Preclinical models proved that adoptive transfer of Tregs or the use of compounds that can favor their function in vivo are effective for prevention and treatment of graft-vs.-host disease (GvHD). Following these findings, Treg-based therapies have been employed in clinical trials. Adoptive immunotherapy with Tregs effectively prevents GvHD induced by alloreactive T cells in the setting of one HLA haplotype mismatched hematopoietic transplantation. The absence of post transplant pharmacologic immunosuppression unleashes T-cell mediated graft-vs.-tumor (GvT) effect, which results in an unprecedented, almost complete control of leukemia relapse in this setting. In the present review, we will report preclinical studies and clinical trials that demonstrate Treg ability to promote donor engraftment, protect from GvHD and improve GvT effect. We will also discuss new strategies to further enhance in vivo efficacy of Treg-based therapies.Since their discovery CD4 They maintain tolerance to self and prevent autoimmune diseases, control excessive immune responses to allergens and pathogens, help maintain a balance with commensal microbial flora and the maternal tolerance to fetus. They have been shown to infiltrate tumors and suppress anti-tumor immunity , thus they can efficiently compete for IL-2 with Tcons and NK cells  with promising results. Allogeneic HCT is a life-saving treatment for high-risk hematologic malignancies . After a+CD25+ T-cell depletion from the bone marrow graft exacerbated GvHD. When additional CD4+CD25+ T cells, either freshly isolated or ex-vivo expanded, were infused, GvHD onset was delayed and even prevented to various degrees  hematopoietic transplantation, the high degree of HLA mismatch triggers strong host-vs.-graft and graft-vs.-host alloresponses. In the early 1990s, the combination of a myeloablative and immunosuppressive conditioning regimen with the infusion of a “mega-dose” of T-cell-depleted HSCs made haploidentical transplantation feasible and effective without the need for any post-transplant GvHD prophylaxis. The major limitation of this approach was delayed post-transplant immune recovery, which resulted in ~40% NRM, mainly due to infections –87. Moresphamide . Treg-mesphamide , 92. A rsphamide . Moreovesphamide , 95. Sucsphamide –102. Anol grafts .+CD25+ Tregs are isolated by a two-step immunomagnetic selection consisting of a negative CD19/CD8 selection, followed by a positive CD25 selection. The purity of CD4+FOXP3+ Tregs is around 70–80%. 2 × 106 Tregs/kg are infused 4 days before 1 × 106 Tcons/kg and about 10 × 106 CD34+ cells/Kg. No pharmacological GvHD prophylaxis is given. The first study reported results in 28 patients with hematologic malignancies (24 in any complete remission and 4 in relapse at transplant). They received a conditioning regimen with TBI, thiotepa, fludarabine, and cyclophosphamide. Twenty-six patients engrafted. Treg/Tcon adoptive immunotherapy was associated with rapid reconstitution of B cells and of T cells with a wide repertoire. Compared with standard T-cell depleted haploidentical transplantation, reconstitution of pathogen-specific CD4+ and CD8+ T cells, and of mature NK cells was faster. The incidence of CMV reactivation was markedly reduced, with no CMV-related deaths. Two patients developed ≥grade 2 acute GvHD and no patient developed chronic GvHD. One patient (in chemoresistant relapse at transplant) relapsed. Thirteen patients died of NRM, 4 of them because of extra-hematological toxicity , several protocols of expansion under good manufacturing practice have been developed and tested in clinical trials . Purity of CD4+FOXP3+ Tregs was over 90%. The first cohort of 5 patients received frozen Tregs but showed signs of GvHD, consistent with the reduced functionality of Tregs after cryopreservation and thawing . When complexed with IL-2, this antibody preferentially triggered the proliferation of Tregs , which strongly stimulate Treg proliferation while weakly affect CD4+ T cell proliferation  . MacDonavant CAR . A similvant CAR . Anothervant CAR .Finally, several compounds are under investigation for the prevention and treatment of GvHD, such as Janus-activated kinase inhibitors and others. They share the ability to preferentially inhibit Tcon function while sparing Treg suppressive activity to various degrees . Thus, Tin vivo (Treg-based therapies have provided promising clinical responses in allogeneic HCT. These treatments have proven to be safe and are not associated with the side effects that could have been anticipated, such as increased susceptibility to infections and leukemia relapse. Efforts are ongoing to improve the effectiveness of these approaches, whether they are based on adoptive transfer of freshly isolated, expanded or modified Tregs or on the induction of Treg expansion and function in vivo .To these days, treatment of patients with chemoresistant leukemia is still largely ineffective. In the present review, we discussed that Treg-adoptive transfer allows for a strong Tcon-mediated GvT effect while controlling GvHD. Although it requires further optimization, we believe this strategy is close to become an effective treatment for these patients.AM and SP wrote the manuscript. AV reviewed the manuscript. AP wrote and reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NSCLC lines showed a mean IC50 of 1.15 µM for fascaplysin. Analysis of signal transduction mediators point to an ATM-triggered signaling cascade provoked by drug-induced DNA damage. Fascaplysin reveals at least an additive cytotoxic effect with cisplatin, which is the mainstay of lung cancer chemotherapy. In conclusion, fascaplysin shows high activity against lung cancer cell lines and spheroids of SCLC CTCs which are linked to the dismal prognosis of this tumor type. Derivatives of fascaplysin may constitute valuable new agents for the treatment of lung cancer.Lung cancer is a leading cause of tumor-associated mortality. Fascaplysin, a bis-indole of a marine sponge, exhibit broad anticancer activity as specific CDK4 inhibitor among several other mechanisms, and is investigated as a drug to overcome chemoresistance after the failure of targeted agents or immunotherapy. The cytotoxic activity of fascaplysin was studied using lung cancer cell lines, primary Non-Small Cell Lung Cancer (NSCLC) and Small Cell Lung Cancer (SCLC) cells, as well as SCLC circulating tumor cell lines (CTCs). This compound exhibited high activity against SCLC cell lines (mean IC Among malignant diseases, lung cancer is the leading cause of mortality . NSCLC cFascaplysinopsis Bergquist sp. sponge which was isolated by Roll et al. in 1988 [50 of 0.35 μM), and accordingly, drug-treated cancer cell lines arrested preferentially in the G0/1 cell cycle phase [50 of >100 μM for CDK1, >50 μM for CDK2 as well as 20 μM for CDK5 [The marine drug fascaplysin  is a red bis-indole alkaloid of the  in 1988 ,11. Cyclle phase . Minor afor CDK5 . In addifor CDK5 . Non-plafor CDK5 .50 values 0.6–4 μM) [Cytotoxicity tests showed broad activity of fascaplysin towards a panel of 36 cancer cell lines (IC.6–4 μM) . Antican.6–4 μM) . Fascapl.6–4 μM) . In expe.6–4 μM) . Angioge.6–4 μM) .50 values measured in SCLC cell lines were found to be similar to the two chemoresistant NSCLC cell lines H1299 and A549 and the chemosensitive H23 cell line, respectively.SCLC responds to first-line chemotherapy with platinum-based drugs/etoposide, but relapses early with topotecan remaining as the single approved therapeutic agent . We haveIn the present work, the investigation of the cytotoxic effects of fascaplysin is extended to include single cell suspensions and spheroids of SCLC circulating tumor cells (CTCs) and several NSCLC cell lines. Our lab has established a panel of 6 CTC SCLC cell lines derived from the blood samples of distinct patients with extended disease SCLC . Further50 values of breast cancer and ovarian cell lines (range: 0.48–1.21 µM), SCLC cell lines (range: 0.2–1.48 µM) and NSCLC cell lines (range: 0.63–2.04 µM). Whereas SCLC and breast/ovarian cancer cell lines exhibited similar mean IC50 values (0.96 ± 0.5 versus 0.89 ± 0.45 µM), NSCLC cell lines proved to be less sensitive (1.15 ± 0.59 µM). SCLC26A and S457 are primary SCLC cell lines derived from pleural effusions of patients before and after therapy failure, respectively. The nonmalignant HEK293 cell line showed an IC50 value of 1.6 ± 0.42 µM. BH295 and IVIC-A are primary NSCLC cell lines derived from pleural effusions of patients with ALK and EGFR TKI resistance. Numrical values of the IC50 data are presented in The chemosensitivity of a range of cancer cell line to fascaplysin was measured in MTT cytotoxicity assays. 