However, due to the higher total tissue weight, a higher number of treatment options could be tested using surgical specimen: a mean of 8 (range 1–23) for the surgical specimen and 2 treatment options (range 1–9) for the biopsy samples . Using regression analysis over all samples, it was determined that a minimum of 69 mg of tissue is required to test a minimum of three different treatment options (p < 0.001).The amount of ATP quantified for solvent control samples  in the assay was measured to determine any differences in metabolic activity not associated with cytostatic treatment. Comparison of the tissue-derived spheroids revealed differences between biopsy and surgical samples in metabolic activity, biopsy samples reaching a mean of 0.479 cps/cell compared to a mean of 1.055 cps/cell for surgical samples . The mean cps value relative to the number of cells per spheroid of the primary tumor spheroids was 0.628 cps; recurrent tumors achieved higher values with a mean of 1.022 cps, the difference was however not statistically significant. Spheroids generated from breast cancer cell lines showed much higher metabolic activity values with a mean of 26.55 cps . The highest values of metabolic activity was observed for MCF7 (36.85 cps/cell), followed by T-47D (32.36 cps/cell), HCC1937 (28.10 cps/cell), and HCC1143 (3.76 cps/cell), the difference between the cell lines was not significant.In order to determine the effect of cell number in tissue-derived spheroids on metabolic activity data was collected using <10,000, 10,000–20,000, >20,000 cells per spheroid. As shown in Fig. Collectively, the tissue-derived spheroids showed a high variability in response to cytostatic treatment in vitro, regardless of primary or recurrent disease. Individual results are shown in Fig. Data shown in Fig. In order to better identify patient subgroups responding best to individual treatment regimen, the results from the patient-derived spheroids were grouped according to the clinical factors reported in Table As shown in Table Treatment efficacy to Doc was greater with higher metabolic activity (p < 0.05). For all other tested compounds no significant correlation between these two factors was found. However, an effect was seen when comparing treatment efficacy with a metabolic activity above and below the observed median of 0.169 cps/cell Table . TreatmeContrary data was found for the cell lines, both hormone receptor positive/low grade cell lines consistently responding better to treatment in vitro. This effect was seen for Doc , EC-Pac , DocAC , and EC-Doc .A grouped comparison of receptor positive or negative tissue with the respective HR+ or HR− cell line spheroids showed no difference in the treatment with taxanes. However, for all other tested compounds drug efficacy was significantly higher in tissue spheroids regardless of hormone receptor status. Exception was the treatment efficacy for 5-FU resulting in a higher drug response in HR− tissue spheroids compared to HR− cell line spheroids (p < 0.05). Grouped comparison according to grading revealed tissue spheroids responding much better to treatment compared to the respective cell line spheroids with the same grading. Taxane treatment was not significantly more effective for either high- or low-grade tissue spheroids versus cell lines. Again, 5-FU was only significantly more effective for high-grade tissue spheroids compared to the matched cell lines .An overview of the assay data from all tested patients is shown in Fig. Detailed treatment options were further tested with spheroids generated from five different patients with disseminated tumors Fig. . ResultsIn this study, tissue-derived spheroids from HER2 negative patients were studied to determine differences in the observed treatment efficacy for the most frequently applied treatment schemes in the primary, recurrent, and metastatic setting and compared to results obtained from spheroids generated from HER2 negative breast cancer cell lines.Regardless of the tested compound(s) and clinical characteristics, the range in treatment response for patient tissue-derived spheroids was highly heterogeneous. However, the mean values are representative of current international guideline recommendations , 56, 57.Comparing the results obtained from tissue-derived spheroids according to hormone receptor status showed that a higher treatment efficacy to anthracycline-taxan combination treatment was found for hormone receptor negative tissue samples. This difference in response confirm results published by Kaufmann et al.  which alOther differences found in treatment efficacy between clinical subgroups were not statistically significant, mainly due to the large range in the collected data and the small sample size for individual subgroups. A fact reflecting this high heterogeneity in the treatment efficacy found for the tissue spheroids is that each patient tumor showed a distinct response pattern as seen in the heat map in Fig. In general, cell line spheroids were consistently more resistant to in vitro treatment compared to tissue-derived spheroids. Treatment efficacy data was similar to other data previously reported in 3D cell culture , 63. TheRegarding the tissue-derived spheroids, the observed between patient, as well as intrapatient heterogeneity between tumor locations show the necessity for a more individualized treatment algorithm or diagnostic assay. The question remains for the ideal model to test treatment efficacy, currently no method is without its drawbacks, and however it is important to consider all aspects of tumor biology, as well as the tumor microenvironment , 65. CurCurrently further data is necessary to determine an association with prognostic factors regarding survival and an additional interventional study to determine the independent prognostic and predictive power of the breast cancer spheroid model. Data obtained through these additional studies may aid in bridging the gap between guideline-directed treatment recommendations and tumor-directed therapeutic needs using a more representative in vitro model.The current study was conducted to provide additional data to validate the breast cancer spheroid model and its predictive potential regarding clinical treatment outcome for HER2 negative breast cancer patients. Tissue-derived spheroids treated in vitro with guideline recommended cytostatic drugs and drug combinations recapitulate clinical findings for HER2 negative patients. These findings are in stark contrast to HER2 negative breast cancer cell lines suggesting an impact of the stromal microenvironment in drug response. Taken together, comparison between tissue spheroids and cell line spheroids underlines that the tissue-derived breast cancer spheroid model has the advantage of representing the individual patient tumor much more closely in comparison to established breast cancer cell lines and may be more suited for preclinical drug testing.


Variations in biochemical features are extensive among cells. Identification of marker that is specific for each cell is essential for following the differentiation of stem cell and metastatic growing. Fourier transform infrared spectroscopy (FTIR) as a biochemical analysis more focused on diagnosis of cancerous cells.  In this study, commercially obtained cell lines such as Human ovarian carcinoma (A2780), Human lung adenocarcinoma (A549) and Human hepatocarcinoma (HepG2) cell lines in 20 individual samples for each cell lines were used for FTIR spectral measurements. Data dimension were reduced through principal component analysis (PCA) and then subjected to neural network and linear discrimination analysis to classify FTIR pattern in different cell lines. , as well as amide I and amid II bands of proteins. The PCA-ANN analysis provided over 90% accuracy for classifying the spectrum of lipid section in different cell lines. This work supports future study to establish the data bank of FTIR feature for different cells and move forward to tissues as more complex systems.The results showed dramatic changes of FTIR spectra among different cell types. These appeared to be associated with changes in lipid bands from CH2 symmetric and asymmetric bands These sources of variation causes different biochemical matrix in cells which are relevant for different studies  has been employed as a data dimension reduction model. Reduced data matrix was forwarded to linear discriminate analysis (LDA) and artificial neuronal network (ANN) as most recent developed models to discriminate different cell lines.Cell lines2. All cells have been used at almost the same life time i.e. after two passages. A2780 (human ovarian carcinoma) and A549 (human lung carcinoma) and HepG2 (human liver carcinoma) cell lines were obtained from Pasture Institute National Cell Bank of Iran . All cell lines were grown in RPMI-1640 medium and supplemented with 10% heat inactivated fetal bovine serum, antibiotics: penicillin, streptomycin . Cells were maintained at 37 °C in humidified atmosphere containing 5% COCell preparation for spectroscopy2 flasks with fresh medium to reach the logarithmic phase of growth curve. After that cell were washed twice in saline (0.9% NaCl), suspend and centrifuged at 1000 rpm for 5 min, then resuspended in saline to obtain a concentration of 1 × 105 cells. 