50 values of the other lines were equal or below 0.5 µM. A comparison of the ratios of IC50 values of single cell suspensions and tumorospheres for cisplatin and fascaplysin demonstrates that for fascaplysin, the differences in chemosensitivities between these 2D- and 3D-cultures are much less than for the platinum drug  and increased phosphorylation of STAT4, eNOS, c-Jun, and p27(T157) in the case of fascaplysin-pretreated NCI-H526, and numerous increases of phosphorylation in A549 cells, with the exception of decreases in p70 S6 kinase, STAT4, and p53(S392) . PhosphoThe signal transduction mediators related to fascaplysin-induced alterations in NCI-H526 and A549 cells are depicted schematically in Combination indices (CI) were calculated using the Chou-Talaly method, indicating synergy at values <1 . For NCI50 values for the combinations using the concentrations as indicated revealed synergistic interactions. The CI values ranged from 0.26–0.76 for PC-9 fascaplysin-cisplatin and from 0.1–0.94 for A549 fascaplysin-etoposide, respectively. Data are shown as mean ± SD, the initial concentrations were titrated in 9 two-fold dilution steps.For the NSCLC cell lines PC-9 and A549, combinations of fascaplysin and cisplatin or etoposide were tested in cytotoxicity tests. For both tests, ICLung cancer is the leading cause of cancer-related mortality in both men and women worldwide . TargeteDNA damage response is triggered when sensor proteins ATM (ataxia telangiectasia mutated) and ATR  detect structural distortions or breaks . After DIn conclusion, fascaplysin shows marked anticancer activity in NSCLC and SCLC cells independently of the function of the CDK4 pathway, thus pointing to direct effects on DNA and the transcription of various proteins. The mechanisms of the antitumor effect of fascaplysin demonstrated on several carcinoma models indicate that fascaplysin is close to some drug groups such as intercalating agents, inhibitors of serine-threonine, and tyrosine kinases. Additionally, fascaplysin increases phosphorylation of AKT/PKB and adenosine monophosphate-activated protein kinase (AMPK), which feature anti-apoptotic or pro-survival functions in cancer . In deta50 value in normal cells, as well as no apparent toxicities in murine xenograft models at therapeutic doses [Relapsed SCLC is resistant to a wide range of drugs and clinical trials have not led to improvements in survival rates over recent decades. Chemoresistance of SCLC seems to be related to the formation of large spheroids, termed tumorospheres, which limit drug access and contain quiescent and hypoxic tumor cells which are less sensitive to chemotherapeutics. Such 3D-structures were demonstrated to show increased resistance to cisplatin, etoposide, topotecan, and epirubicin when compared to the same SCLC CTC cells in form of single cell suspensions. In particular, fascaplysin is cytotoxic against SCLC CTC tumorospheres which exhibit high chemoresistance against a range of commonly-administered chemotherapeutics. Fascaplysin-induced cell death of outer SCLC CTC cell layers seems to trigger the elimination of the whole spheroid. Especially in SCLC cells, the induction of ROS by fascaplysin is expected to exert increased damage due to the small volume of the cytoplasmic fraction . It shouic doses .Unless otherwise noted, all chemicals were obtained from Sigma-Aldrich . Dulbecco’s phosphate buffered saline (PBS) was purchased from Gibco/Invitrogen . Compounds were prepared as stock solutions of 2 mg/mL in either DMSO or in 0.9% NaCl solution (cisplatin), sterilized by filtration in case of cisplatin, and aliquots stored at −20 °C.The A549 NSCLC A549 (Rb/p53 wild-type) and NCI-H526 SCLC A549 (Rb protein not expressed/p53 wild-type) cell lines were obtained from the American Type Culture Collection , as well as the other cell lines except primary lines and all SCLC CTCs established in our lab . Cells w7 cells/mL lysis buffer were solubilized under permanent shaking at 4 °C for 30 min, and aliquots of the lysates were stored frozen at −80 °C. After blocking, membranes with spotted catcher antibodies were incubated with diluted cell lysates at 4 °C overnight. Thereafter, cocktails of biotinylated detection antibodies were added at room temperature for 2 h. Phosphorylated proteins were revealed using streptavidin-HRP/chemiluminescence substrate  and detection with a Molecular Imager VersaDoc MP imaging system . Images were quantified using the ImageQuant TL v2005 software  and Microsoft Excel software . The different Western blot membranes were normalized using the 3 calibration spots included. Signaling pathways affected by fascaplysin in NCI-H526 and A549 lung cancer cells were produced using Power Point software .Relative protein phosphorylation levels of 38 selected proteins were obtained by analysis of 46 specific phosphorylation sites using the Proteome Profiler Human Phospho-Kinase Array Kit ARY003  in duplicate tests according to the manufacturer’s instructions. Briefly, cells were rinsed with PBS, 1 × 104 cells in 200 µL medium were treated for four days with twofold dilutions of fascaplysin or cisplatin, respectively in 96-well microtiter plates in quadruplicate . For SCLC CTC tumorospheres, an equivalent number of cells in form of spheroids were tested as described [Aliquots of 1 × 10escribed . The plat test for normally distributed samples . Values are shown as mean ± SD.Statistical analysis was performed using Student’s CDKs are a group of serine/threonine kinases which are critical in the regulation of the cell cycle. A major role of CDK-4 is the phosphorylation of Rb, which is inhibited by fascaplysin and a range of other compounds. Mutations in Rb, along with those of cyclin D and p16(INK4a), has been seen frequently during tumorigenesis of cancers. Investigation of a part of the kinome of NCI-H526 SCLC and A549 NSCLC cell lines reveals different responses to treatment with fascaplysin, most likely to be connected to the Rb phenotype. In NCI-H526 cells, fascaplysin sensitivity is determined by the absence of the CDK4–Rb pathway and DNA damage in combination with putative CDC25 inhibition, whereas in A549, inhibition of CDK4 seems to be the major effect with distinct and small effects on phosphoproteins. Fascaplysin exhibits marked anticancer activity against permanent and primary SCLC and NSCLC cells, with cytotoxic effects against SCLC CTC tumorospheres that are far superior to those of other therapeutics. Therefore, fascaplysin and derivatives with a better clinical profile may constitute valuable agents for lung cancer therapy.