10 μL of each cell suspension was placed on a zinc selenide sample carrier which was dehydrated in a vacuum cabin (0.8bar). These plates were then used for FTIR spectroscopy. The following procedure was similarly applied for all cell lines. Cells were trypsinized from the original flask and seeded in 25 cmFTIR spectroscopy-1 for every wave number between 400 and 4000 cm-1. These experimental conditions were kept constant for all measurements. Each single spectrum was baseline corrected and then all wave number normalized in order to better comparison for the range spanning from 0 to 1.For FTIR studies, thin dried films of cell suspension was used on the Zinc selenide window by using a WQF-510  spectrometer, equipped with a KBr beam splitter and a DLaTGS (deuterated Lantanide triglycine sulphate) detector. The whole system was continuously purged with N2 (99.999%). In each spectrum, 100 scans were collected at a resolution of 4 cmRecovery of drying techniqueDrying of cells in vacuum of 0.8 bars for different times was assessed by FTIR analysis. Remained water in dehydrated sample was evaluated by Differential Scanning calorimetry analysis (DSC).Temperature program was changed from -20 to 100 °C with the rate of 5 °C/min.Data analysisData set-1 have been used in this study as the dataset  between 1000-3000 cm dataset , 26 DistPrinciplecomponent analysisPCA is a well-known method of dimension reduction. The basic idea of PCA is to reduce the dimensionality of a data set, while retaining as much as possible the variation present in the original predictor variables. In mathematical terms, PCA maximizes the variance of a linear combination of the original predictor variables .PCA scores from various PCs were examined to give best separation between cell lines. PCA was used for preliminary data reduction and then output processed with artificial neural networks (ANN) and linear discrimination analysis (LDA).Artificial neural networksArtificial neural networks (ANN) are computerized mathematical models designed to mimic the architecture of the brain. They are able to detect non linearity, making them capable of learning and adaptability . The netMultiple layer perception neuronal networks were designed according to MATLAB. The output of PCA analysis has been used for input layer of ANN model. The output layer consisted of three output neurons, one to classify the A549 category and the others for HepG2, and A2780 data.Linear discriminate analysisThe basic theory of linear discriminate analysis is to classify the dependent variable by dividing an n-dimensional space into two regions that are separated by a hyperplane . The datRecovery of drying technical-1 was omitted in 4 min of vacuum drying. The remained water in dehydrated cell suspension was estimated by DSC analysis (10 μL of A2780 cell suspension was placed on a zinc selenide sample carrier which was dehydrated in a vacuum cabin (0.8bar) for different time. analysis . There iSpectrum alteration-1. These spectra represent average spectra from the 20 individual of the same cells. The normalized FTIR spectra in this region showed alterations in different spectrum areas. There is a peak about 1636 cm-1 in the spectra of the three cell lines. This peak is attributed amid I structure 2 symmetric and asymmetric stretching vibration bands shift to higher wave numbers in A549 cells. Moreover, CH3 stretching vibrations bands at 2950 cm-1  was investigated (Determination of cell-types with immunocytochemistry methods has been reported frequently -8. This stigated , 35. In Although cellular biomolecules are varied but thorough a spectroscopy analysis, such as FTIR, may be capable of detecting these variations as early as in the first hours of sampling. Sixty individual FTIR spectra of A2780, A549 and Hepg2 cell lines forwarded to supervised models for finding pattern of cells. Since several studies used FTIR analysis in cell biology -17, one -1 in the spectra of the three cell line related to β-sheet secondary structure of amid I (-1 in two cell lines but not in the ovarian cell. The bands at 1620 cm-1are assigned to aggregated strand structures of amid I (The results exhibited dramatic change as marker for cell-type identification. There is a peak about 1636 cmf amid I  where cof amid I . Moreove-1 is more accurate than the others when 95% of FTIR data set was anticipated exactly. From our results correct classification provided by the PCA-LDA models were less accurate than those provided by PCA-ANN analysis. Our results demonstrate that it is possible to classify different cell lines based on the analysis of FTIR spectra markers using multivariate PCA-ANN model. PCA-ANN model classifies different FTIR region between 85% to 93% correctly. Comparison of the 20 PCA-ANN models indicates that uses of variable in the region of 2500-3000 cm-1 was classified as a data region with less variability among different series, and therefore very much suitable for the discrimination of different cell lines using both PCA-ANN and PCA-LDA models. It is therefore acceptable to conclude that this segment of FTIR data, which related to protein structure of cells (As is shown in our results, FTIR data set between 1500-2000 cmof cells , is a go