Whether adding a second anti-neoplastic drug may augment response towards Gatipotuzumab, has not been elucidated so far. Since it is known that anti-MUC1 antibodies may alter estrogen receptor activity in breast cancer, this potential interplay was investigated in OC. The correlation between TA-MUC1, estrogen receptors (ERs) and another 12 protein markers as well as their correlation with clinico-pathological parameters in 138 ovarian cancer cases was studied. Finally, Gatipotuzumab and 4-Hydroxy-TTamoxifen (4-OHT) as well as the combination of both was tested for its impact on cell viability in COV318, OV-90, OVCAR-3, and SKOV-3 cells. A strong positive correlation between TA-MUC1 and ERs was detected in OC tissue. Those cases missing ERs but staining positive for TA-MUC1 had significantly reduced overall survival. The combination of 4-OHT and Gatipotuzumab significantly reduced cell viability and was more effective than treatment with Gatipotuzumab alone. Co-stimulation with Gatipotuzumab enhanced the efficacy of 4-OHT in OVCAR-3 and SKOV-3. The data suggest an interplay of TA-MUC1 and ERs in OC. Whether the combination of Gatipotuzumab and TTamoxifen may enhance efficacy of either of the two drugs  O-glycosylated threonine residue [MUC1 is an abundantly expressed transmembrane glycoprotein that undergoes several glyco-modifications during the process of malignant transformation, leading to the exposure of tumor-specific, novel carbohydrate epitopes ,3. Struc residue . Due to  residue ,6. This  residue .So far, two clinical trials have tested Gatipotuzumab for its efficacy in patients with advanced cancer. Fiedler et al. recently published a phase I dose-escalation trial where Gatipotuzumab was applied to patients with advanced cancer, who had failed to respond to standard therapy any longer . PatientTM platform. A “human-like” glycosylation of the now fully humanized antibody increases immunogenicity of the antibody plus TA-MUC1 complex and hence, triggers a stronger ADCC/ADCP response  and phagocytosis (ADCP) leading to cytolysis of the cancer cell . Meanwhiesponse  was significantly more common in serous OC (p = 0.004) but was not related to the remaining pathological parameters. Presence of TA-MUC1 and at the same time loss of GPER (TA-MUC1pos + GPERneg.) characterized a subgroup of patients that were significantly more often staged as FIGO III/IV (p = 0.004) as compared to the remaining cases. In addition, patients with this staining pattern were more often diagnosed with positive lymph nodes (p = 0.024) or high-grade cancer (p = 0.004) (Co-expression of TA-MUC1 and GPER was detected in 94 (68.1%) cases, while 23 (16.7%) and 15 (10.9%) samples expressed either solely TA-MUC1 or solely GPER, respectively. Six (4.3%) patients were found to be double negative. Co-expression of TA-MUC1 and GPER (TA-MUC1= 0.004) .p = 0.037) or high grade (p = 0.007)  cases was found to solely express ERα, while 79 (57.2%) only expressed TA-MUC1. Double positivity for ERα and TA-MUC1 was observed in 38 (27.5%) cases. Just 15 (10.9%) samples expressed neither ERα nor TA-MUC1. Clinicopathological characteristics as split up by the four-dimensional TA-MUC1—ERα scores were tested for statistical associations. Co-expression of TA-MUC1 and ERα was not statistically associated to the clinicopathological data tested. TA-MUC1 positivity without co-expression of ERα was more common in tumors of serous histology (= 0.007) .pos + GPERneg + ERαneg, i.e., expression of TA-MUC1 without co-expression of any of the two ERs, was detected in 18 cases and was associated with advanced FIGO stage (p = 0.004), cancer spread to retroperitoneal lymph nodes (p = 0.021) and poor histologic differentiation (p = 0.011) (The IHC staining pattern TA-MUC1= 0.011) .p = 0.022) and to the whole group of remaining cases (p = 0.02)  A. Within = 0.02) A, B. Fou = 0.02) C. Patienp = 0.006; 72 h: 0.58-fold, p = 0.006) but was less efficient in the remaining cell lines. 4-OHT, which is the active metabolite of Tamoxifen, has been demonstrated to act as a selective estrogen receptor modulator on ERα and as an activator of GPER. Viability of all cell lines was significantly reduced by 4-OHT at both timepoints. 4-OHT as a single agent proved to be most potent in COV318 at the 72 h timepoint yielding a reduction of cell viability down to 0.08 of control level (p < 0.001). Finally, co-stimulation using both Gatipotuzumab and 4-OHT was performed over a 48 h and 72 h period, respectively. Again, co-stimulation revealed a significant reduction of viability in all the four cell lines tested with the strongest effect (as compared to control) being present in COV318 at 72 h . Viability of cells that had undergone co-stimulation was compared to those that had been treated with a single substance only. The combination of 4-OHT and Gatipotuzumab significantly reduced viability of all cell lines as compared to samples that had been treated with Gatipotuzumab alone . In OVCAR-3 and SKOV-3 the overall net effect of 4-OHT and Gatipotuzumab co-stimulation was numerically slightly higher than the sum of individual effects of any of the two substances alone . However, this difference was not significant.OVCAR-3, SKOV-3, OV-90, and COV318 cells were employed to evaluate the effect of Gatipotuzumab, 4-Hydroxy-Tamoxifen (4-OHT), and the combination of the two on cell viability . TA-MUC1ab alone A–D. ExceCell lines were co-stained for TA-MUC1 and GPER. Both proteins were found to be co-expressed in the large majority of cells. TA-MUC1 showed a uniform cytoplasmic staining while GPER was found to be distributed in a web like cytoplasmic pattern. Both TA-MUC1 and GPER showed partial membrane staining, especially in OVCAR-3 and SKOV-3 . RegardiThis study found the Gatipotuzumab epitope TA-MUC1 to be closely correlated to ER expression in OC tissue. Furthermore, when OC cell lines were exposed to Gatipotuzumab and Tamoxifen, the combination of both drugs significantly decreased cell viability in all the four cell lines tested and was superior as compared to Gatipotuzumab alone. Further, co-stimulation with Gatipotuzumab enhanced the efficacy of 4-OHT in OVCAR-3 and SKOV-3. Both GPER and ERα were closely correlated to membrane expression of TA-MUC1 in the patient cohort investigated in the current study. Although a statistical correlation by far does not prove functional interaction, it may suggest a potential crosstalk of TA-MUC1 and ERs. As GPER and TA-MUC1 are both localized on the cell membrane and in the cytoplasm, we performed double immunofluorescence and found co-localization of the two. Though a potential physical interaction would require this finding to be backed up by e.g., immunoprecipitation or a proximity ligation assay, we herein provide initial evidence of a potential interplay of TA-MUC1 and GPER. Since both drugs have already been administered to OC patients in clinical trials, a potential functional interaction of the two might be of clinical interest. Hence, further research should be done thus to clear whether a functional or even physical interaction of TA-MUC1 and GPER may exist. There are several lines of evidence supporting this hypothesis. With respect to signaling pathways, the C-term of MUC-1 was found to detach from the cell membrane and to translocate to the nucleus. Nuclear MUC-1-C binds ERα, thus attenuating Tamoxifen induced changes on ERα dependent transcription . As a sein vivo model of the disease or in humans since, e.g., immune related anti-tumor effects—an important pillar of Gatipotuzumab’s mode of action—are fully excluded in a 2D cell culture setup. Anyhow, as the aim of the current study was to elucidate potential interplay of TA-MUC1 and ERs, the cell culture system was judged to be the most straight forward and most adequate model to pursue this question.Tamoxifen is known to activate GPER and to selectively modulate ERα . The curin vivo models. Furthermore, TA-MUC1, GPER, or ERα negative OC cell lines, knock down models and analysis of downstream pathways need to be employed thus to investigate potential interplay of the three proteins from a mechanistic point of view. Nevertheless, correlation and co-localization of ERs and TA-MUC1 suggests that the effects observed in SKOV-3 and OVCAR-3 may be caused by direct or indirect crosstalk of TA-MUC1 and ERs.Interestingly, the anti-proliferative activity of Gatipotuzumab could be enhanced by co-stimulation with Tamoxifen. Regarding the OVCAR-3 and SKOV-3 cell lines, the anti-proliferative action of both drugs in combination was greater than the sum of individual effects of any of the two drugs alone—though this was not statistically significant. Since such effects were only observed in two out of four cell lines, they need to be interpreted with care. Whether Tamoxifen indeed facilitates action of Gatipotuzumab (and vice versa) remains to be proven by independent experiments and pos + ERαneg + GPERneg) was positively correlated to advanced FIGO stage, high grade and cancer spread to retroperitoneal lymph nodes  tissue was retrieved from the histopatholgical archive of the Department of Gynaecology and Obstetrics, Ludwig-Maximilians-University, Munich, Germany. Tissue had been collected from 138 patients who had undergone surgery for OC from 1993–2002 at Department of Gynaecology and Obstetrics, Ludwig-Maximilians-University, Munich, Germany. Clinicopathological data were gathered from patients’ charts, pathology reports or from the Munich Cancer Registry and are displayed in The tissue samples used for the current analysis were retrieved from the archive of the Department of Gynaecology and Obstetrics, Ludwig-Maximilians-University, Munich, Germany. Tissue had been collected for routine diagnostics at the time the patients had been treated at our institution (1990–2002). When this retrospective study was initiated all diagnostic procedures had already been fully completed, the tissue samples were classified as left-over material and underwent irreversible anonymization. Under these circumstances no individual written informed consent was needed as per declaration of the Ethics Committee of the Ludwig-Maximilians-University. The study was approved by the Ethics Committee of the Ludwig-Maximilians-University  and 18-392 (approved on 25 June 2018)) and was performed according to the standards set in the declaration of Helsinki 1975. All researchers were blinded from patient data during experimental analysis.neg + GPERpos), for TA-MUC1 only (TA-MUC1pos + GPERneg), for both biomarkers (TA-MUC1pos + GPERpos) or for neither of the two (TA-MUC1neg + GPERneg). The same was done in case of ERα, respectively. Finally, the feature TA-MUC1pos + GPERneg + ERαneg was studied.Immunohistochemical data of TA-MUC1 and GPER/ERα staining regarding this panel were retrieved from the laboratory archive as previously published ,31,36. Al-glutamine. Cell culture media was supplemented with 10% v/v fetal calf serum  and contained no antibiotics and antimycotics. Cell lines were routinely checked for absence of mycoplasma contamination by performing a commercially available PCR based mycoplasma test kit . Cells were cultured in a humidified atmosphere at 37 °C and 5% CO2.COV318, OV-90, OVCAR-3, and SKOV-3 were purchased form the American Tissue Culture Collection (ATCC) and from the European Collection of Cell Cultures (ECACC). All cell lines were grown in DMEM containing stable Cells were seeded on glass slides in quadriperm dishes. After a 36 h incubation period, attached cells were fixed in acetone solution for 5 min. Acetone was discarded and slides were washed three times in PBS. Cells were then blocked with Ultra V-Block for 15 min. Primary antibodies were applied at the following concentrations: rabbit anti GPER  1:400, Gatipotuzumab  1:400. Following an incubation period of 16 h at 4 °C, slides were washed using PBS and incubated with secondary antibodies . Secondary antibodies were diluted 1:500 and 1:250, respectively and incubated for 30 min in the dark. Following washes streptavidin-Cy2 solution was applied and incubated for 30 min in the dark. Finally, slides were washed three times, air-dried and mounted with VectaShield mounting medium containing DAPI . Immunofluorescence was visualized by using a Zeiss fluorescence microscope and processed with the Zeiss AxioVison Rel. 4.8 .SKOV-3 and OVCAR-3 were seeded in 96 well plates at a concentration of 3000 cells per well. After cells had attached culture media was changed to media containing either carrier solution, Gatipotuzumab , 4-OHT  or the combination of Gatipotuzumab  and 4-OHT . Cells were incubated for 48 h and 72 h, respectively. Following the stimulation period, 10 µL of WST solution  were added to each well. Plates were incubated for another 30 min before the plate was read on a microplate reader at 580 nm. Experiments were performed three times in different passages of cells achieving similar results.t-test. p values lower than 0.05 were considered significant.Data were tested for statistical significance by employing the IBM statistic package SPSS (version 25) and by Microsoft Excel. SPSS was used to plot Kaplan–Meier graphs and box plots. Survival times were compared using log-rank statistics. Cell culture data were tested for statistical significance by using Student’s


Unlike sHCC74 and SAR-HCV, both sHCC29 and sHCC63 had no detectable surface HLA class I antigen expression, alongside undetected intracellular β2-microglobulin (β2m) and marked HLA class I heavy chain and selective antigen-processing machinery (APM) component downregulation. The loss of β2m in sHCC29 and sHCC63 was caused by a >49 kb deletion across the B2M locus, while their downregulation of APM components was transcriptional, reversible by IFN-γ only in several components. β2m was also undetected in the primary HCC lesions of the patients involved, indicating its in vivo relevance. We report for the first time HLA class I antigen loss with underlying B2M gene deficiency and APM defects in 50% (2 of 4) of the sHCC cell lines tested. These findings may have implications for a proper design of T cell immunotherapy for the treatment of sHCC patients.Limited information is currently available concerning HLA class I antigen abnormalities in sarcomatoid hepatocellular carcinoma (sHCC). Here, we have analyzed the growth characteristics and HLA class I antigen status of four sHCC cell lines ; the first three were newly established in this study. Among the four, sHCC29 showed the highest growth rate  Hepatocellular carcinoma (HCC) is the 6th most common solid tumor and the third leading cause of cancer-related death worldwide , 2. AlthIt has been known for some time that malignant cells can have impaired HLA class I antigen expression during the course of tumor progression –15. HLA 2mβ gene, associated with downregulation of several components in the HLA class I antigen presentation pathway.The HLA class I antigen status in tumor cells may represent a key variable for the efficacy of therapy that relies on CTLs to eliminate the tumor cells . PrimaryThe clinical histories of patients from whom the three newly established sHCC cell lines originated are as follows.A 58-year-old woman with history of liver cancer originating from chronic hepatitis B was admitted to Taipei Tzu Chi Hospital, Taiwan, in August 2010, with chief complaints of yellowish discoloration of the skin and tea-color urine for one week. She has received transcatheter arterial chemoembolisation (TACE) three months prior to admission to this hospital. Laboratory tests showed elevated serum total bilirubin (5.1 mg/dL), alpha-fetoprotein (182.7 ng/mL), and carbohydrate antigen 19-9 (70.5 U/mL) levels. The carcinoembryonic antigen (CEA) serum level was within the upper limit of normal. Computed tomography revealed a hypervascular mass in the liver hilum measuring 4 × 5 cm. The patient underwent left lobectomy, and pathology showed proliferation of spindle-shaped hepatocellular carcinoma cells. The patient has survived for 5 additional years without tumor recurrence as of this writing.A 39-year-old man with a history of HBV-related cirrhosis was referred to Hualien Tzu Chi General Hospital, Taiwan, in May 2011, diagnosed as having liver cancer without any previous treatment for HCC. The alpha-fetoprotein serum level was 123.5 ng/mL. Both the carbohydrate antigen 19-9 and CEA serum levels were within the normal range. The combination of computed tomography with hepatic arteriography and arterial portography (CTHA/CTAP) showed a huge hypervascular tumor in the right lobe of the liver. The patient underwent liver resection. Histological examination revealed spindle-shaped sarcomatoid carcinoma cells with unclear trabecular and pseudoglandular structures. However, the tumor relapsed in the residual liver 5 months after surgery, and despite TACE therapy, the patient died one year later.A 72-year-old woman was admitted to Hualien Tzu Chi General Hospital, Taiwan, and diagnosed as having liver cancer originating from chronic hepatitis C. The alpha-fetoprotein, carbohydrate antigen 19-9, and CEA serum levels were all within the upper limit of normal. Computed tomography showed a hypervascular tumor in the caudate lobe of the liver measuring 5 × 6 cm. She underwent segmentectomy, and pathology revealed spindle-shaped sarcomatoid carcinoma cells. One year after surgery, the tumors relapsed in the residual liver. The patient began with TACE therapy. Unfortunately, she died two years later.in vitro. Cultured cell lines between 18 and 24 passages were used in the subsequent studies.Three sHCC cell lines were established from HCC tissues during surgery performed at Hualien Tzu Chi Hospital with patients' consent. The study has been approved by the Research Ethics Committee in Tzu Chi General Hospital (IRB 101–62). The three sHCC cell lines  were established according to standard procedures. Essentially, the minced HCC tissues were pretreated with collagenase, washed, and cultivated on the mitomycin C-treated NIH/3T3 feeder layer for 4 to 6 passages to select the HCC cell lines. Homogenous HCC cell populations were obtained, and sustained proliferation was observed over 30 passages in culture The fourth sHCC cell line known as SAR-HCV cell line was previously established from a malignant liver lesion of a 68-year-old male patient infected with HCV. Its initial characterization has been reported previously , 24.n = 5/group). The experimental protocol was approved by the Animal Ethics Committee, Chang Gung University, Taoyuan, Taiwan. Single cell suspensions of the three cell lines were prepared from monolayer cultures by light trypsinization and washing in phosphate-buffered saline (PBS). Cells were then counted, and 107 cells/0.1 mL PBS were inoculated into the flank of each mouse. The animals were examined every 2 days to monitor the growth of tumors. The volume of palpable tumor nodules was calculated according to the formula volume(mm3) = 0.4 × a × b2, where (a) is the major diameter, and (b) is the minor diameter perpendicular to the major one.Six-week-old female Non-Obese Diabetic (genetic background)/Severe Combined Immunodeficiency (SCID) mice were purchased from BioLASCO, Taiwan Co. Ltd., Taipei, Taiwan. Tumorigenicity assays of the three cell lines  were carried out in such mice in this study .Recombinant human IFN-β2m-associated HLA-A, HLA-B, HLA-C, HLA-E, and HLA-G heavy chains β2m-peptpatients  cell-baspatients ; all of