Background: ACKR2 is an atypical chemokine receptor that is biologically distinct from conventional chemokine receptors.Results: An N-terminal sulfated tyrosine motif is essential for ligand binding by ACKR2.Conclusion: The structure/function basis for ligand binding by ACKR2 is similar to that of conventional chemokine receptors.Significance: ACKR2 N-terminal peptides may be therapeutically useful as pan-chemokine blockers in inflammatory pathologies. The atypical chemokine receptor, ACKR2 is a pivotal regulator of chemokine-driven inflammatory responses and works by binding, internalizing, and degrading inflammatory CC-chemokines. ACKR2 displays promiscuity of ligand binding and is capable of interacting with up to 14 different inflammatory CC-chemokines. Despite its prominent biological role, little is known about the structure/function relationship within ACKR2, which regulates ligand binding. Here we demonstrate that a conserved tyrosine motif at the N terminus of ACKR2 is essential for ligand binding, internalization, and scavenging. In addition we demonstrate that sulfation of this motif contributes to ligand internalization. Furthermore, a peptide derived from this region is capable of binding inflammatory chemokines and inhibits their interaction with their cognate signaling receptors. Importantly, the peptide is only active in the sulfated form, further confirming the importance of the sulfated tyrosines for function. Finally, we demonstrate that the bacterial protease, staphopain A, can cleave the N terminus of ACKR2 and suppress its ligand internalization activity. Overall, these results shed new light on the nature of the structural motifs in ACKR2 that are responsible for ligand binding. The study also highlights ACKR2-derived N-terminal peptides as being of potential therapeutic significance. The human acute monocytic leukemia cell line, THP-1, was maintained in RPMI 1640 (Sigma), 10% FCS, 4 mm glutamine, and streptomycin and penicillin . All cultures were incubated at 37 °C with 5% CO2 and 95% humidity. Human dermal-derived lymphatic endothelial cells  plus 10% FCS, 4 mal cells , human kal cells , BeWo  were introduced into human ACKR2 cDNA by PCR to generate HA-ACKR2 as described . ProductPlasmids were stably transfected into HEK 293 cells using the Effectene® transfection reagent kit (Qiagen). Stable transfectants were selected in 1.6 mg/ml G418 . High ACKR2-expressing cells were separated from low expressing and nonexpressing cells by adding anti-HA biotin (Miltenyi Biotec) to cells and mixing with anti-biotin MicroBeads (Miltenyi Biotec). Cells were run through MACS separation columns (Miltenyi Biotec) attached to a MidiMACS separator (Miltenyi Biotec), and eluates were collected. Expression of ACKR2 was verified by flow cytometry using anti-ACKR2 monoclonal antibody and FITC-labeled secondary antibody (R&D Systems) or alternatively biotinylated anti-HA antibody (Miltenyi Biotec) and phycoerythrin-streptavidin-labeled secondary antibody (R&D Systems).2 and subsequently washed twice in ice-cold FACS buffer. DRAQ7  was added to each cell suspension to identify nonviable cells. Fluorescence intensity of the cells was acquired on a MACSquant analyzer (Miltenyi Biotec) and analyzed using MACSquant software.Either CCL2 or CCL22  was added to cells. Cells were incubated for different time periods at 37 °C in 5% CO4 cells/well) in media. The following day, biotinylated human CCL2 (bio-CCL2) (Almac) was added to the media at a concentration of 50 μg/ml, and cells were incubated at 37 °C in 5% CO2. Medium was collected at different time points ranging from 0 to 30 h after the addition of bio-CCL2 and stored at −20 °C before being analyzed by Western blotting.Cells were plated out the night before the assay into a 96-well plate to ∼80% confluency (2 × 10TM epitope tag protein isolation kit (Miltenyi Biotec) with anti-HA MicroBeads. Cell lysates were prepared before magnetic labeling was performed by adding anti-HA MicroBeads to the cell lysates and incubating for 30 min on ice with regular gentle agitation. The mixture was run through a μ-column suspended on a magnet as per the manufacturer's instructions. After several washes to remove any nonspecific material, elution buffer (SDS buffer) heated to 95 °C was added to the column, and samples were collected . Subsequent to elution samples were analyzed by SDS-PAGE before Western blotting with an anti-sulfotyrosine antibody  or an anti-HA antibody .HA-ACKR2 was immunoprecipitated from ACKR2-expressing HEK cells using the μMACSCells were grown in media supplemented with a range of concentrations of sodium chlorate  (Sigma) Cells were plated out the night before in 6-well plates and grown to ∼80% confluency. The following day cells were transfected with TPST-1 siRNA, TPST-2 siRNA, or negative control siRNA using HiPerfect transfection reagent (All from Qiagen). The next day, cells were transiently transfected with the HA-ACKR2 plasmid described above using the Effectene® transfection reagent kit (Qiagen). 24 h later cells were tested for their ability to uptake AF-CCL22 using chemokine uptake assays. Cell lysates were also collected and analyzed by Western blotting using an anti-ACKR2 antibody (Sigma).Quantitative PCR was performed as described previously , 19 usinPeptides representing the first 35 amino acids of the N terminus of the ACKR2 protein (ACKR2-N) were generated by peptide synthesis (Almac Scotland). Two versions of ACKR2-N were generated, with either sulfated or nonsulfated tyrosine residues. These are denoted as ACKR2-N(s) and ACKR2-N(un-s) respectively. A hexahistidine tag was incorporated into the C-terminal tail of both ACKR2-N(s) and ACKR2-N(un-s).TM streptavidin kit (Miltenyi Biotec) was used to ascertain any binding relationship between the two molecules. Samples collected from such experiments were then run on SDS gels and analyzed by Western blotting to examine any co-pulldown of ACKR2-N.Biotinylated chemokines (Almac) were incubated with ACKR2-N(s), and the μMACSSamples were mixed 1:4 with NuPAGE® LDS sample buffer (Invitrogen) and either heated to 95 °C for 5 min or, when analyzing ACKR2 expression, incubated at room temperature for 10 min . SamplesGels were transferred onto a nitrocellulose membrane using an iBlot® system (Invitrogen) according to manufacturer's instructions. Following transfer, the membrane was washed in PBS Tween (PBST) and blocked in 5% Milk/PBST either for 1–2 h at room temperature or overnight at 4 °C. After blocking, the membrane was washed briefly in PBST and incubated with primary antibody overnight at 4 °C. The membrane was subsequently washed four times for 5 min each in PBST and incubated with secondary antibody for 1 h at room temperature. Blots were developed via a chemiluminescence reaction  before being placed between acetate sheets and exposed to x-ray film  in a dark room for varying time periods. Film was then developed in an X-Omat processor .2. In similar experiments Alexa-CCL2 was “precomplexed” with ACKR2-N by incubating 1.0 μg of ACKR2-N(s) or ACKR2-N(un-s) with 60 ng of Alexa-CCL2 in a total volume of 20 μl of PBS for 15 min at room temperature before addition to wells. Cells were subsequently analyzed on a MACSquant analyzer (Miltenyi Biotec).Cells were treated as detailed previously; however, 1 μg of ACKR2-N(s) or ACKR2-N(un-s) (Almac) was added to wells simultaneously with 60 ng/well of Alexa-CCL2 before incubation for 1 h at 37 °C in 5% CO5 cells/well) in regular media. The following day staphopain A (Sigma) was added to (2 μm) to wells, and the cells were incubated for various time periods ranging from 15 min to 2 h at 37 °C in 5% CO2. Afterward cells were scraped, and cell lysates were prepared using cell lysis buffer (Miltenyi Biotec). Samples were then analyzed by Western blotting. For chemokine uptake assays cells were washed twice with PBS before scraping off and resuspending in complete medium and adding 25 nm Alexa-CCL22. Cells were incubated for 1 h at 37 °C in 5% CO2 and subsequently washed twice in ice-cold FACS buffer. DRAQ7 (BioStatus) was added to each cell suspension to identify nonviable cells. The fluorescence intensity of the cells was measured on a MACSquant analyzer (Miltenyi Biotec).Cells were plated out the night before the assay into 6-well plates to about 80% confluency . The raw data were analyzed using the integrated MARS data analysis software (BMG Labtech).Chinese hamster ovary (CHO) K1 cells expressing ACKR2 were plated out the night before on black 96-well plates to ∼80% confluency. The following day, cells were treated with staphopain A (Sigma) in PBS at concentrations of 0.5 and 2 μp < 0.05 being regarded as significant. Individual statistical tests used are noted in the relevant figure legends.The data were analyzed using GraphPad Prism (Version 5) software  with A, we generated a mutant version of ACKR2 (henceforth referred to as mutant 1) in which all three conserved