FN1) expression was a predictive biomarker in some patients with chemotherapy-resistant RCC, which especially correlated with two starvation-resistant RCC cell lines. These results indicate these cell lines emulate chemotherapy-resistant RCC and might be useful in the search for markers to predict poor prognosis and in the development of therapeutic agents and their index markers for chemotherapy-resistant RCCs.Starvation-resistant renal cell carcinoma (RCC) cell lines are considered dormant-state cells that survive even under glucose starvation. The cellular biological and global transcriptional analysis using these cells identified potential markers of chemotherapy-resistant RCC and therapeutic agent candidates. Recently, we showed that ARL4C was a predictive biomarker for poor prognosis in patients with chemotherapy-resistant RCC by the global transcriptional analysis of patient primary tissues. The objective of this study was to identify the characteristics of chemotherapy-resistant RCC by the global transcriptional analysis of primary tissues of patients with RCC and RCC cell lines. The connective global transcriptional analysis showed that two starvation-resistant RCC cell lines, SW839 and KMRC-1, were strongly correlated to tissues of patients with chemotherapy-resistant RCC and showed high expressions of invasive- and proliferation-related genes. We found fibronectin ( Renal cell carcinoma (RCC) is the most common renal malignancy and its incidence is currently increasing . More thTNFSF10/TRAIL) [ARL4C, one of these seven genes, was demonstrated to be a useful predictor of a poor prognosis in patients with chemotherapy-resistant RCC by global transcriptomic analyses. Cases with high ARL4C expression were associated with a significantly shorter survival periods than in the cases with low ARL4C expression , and Cox univariate and multivariate analyses showed that high ARL4C expression accurately predicted poor survival in this cohort . Moreover, these finding were independently confirmed by clinicopathological analyses of another clinical cohort [ARL4C expression are resistant to treatment with tyrosine kinase and mTOR inhibitors. Therefore, novel therapeutic agents targeting other molecules will be required for the treatment of RCC patients.Previously, we demonstrated the presence of two types of cells in RCC involved in carbon metabolism and cell signaling under glucose starvation, which is the major nutrient denied to cells following the inhibition of angiogenesis . These f0/TRAIL)  were pot0/TRAIL) , etomoxi0/TRAIL) , and che0/TRAIL) , may be l cohort . HoweverIn this study, we identified the characteristics of chemotherapy-resistant RCCs by the global transcriptional analysis of primary tissues from patients with RCC and RCC cell lines. We showed that starvation-resistant cells might be useful for the development of predictive biomarkers for a poor prognosis, the development of therapeutic agents, and index markers in patients with chemotherapy-resistant RCC.2 atmosphere.Three starvation-resistant RCC cell lines  and four starvation-sensitive RCC cell lines  were used in this study. These cell lines were purchased from either the American Type Culture Collection, Riken Cell Bank, Cell Resource Center for Biomedical Research in Tohoku University  or the Japanese Collection of Research Bioresources (Japan). All cell lines were maintained in RPMI 1640 , containing 25 mM glucose, supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C in a humidified 5% COTotal RNA was extracted from seven RCC cell lines using the Trizol Plus RNA Purification kit . Total RNA was quantified using a Bioanalyzer  according to the manufacturer’s instructions. The RNA Integrity Numbers of all prepared total RNA samples were over 8.0.A library of template molecules for high-throughput DNA sequencing was converted from total RNA using the TruSeq RNA Sample Prep Kitv2  according to the manufacturer’s protocol. The library was quantified using a Bioanalyzer (Agilent) following the manufacturer’s instructions. The library (4 pM) was subjected to cluster generation on a Single Read Flow Cell v4 (TruSeq SR Cluster Kit v2-cBot-GA) with a cBot generation instrument (Illumina). Sequencing was performed on a Genome Analyzer GAIIx for 58 cycles using Cycle Sequencing v5-GA reagents (Illumina).https://www.r-project.org/) using genes.count_table following Cuffnorm were used to identify the correlation rates and the principal component analysis of 50 samples containing 43 primary tissues of patients with RCC and seven RCC cell lines, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and gene ontology (GO) analysis were performed using DAVID Bioinformatics Resources 6.8 [Image analysis and base calling were performed using Real Time Analysis version 1.13 (Illumina). The sequence libraries for each sample were processed using CASAVA Software 1.8.2 (Illumina) to produce 51-bp sequence data in fastq format. The fastq files were processed using Cutadapt version 1.2.1  with optrces 6.8 . The datBox and whisper plots were generated using a function of boxplot in free software R.ROC curve analysis was performed using the ROCR package in free software R and the maximum accuracy was calculated.t-tests with a pooled standard deviation (SD) was used to compare the means of multiple groups. A p value < 0.05 denotes statistical significance.Data were reported as the mean ± standard error (SE). The values were derived from at least three experiments. Statistical analyses were performed using R. One-way factorial analysis of variance (ANOVA) accompanied by pair-wise comparisons using Global transcriptional analysis was performed using seven RCC cell lines and 43 primary tissues of patients with RCC using previous data deposited in the DNA Data Bank of Japan (DDBJ) Japanese Genotype-phenotype Archive for genetic and phenotypic human data under accession number JGAS00000000149. This data contained 43 primary tissues and showed the following as previously reported [FN1  and SW839 and KMRC-1, relative to Group B containing the other 45 samples. We analyzed genes that were differentially expressed between Group A and B using Cuffdiff and identified 164 genes that were significantly up-regulated in Group A compared with Group B . These 1 Group A . These rFN1 was the highest principal component loading gene  had significantly higher FN1 expression compared with samples in Group q1 surviving ≥ 5 years without metastases  of 3760.31. This cut-off FPKM value for poor prognosis  separated Subgroup q3A from the other groups. Therefore, the maximum accuracy of predicting patient mortality in 16 patients with metastasis was low (0.625). However, FN1 predicted the development of metastasis in 43 patients with a high maximum accuracy of 0.814 based on the lower cut-off FPKM value of 750.53. These results showed that FN1 might be a useful predictive biomarker for poor prognosis and the development of metastasis in patients with RCC. However, FN1 might be a specific index marker of Subgroup q3A, which formed part of the poor prognosis patient group with chemotherapy-resistant RCC.ing gene  and conting gene . Therefotastases . In addiroup q3B . Moreoveroup q3B  showed tThis study showed that starvation-resistant RCC cell lines, SW839 and KMRC-1, were strongly correlated and emulated primary tissues from poor prognosis patients with chemotherapy-resistant RCC, and indicated their characteristics of chemotherapy-resistant RCC, which was useful for the search of markers to predict poor prognosis and the development of therapeutic agents and their index markers for chemotherapy-resistant RCCs.The global transcriptional analysis of 43 primary tissues from patients with RCC and seven RCC cell lines showed that starvation-resistant RCC cell lines, SW839 and KMRC-1, were strongly correlated to primary tissues of poor prognosis patients with chemotherapy-resistant RCC, especially three of nine primary tissues from RCC patients. Their cell lines and tissues formed a group (Group A) that had a common malignant phenotype of maintaining invasive and proliferating activities compared with other cell lines and tissues as determined by their gene expression pattern. We previously reported that starvation-sensitive RCC cell lines showed G2/M-phase arrest under glucose deprivation leading to cell death, but that starvation-resistant RCC cell lines containing SW839 and KMRC-1 survived under glucose deprivation without the induction of G2/M transition-arrest . This reFN1, an ECM-related gene, compared with other cell lines and tissues. Therefore, this study indicates that FN1 expression might be a useful marker to predict a poor prognosis as previously reported [FN1 expression was a predictive biomarker of a group of poor prognosis patients with chemotherapy-resistant RCC, termed Subgroup q3A. Makers for specific subgroup of patients will be useful in combination with markers used to cover total patients. For patients with RCC, FN1 expression will be useful in combination with a predictive biomarker of poor prognosis such as ARL4C, which predicts patient mortality caused by RCC within 2 years after the initial diagnosis of metastasis with a high accuracy of 97.7% [SW839, KMRC-1, and primary tissues from poor prognosis patients with chemotherapy-resistant RCC up-regulated expression of reported . Howeverof 97.7% . SW839 aStarvation-resistant RCC cell lines, including VMRC-RCW as well as SW839 and KMRC-1, were previously reported to undergo cell death induced by buformin (a biguanide) , 11, etoCD274 (PDL1), a target of checkpoint inhibitors, was an up-regulated gene in Group A compared with Group B  and SW839 and KMRC-1. bThe other 45 samples.(DOCX)Click here for additional data file.