N-terminal tyrosine residues, as well as a fourth unique to higher order primate ACKR2, within this motif were mutated to phenylalanines. This construct was stably transfected into HEK cells and compared with HEK cells expressing WT ACKR2 for ligand binding and internalizing ability. Both molecules had an HA tag at the extreme N terminus allowing for flow cytometric assessment of expression levels in the stable clones. As shown in Fig. 2B, transfected HEK cells expressed similar levels of cell surface WT ACKR2 and mutant 1. Next we assessed the relative ability of WT ACKR2 and mutant 1 to internalize ligand by analyzing uptake of Alexa-CCL2 by flow cytometry. As shown in Fig. 2C, it is clear that untransfected HEK cells do not internalize Alexa-CCL2 (panel i). However, whereas WT ACKR2 internalizes it avidly , similar uptake is not seen with mutant 1 . Indeed on multiple repeat experiments, shown in Fig. 2D, it was clear that mutant 1 displayed a significantly reduced ability to internalize ligand even at very high chemokine concentrations. Finally, we examined the ability of WT ACKR2 and mutant 1 to degrade ligand. As shown in Fig. 2E, whereas HEK cells expressing WT ACKR2 substantially degraded biotinylated CCL2 over 24 h, as detected by Western blotting using a streptavidin detection system, two clones of HEK cells expressing mutant 1 did not. Thus N-terminal tyrosine residues are essential contributors to the ability of ACKR2 to internalize and degrade chemokines.Alignment of the primary sequences of the N terminus of ACKR2 from a variety of mammalian species  revealed32–Fig. 2A, both a pool of mutant 1 transfected HEK cells and two separately isolated clones displayed markedly reduced sulfation compared with WT ACKR2. Note that the low level staining seen in the mutant 1 tracks is seen routinely, and thus we presume it to be background. To further implicate sulfation in ligand internalization by ACKR2, we incubated HEK cells expressing WT ACKR2 in 30 mm sodium chlorate, which blocks protein sulfation, and assessed the impact of this on ligand uptake. As analyzed by flow cytometry and as shown in Fig. 3B, growth in the presence of sodium chlorate partially but significantly reduced the ability of HEK cells expressing ACKR2 to internalize ligand  in a time-dependent manner. To further examine the ability of sodium chlorate to reduce ligand binding and internalization by ACKR2, we tested higher concentrations. As shown in Fig. 3C (panel i), although concentrations of sodium chlorate as high as 100 mm partially reduced ligand binding by ACKR2, concentrations of 150 mm induced a considerably more marked and significant reduction. This was also shown by flow cytometry  and Western blotting . Thus both mutation of N-terminal tyrosine residues and treatment of ACKR2-expressing cells with high concentrations of sodium chlorate demonstrate the involvement of tyrosine sulfation in ligand binding and internalization by ACKR2.The inability of the tyrosine mutant variant of ACKR2 to internalize ligand suggested that sulfation of these residues may, as has been reported from conventional chemokine receptors, be essential for ligand binding and uptake , 33, 35.Fig. 3A, although very little expression of TPST1 was detected in any of the cell types examined, the strongest expression of TPST2 was detected in lymphatic endothelial cells and placenta, both sites of high ACKR2 expression  and protein  levels. To examine the importance of these tyrosylsulfotransferases for ACKR2 sulfation and function in transfected HEK cells, we pretreated HEK cells with siRNA to either, or both, enzymes and then transiently transfected these cells with ACKR2 and measured ligand binding (using Alexa-CCL22) 24 h later. As shown in Fig. 4C (panel i), treatment with siRNA to either enzyme individually had no significant effect on ligand binding by ACKR2; however, simultaneous treatment with siRNA to both enzymes significantly reduced ligand binding. This treatment was not associated with any reduction in ACKR2 protein expression  and suggests that both enzymes are independently able to contribute to sulfation of and therefore ligand binding by ACKR2.Tyrosine sulfation is dependent on the expression of tyrosylsulfotransferases, of which two are known to be expressed and functioning in mammalian cells . To gainFig. 4pression –17, suggA, flow cytometry for the HA tag revealed similar levels of cell surface expression of each of the four mutants. As above, ligand internalization was assessed by flow cytometry to examine the ability of the WT and mutant variants to take up Alexa-CCL2. Quantitation of multiple flow cytometry experiments  demonstrated again the profound inability of mutant 1, the full tyrosine variant of ACKR2, to internalize ligand. These data further demonstrated that mutation of the individual conserved tyrosine residues did not significantly impair ligand internalization compared with the WT receptor. In contrast, and as shown in Fig. 5C, phenylalanine mutation of the nonconserved tyrosine residue found in human ACKR2 (mutant 5) in fact led to significantly enhanced ligand internalization, suggesting that this may represent an evolutionary adaptation in human ACKR2 that alters the ability of ACKR2 to bind ligand.We next performed more specific mutagenesis studies in which individual tyrosines within the conserved motif were mutated to phenylalanines , it was clear that introduction of the single residues into the fully mutated motif is sufficient to at least partially rescue ligand uptake by ACKR2.Next we carried out the reciprocal experiment in which we introduced individual tyrosines into the fully tyrosine mutated variant of ACKR2 (mutant 1) to see whether single tyrosine residues were sufficient to rescue ligand internalization by this mutant. These mutants were stably transfected into HEK cells as above, and as shown in Fig. 5Thus together these data demonstrate that although tyrosine residues are essential for efficient ligand internalization, this cannot be accounted for on the basis of a single tyrosine residue within the conserved motif. Indeed, the fact that reintroduction of single tyrosines was not sufficient to fully reconstitute ligand binding and internalization indicates a requirement for multiple N-terminal tyrosine resides for full ACKR2 function.A). Two versions were generated: one was sulfated on tyrosine residues (ACKR2-N(s)), and the other was left unsulfated (ACKR2-N(un-s)). The sulfated variant was generated by tyrosine sulfation post-peptide synthesis, which yielded peptide species displaying variable tyrosine sulfation patterns . Initial assessment of the ability of the sulfated peptide to bind ligand involved the use of biotinylated chemokine ligands and streptavidin beads to measure the ability of ligand to interact with and therefore pull down the ACKR2 peptides. This assay involves the eventual detection by Western blotting of the peptide using anti-ACKR2 antibodies. As shown in Fig. 6C (panel i) and as quantified by densitometry in Fig. 6C (panel ii), although biotinylated CCL2 was able to pull down the sulfated ACKR2-N peptide (lane 1), this was not seen to the same extent with biotinylated CCL19 (lane 2), a homeostatic CC-chemokine that does not bind to ACKR2. In fact the amount of ACKR2-N pulled down by biotinylated CCL19 was no greater than that pulled down by nonspecific interaction of ACKR2 with the streptavidin beads (lane 3). These data therefore confirm the relative selectivity of the peptide for inflammatory CC-chemokines.To further examine the importance of tyrosine residues and sulfation status for the ability of the N terminus of ACKR2 to interact with ligand, we generated a 35-amino acid peptide (ACKR2-N) corresponding to the extreme N terminus of ACKR2 A. Two veD, the sulfated peptide, but importantly not the unsulfated peptide, was able to significantly impair binding and internalization of Alexa-CCL2 by ACKR2 transfected HEK cells. In addition, the sulfated peptide was able to significantly impair the binding of Alexa-CCL2 to CCR2 on THP1 cells . Notably, the ability of the peptide to inhibit ligand internalization by ACKR2 was unaffected by precomplexing with ligand . Importantly this precomplexing also did not enhance the neutralizing activity of the unsulfated peptide. Thus a sulfated peptide derived from the ACKR2 N terminus is capable of inhibiting ligand interactions with ACKR2 and its cognate signaling receptor, CCR2.Next we examined the ability of the peptide, in both sulfated and unsulfated forms, to interfere with the ability of the inflammatory CC-chemokine CCL2 to bind to either ACKR2 (in heterologous transfectants) or to its cognate signaling receptor CCR2 (in THP1 cells). As shown in Fig. 6A and quantified in Fig. 7B, HEK cells expressing ACKR2 produced a variety of full-length and truncated variants detected using this antibody. Importantly one of these variants of ∼38 kDa, equivalent to the size observed previously for truncated ACKR2, appeared in increasing amounts following exposure of the cells to staphopain A. Attempts to formally confirm the molecular nature of the truncated species using mass spectrometry were unsuccessful, possibly because of the previously reported difficulties in obtaining mass spectrometry data from ACKR2 . To confirm this in a separate cell line, we transfected CHO cells with ACKR2  and examined ligand