PQR620 was largely cytostatic, but the combination with the BCL2 inhibitor venetoclax led to cytotoxicity. Both the single agent and the combination data were validated in xenograft models. The data support further evaluation of PQR620 as a single agent or in combination with venetoclax.The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling cascade is an important therapeutic target for lymphomas. Rapamycin-derivates as allosteric mTOR complex 1 (TORC1) inhibitors have shown moderate preclinical and clinical anti-lymphoma activity. Here, we assessed the anti-tumor activity of PQR620, a novel brain penetrant dual TORC1/2 inhibitor, in 56 lymphoma cell lines. We observed anti-tumor activity across 56 lymphoma models with a median IC The high frequency of genomic alterations in lymphomas affecting genes coding for proteins involved in the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway ,2 highlin = 56) derived from lymphomas. The compound showed potent anti-proliferative activity in most of the tested cell lines . The most sensitive subtype was mantle cell lymphoma (MCL)  (p = 0.0095)  vs. 136 nM ; p = 0.0007). The anti-tumor activity of PQR620 appeared mostly cytostatic. Apoptosis induction was only seen in 8/56 cell lines  without association with histotype, BCL2, MYC or TP53 status  . Convers 0.0095) . When we3 status . PQR620 3 status .3 tumors and were carried out for 21 days. PQR620 determined a decrease of the tumor volumes in comparison with controls from day 12 (p < 0.05)  DLBCL RI-1 cell line. Treatments with PQR620 ) started with 100–150 mm < 0.05) . There wDue to the low induction of apoptosis after PQR620 as a single agent, we assessed the combination of the dual TORC1/2 inhibitor and the BCL2 inhibitor venetoclax in four DLBCL cell lines. The combination was in vitro synergistic in terms of anti-proliferative effect, as shown by exposing the cell lines to increasing doses of PQR620 and venetoclax as single agents or in combination . The addp < 0.001), resulting in an eradication of the implanted tumors in the absence of toxicity. The treated/control ratio (T/C) was <10% and, according to the U.S. National Cancer Institute (NCI) rules, the drug combination is declared as very active [Based on the in vitro data, the activity of PQR620  in combination with venetoclax  was evaluated in an in vivo model using the germinal center B-cell type (GCB)-DLBCL cell line SU-DHL-6, bearing the t chromosomal translocation . PQR620 y active .50 value of 250 nM after 72 h of exposure, lower than what is reported in 66 cell lines derived from various solid tumors [PQR620 is a novel dual TORC1/2 inhibitor with higher affinity for the enzymatic catalytic domain than previously reported second-generation compounds, such as INK128, CC223 and AZD2014 . Here, wd tumors .PQR620 was largely cytostatic, as is the case for other mTOR inhibitors in lymphomas ,25,26. HFor novel compounds, it is useful to identify groups of patients that might benefit the most from the treatment. Genomics studies have identified DLBCL subgroups with a constitutively activated PI3K/mTOR pathway: GCB- and ABC-DLBCL, belonging to the B-cell receptor signaling cluster , with GCThe anti-tumor activity of PQR620 was particularly high in the MCL cell lines. This observation is interesting, since MCL is the histotype in which first generation mTOR inhibitors have been mostly clinically developed ,8,37,38 TP53. The contribution of TP53 in the response to mTOR inhibitors is not defined, but it is certainly relevant in particular conditions [TP53 mutations and deletions, do not represent the best target population for PQR620.The second group with decreased PQR620 sensitivity was represented by mutated DLBCL cell lines bearing an inactive nditions ,42,43. Dnditions , indicatnditions , enrichePQR620 was designed starting from the dual PI3K/mTOR inhibitor bimiralisib (PQR309) ,45, incrBCL2, MYC and TP53 was defined as previously reported [X2 test or Fisher’s exact test (two-tailed), as appropriate. Binomial exact 95% confidence intervals (95% C.I.s) were calculated for median percentages. Differences in IC50 values among lymphoma subtypes were calculated using the Wilcoxon rank-sum test. Statistical significance was defined by p values of 0.05 or less. The synergism of drug combination was calculated by using the Chou–Talalay Index, as previously done [Established human cell lines were cultured as previously described  and theireported . PQR620 reported . Apoptosreported  and it wreported ,50. The sly done . StatistPrkdcscid/NCrHsd) mice and analysed as previously described [Xenograft experiments with the RI-1 and the SU-DHL-6 cell lines were performed using NOD-Scid (NOD.CB17-escribed . Mice maescribed . In vivoescribed .In conclusion, PQR620 showed in vitro and in vivo anti-tumor activity in lymphoma models as a single agent and in combination with venetoclax, supporting its further development.