binding and internalization following treatment with 0.5 and 2 μm staphopain A. In these experiments, ligand binding and uptake were assessed using a fluorescence-based assay adapted for 96-well plate format. As shown in Fig. 7D (panel ii), staphopain A treatment resulted in more marked inhibition of ligand uptake and internalization in this cell line than was seen with the transfected HEK cells. Specifically, at 2 μm, staphopain A was able to reduce ligand binding and uptake by ACKR2-expressing CHO cells by ∼50%. These data therefore suggest that ACKR2 is a natural substrate for staphopain A, which results in cleavage of the ACKR2 N terminus.We have previously reported that, in heterologous transfectants, ACKR2 is subject to N-terminal processing giving rise to a 38-kDa truncated receptor that has lost approximately the equivalent of the 35-amino acid peptide generated in this study . There hFig. 7om ACKR2 . In addim), and this probably relates to the extremely long half-life of the ACKR2 protein (m) resulted in more substantial and highly significant inhibition of ligand uptake and internalization by ACKR2-expressing HEK cells. Importantly the roles played by sulfated tyrosines are further confirmed by the use of peptides derived from the ACKR2 N terminus. Notably a sulfated variant of the peptide is able to bind ligand and thereby inhibit ligand binding to ACKR2 or the signaling receptor CCR2, whereas the unsulfated variant is not. The ability of a peptide from the ACKR2 N terminus to bind ligand suggests that this site is of general importance for chemokine ligand binding to receptors because similar peptides from other conventional chemokine receptors also display ligand binding properties (Although a large number of studies have described the importance of tyrosine sulfation for chemokine receptor ligand binding and function, there have been few studies  into the protein , 48. Notoperties , 49, 50.Staphylococcus aureus protease staphopain A to yield an N-terminally truncated variant of ∼38 kDa. In keeping with the importance of this region for ligand binding, staphopain A treatment of ACKR2-expressing cells also significantly reduced ligand internalization. This reduction was modest in ACKR2-expressing HEK cells but was considerably more marked in ACKR2-expressing CHO cells. These differences may relate to the relative recycling ability of ACKR2 from the extensive intracellular stores in HEK cells and CHO cells but may also be explained by differences in cell surface glycocalyx density and thus access for the enzyme to ACKR2. Despite this functional evidence of truncation, which supports the Western blot data, attempts at obtaining mass spectrometry confirmation of the molecular nature of the truncated ACKR2 product were unsuccessful. We have previously reported difficulties in mass spectrophotometric analysis of ACKR2 (We have previously reported that, in cells expressing ACKR2, the protein is detected in two processed forms. The first is the full-length protein, and the second is an N-terminal variant truncated by ∼35 amino acids. We have proposed that this may represent shedding, from the seven-transmembrane base of ACKR2, of a peptide capable of binding ACKR2 ligands and such a function of this peptide is supported by the current study. This peptide may therefore act in the vicinity of ACKR2-expressing cells as a motile blocker of inflammatory CC-chemokine function. Thus far we have not identified any mammalian proteases capable of generating such a peptide from intact ACKR2. However, previous reports of the ability of bacterially derived proteases to cleave the N terminus of other chemokine receptors, most notably CXCR2 , prompteof ACKR2  and propof ACKR2 , ACKR2 hin vivo testing.The ability of the ACKR2-derived peptide to inhibit the binding of CCL2 to its cognate receptor, CCR2, suggests that this may have some therapeutic benefit. This would be particularly important if the ACKR2-derived peptide was able to bind all ACKR2 ligands. Notably, despite it being over two decades since the initial cloning of chemokine receptors, there are no small molecule blockers of chemokine receptor function licensed for use in inflammatory pathologies. This inability to therapeutically utilize inflammatory chemokine receptor blockers most likely reflects the highly redundant nature of the inflammatory chemokine response , 53. In In conclusion, therefore, we provide for the first time evidence of the importance of N-terminal tyrosine residues for ACKR2 function. We highlight this area as being essential for ligand internalization and scavenging and demonstrate the ability of a peptide derived from this region to bind inflammatory CC-chemokines.