To examine different features in tumorigenicity of various hiPSCs, 10 commonly available hiPSC lines were subjected to in vivo tumorigenicity testing. Transplanted hiPSC lines showed remarkable variation in tumor incidence, formation latency, and volumes. Most of the tumors formed were classified as immature teratomas. However, no signs of malignancies, such as carcinoma and sarcoma, were recognized in the tumors. Characteristics associated tumorigenicity of hiPSCs were investigated with microarray analysis, karyotype analysis, and whole exome sequencing. Gene expression profiling and pathway analysis supported different features of hiPSC lines in tumorigenicity. hiPSC lines showed chromosomal abnormalities in some lines and 61–77 variants of cancer-related genes carrying effective nonsynonymous mutations, which were confirmed in the COSMIC databases. In this study, the chromosomal abnormalities and cancer-related gene mutations observed in hiPSC lines did not lead to the malignancy of tumors derived from hiPSCs. Our results suggest that the potential tumorigenicity risk of hCTPs containing residual undifferentiated hiPSCs is dependent on not only amounts of undifferentiated hiPSCs but also features of the cell lines used as raw materials, a finding that should be considered from the perspective of quality of hCTPs used.Human induced pluripotent stem cells (hiPSCs) represent promising raw materials of human cell-based therapeutic products (hCTPs). As undifferentiated hiPSCs exhibit intrinsic tumorigenicity properties that enable them to form teratomas, hCTPs containing residual undifferentiated hiPSCs may cause tumor formation following transplantation. We first established quantitative and sensitive tumorigenicity testing of hiPSCs dissociated into single cells using NOD/Shi-scid IL2Rγ Practically, several clinical trials and studies using hPSC-derived hCTPs have already started to treat spinal cord injuries, age-related macular degeneration, heart failure, and type I diabetes around the world )2. The engraftment was determined according to progressive nodule growth at the injection site. Mice were euthanized and necropsied when tumors grew greater than 10% of their body weight or when a sign of deconditioning was noted according to the categories of Scientists Center for Animal Welfare (SCAW) . Low power view (× 1.25) of teratoma represents two or three germ layer components . Higher power view (× 10) shows mesodermal cartilage and endodermal intestinal tract-like duct (B), ectodermal glial tissues, melanocytes and choroid-like tissues (D), ectodermal glial tissues (F), mesodermal smooth muscle and endodermal intestinal tract-like duct (H), ectodermal choroid-like tissues and immature neuroepithelia (J), ectodermal choroid-like tissues and melanocytes (L), ectodermal stratified squamous epithelia and endodermal duct structures (N), ectodermal glial and neural cells and mesodermal blood vessels (P), endodermal duct structures accompanied with intestinal and respiratory epithelium-like cells (R), and mesodermal smooth muscle and endodermal intestinal tract structures (T).NOG mice were subcutaneously injected with 3 × 10(PDF)Click here for additional data file.S2 FigA set of 16,454 probes on GeneChip Human Genome U133 Plus 2.0 Array was statistically identified with significantly different expression levels among 10 hiPSC lines . Hierarchical clustering analysis was performed using R version 3.5.1 software.(TIFF)Click here for additional data file.S3 FigTranscript expression of OCT3/4 (A), NANOG (B), SOX2 (C), and LIN28A(D) in 10 hiPSC lines is shown with microarray data. Data are represented as mean ± SD (n = 3).(TIFF)Click here for additional data file.


These plants are widely used in the Moroccan traditional folk medicine. The cytotoxic effect was explored against two cancer cell lines, Vero and RD, using the colorimetric MTT assay. The obtained results showed that the cytotoxicity differed according to the used extract with an efficient effect of Cotula cinerea extracts compared to Salvia verbenaca. A potent cytotoxicity was thus observed for the Cotula cinerea hexane extract which inhibited the growth of RD cell line at the lowest IC50 value (57.21±3.43 µg/mL). This was followed by the ethyl acetate extract and the essential oil with moderate effects against RD cell line and showed IC50 values of 187.52±6.27 µg/mL and 173.05±4.46 µg/mL, respectively. On the other hand, different results were obtained and Cotula cinerea essential oil was the most cytotoxic with the lowest IC50 value (72.72±2.18 µg/mL) against Vero cell line. In the same conditions, higher concentrations were needed in the case of Salvia verbenaca extracts. The results of this study showed thus that Cotula cinerea essential oil and hexane extract showed significant cytotoxic effects against RD and Vero cell lines, respectively, and could be considered as novel source of antitumor agents. This study is expected to be beneficial for clinical and traditional applications for Cotula cinerea as a remedy against cancer and opens new perspectives for further investigations on other types of cancer cell lines.The objective of this work was to investigate the cytotoxicological effect of the extracts (hexane, ethyl acetate, and Cancer is a complex disease caused by several factors such as genetic deregulation, environmental influencing agents, and epigenetic modification factors , 2. AccoTraditional forms of medicine, especially the herbal products deployed for centuries in many parts of the world, could be good alternative for scientific investigation due to their attributes against tumor cell lines. Indeed, plants are rich sources of new compounds from which some secondary metabolites have been shown to possess anticancer properties –7. Furth Cotula cinerea and Salvia verbenaca from Morocco. Cotula cinerea which is of the Asteraceae family is a xerophytic plant widely distributed in sandy and desert grounds [ Salvia verbenaca is a Mediterranean plant currently belonging to the Lamiaceae family. The plant is widely distributed in Morocco and is widely used in Moroccan traditional folk medicine as a cholagogue, antiseptic, diuretic, and astringent [ Cotula cinerea and Salvia verbenaca. This work was therefore initiated in order to evaluate the in vitro antiproliferative effect of Cotula cinerea and Salvia verbenaca extracts and essential oil against tumor RD and Vero cell lines.In this work we were interested in exploring the potential cytotoxicological effect of grounds . Popular grounds . Salvia tringent . However Cotula cinerea were collected in March 2014 in the south of Morocco from the region of Dakhla-Oued Ed-Dahab (province of Aousserd). Those of Salvia verbenaca were collected on March 2015 at the locality called Ain Aouda, in the Rabat region, Morocco. The plants were identified by Professor Fennane from the Scientific Institute, Mohammed V University in Rabat. Voucher specimens of both plants were deposited in the herbarium of the Botany Department of this institution. The collected aerial parts were left under shade at room temperature until complete drying.The aerial parts of Cotula cinerea and Salvia verbenaca were crushed and 200 g of each plant material was macerated and stirred for 24 hours at room temperature in an ethanol/water  mixture with frequent stirring. The maceration process was repeated twice during 24 h each in order to achieve an exhaustive extraction. After filtration and evaporation of ethanol, the obtained aqueous solution was successively fractionated with hexane, ethyl acetate, and n-butanol. Then, solvents of the different obtained extracts were evaporated and the dried residues were used to explore their cytotoxic effect. The hexane, ethyl acetate, and n-butanol extraction yields were, respectively, 1.00, 3.00, and 4.50% for Cotula cinerea and 1.58, 4.64, and 5.28 for Salvia verbenaca.The dried aerial parts of Cotula cinerea aerial parts was extracted by hydrodistillation using a Clevenger-type apparatus. The extraction was achieved during six hours and the obtained oil (yield 0.92%) was stored in a glass vial until use.The essential oil of2 and 100% relative humidity atmosphere.The two tumor tested cell lines used in this study were RD  and Vero (Monkey kidney cancerous cell lines: ATCC N°CCL-81). These two species were obtained from the National Institute of Health, Rabat, Morocco, and were cultivated as previously described by Aneb et al., 2016 . BrieflyμL medium MEM containing 4.106 cells/mL were placed in each well containing MEM and incubated at 37°C in 5% CO2/humidified air for 24 h. After 24 h incubation, cells were then treated with the essential oil of Cotula cinerea or its extracts  in addition to those of Salvia verbenaca. Briefly, 100 μL of each extract at concentrations ranging from 31.25 to 500 µg/mL was dissolved in DMSO (1%) and was added to each well in triplicate. The microplates were then incubated for 48 h at 37°C in air condition of 5% CO2. Sterile PBS and 1% DMSO (vehicle) were used as negative controls.Before evaluating the antiproliferative activity, the cellular density of each species was determined using light microscopy. When cellular density reached a subconfluence threshold, cells were attached by trypsin-EDTA, washed twice with phosphate buffered saline (PBS), and centrifuged at 150 rpm for 15 min (4°C) and were then suspended in fresh culture medium (MEM). The enumeration of cells was made using Malassez cell count. Before treatment with extracts, 100 µL of MTT (5 mg/mL) was added to each microwell and incubated for 3 hours at 37°C in 5% CO2. Tetrazolium salts were cleaved to formazan and the reaction was stopped by addition of 100 µL of 50% (v/v) isopropanol-10% (w/v) sodium dodecyl sulfate (SDS) mixture to each well in order to dissolve insoluble formazan formed after tetrazolium dye reduction [50) were obtained by modeling the percentage of cytotoxicity versus concentration of extracts. Experimental data were achieved in triplicate and the obtained results were expressed as means ± SD.The evaluation of cell viability has been achieved through the use of MTT 3- assay. The test is based on the biotransformation of MTT into formazan crystals by mitochondrial dehydrogenases of living cells. The absorbance of the formazan will then be determined by spectrophotometry at 550 nm. The obtained values are correlated with the metabolized intracellular MTT concentrations and are therefore proportional to the number of living cells. Briefly, 20 Cytotoxicity (%) = 100 × (absorbance of untreated cells − absorbance of treated cells)/absorbance of untreated cells. Cotula cinerea and Salvia verbenaca. The three extracts  of each plant in addition to the essential oil of Cotula cinerea have then been assayed for their cytotoxic activity against RD and Vero cell lines using MTT assay.In an ongoing program aimed at the exploration of the phytochemical compounds and the biological activities of medicinal plants of Morocco, we were interested, in this work, in the cytotoxic effect ofThe obtained findings for the two cell lines RD and Vero are summarized in Figures  Cotula cinerea compared to Salvia verbenaca. Comparison of the obtained results for both cell lines showed that Cotula cinerea extract activity was higher than its corresponding Salvia verbenaca one. This was observed for both cell lines.For both lines, an increase of the cytotoxicity with the concentration of the essential oil or the used extracts was observed indicating a dose dependent effect. An examination of the obtained results showed that the cytotoxic effects of the investigated plants extracts were different according to the tested cell line with an efficient activity of Cotula cinerea hexane extract showed the higher cytotoxic effect followed by the ethyl acetate, the essential oil, and the n-butanol extract. In the same conditions and for the Vero cell line, the best cytotoxic effect was observed with the essential oil followed by the hexane, the ethyl acetate, and the n-butanol extracts.In the case of the RD cell line, the50 in µg/mL) and the obtained results are gathered in 50 value (57.21±3.43 µg/mL) was observed with the Cotula cinerea hexane extract indicating an important antiproliferative effect. The essential oil and the ethyl acetate extract showed moderate cytotoxic effects . Higher IC50 values (greater than 500 µg/mL) were observed in the case of Cotula cinerea n-butanol and most of the Salvia verbenaca extracts which presented then the weakest cytotoxicity activity.The cytotoxic activity was also expressed in terms of concentration required to inhibit 50% of the tested tumor cell lines  followed by the hexane, the ethyl acetate, and the n-butanol extracts . Higher concentrations were needed in the case of Salvia verbenaca extracts which showed the weakest cytotoxic effects.In the case of the Vero tumor cell line, The noticed differences in the observed cytotoxicity of the investigated mixtures could obviously be due to the phytochemical composition of each extract. The exact mechanism through which the explored extracts act on each tumor cell line is obviously difficult to highlight. Indeed, the explored extracts are complex mixtures containing several phytochemical compounds which could have different biological activities. In addition, these mixtures could act in a synergic or in an antagonism manner. Cotula cinerea and Salvia verbenaca extracts showed the presence of polyphenols such as flavonoids and other secondary metabolites. These compounds could explain the cytotoxic effect of this extract on RD tumor cell line. Indeed, the antitumor properties of phenolic compounds and flavonoids against several cell lines representing numerous human cell lines have been previously reported [A preliminary phytochemical screening of thereported . The exareported . Cotula cinerea essential oil against the Vero tumor cell line could be due to presence of cytotoxic molecules in the oil. Essential oils are complex molecules containing various and several terpenoids compounds belonging mainly to monoterpene and sesquiterpene chemical groups and presenting different functional groups such as alcohols, phenols, ketone, etc. [The antiproliferative activity ofne, etc. . These cne, etc. . Moreove Cotula cinerea essential oil is very complex involving several targeting pathways as previously reported [ Cotula cinerea essential oil can act through various mechanisms. Among these are the activation of the apoptosis via the caspases' family proteins expressions, the modifications in signaling pathways, the interruption of the life cell cycle, the modification in DNA methylation, and histones acetylation of tumor cell lines [ Cotula cinerea in cytotoxic activity.The action and mechanism ofreported . Due to ll lines , 15. The Cotula cinerea essential oil and hexane extract showed significant and encouraging cytotoxic effects against RD and Vero cell lines, respectively, and could then be considered as source of novel antitumor agents. The obtained results raise then the possibility of a potential use of Cotula cinerea as a source of potent anticancer products. This study is expected thus to be beneficial for clinical and traditional applications for Cotula cinerea as a remedy against cancer and opens new perspectives for further exploration on other types of cancer cell lines. Further investigations regarding the identification of bio-actives molecules responsible for these effects are needed. Examination of the cytotoxic activity of the Cotula cinerea individual compounds would give crucial information regarding the exact mechanism of this plant in antitumor activity.Our results indicated that


Burkitt lymphoma (BL) is a highly aggressive B-cell lymphoma associated with MYC translocation. Here, we describe drug response profiling of 42 blood cancer cell lines including 17 BL to 32 drugs targeting key cancer pathways and provide a systematic study of drug combinations in BL cell lines. Based on drug response, we identified cell line specific sensitivities, i.e. to venetoclax driven by BCL2 overexpression and partitioned subsets of BL driven by response to kinase inhibitors. In the combination screen, including BET, BTK and PI3K inhibitors, we identified synergistic combinations of PI3K and BTK inhibition with drugs targeting Akt, mTOR, BET and doxorubicin. A detailed comparison of PI3K and BTKi combinations identified subtle differences, in line with convergent pathway activity. Most synergistic combinations were identified for the BET inhibitor OTX015, which showed synergistic effects for 41% of combinations including inhibitors of PI3K/AKT/mTOR signalling. The strongest synergy was observed for the combination of the CDK 2/7/9 inhibitor SNS032 and OTX015. Our data provide a landscape of drug combination effects in BL and suggest that targeting CDK and BET could provide a novel vulnerability of BL. Gene mutations in BL target essential cancer pathways including e.g. p533, the SWI/SNF complex4 and the transcription factor TCF3 (E2A) or its negative regulator ID3. Pro-survival signals are elicited through phosphatidylinositol-3-OH kinase pathway (PI3K) activation by TCF3/ID3 mutations and tonic B-cell receptor signalling6.Burkitt’s lymphoma (BL) is a highly aggressive non-Hodgkin lymphoma (NHL), which is driven by the characteristic translocation of the MYC oncogene8. Current treatment of BL consists in intensive chemotherapy including combinations of cyclophosphamide, doxorubicin, methotrexate, vincristine and prednisone or combinations of methotrexate, cytarabine, etoposide, ifosfamide and dexamethasone7. Chemotherapy of BL has been successfully combined with the CD20 antibody rituximab.BL can be managed very effectively using intensive chemoimmunotherapy, especially in younger patients7, which underscores the necessity for alternative treatments. These are unlikely to emerge from further chemotherapy optimization. Relapsed or refractory BL has a dismal prognosis and is generally considered incurable. Therefore, platforms to generate rational novel combinations for BL could have immediate clinical consequences and may allow a functional dissection of genotype specific sensitivities. Cell lines provide a robust model for drug response studies and can be used to develop new treatment strategies including combinations. Recent comprehensive large-scale studies provided detailed analysis of tumour specific determinants of drug response based on molecular characterization of cell lines and their pharmacological profiles12. Pharmacological profiling studies identified synergistic drug combinations with ibrutinib in activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL)14 or NF-κB-targeted strategies in mantle cell lymphoma (MCL)15. While previous studies include a large number of cell lines, individual entities were underrepresented, i.e. the number of BL cell lines ranges from as few as 3 up to 11 in the mentioned platforms.However, the elderly and patients with immunodeficiency have an inferior outcomein vitro studies revealed synergistic drug interactions i.e. between PI3K inhibitor and chemotherapy16 as well as mTOR and histone deacetylase inhibitors17. However, currently there are no synergistic combinations of targeted drugs in clinical use, hence arises the necessity for preclinical models to provide rational drug combinations.Previous 18. While activation of MYC in mouse B cells was insufficient for lymphomagenesis, a cooperating mechanism of PI3K activation in BL was identified19.Recent studies provide evidence for dependency of BL on tonic B-cell receptor (BCR) signalling to PI3K21. BRD4 plays an important role in transcription of many genes including MYC22, which is overexpressed in BL, and hence BET inhibition potently suppresses the expression of MYC in leukaemia and lymphoma cell lines leading to induction of cell cycle arrest and apoptosis21.BET family, including BRD2, BRD3, BRD4 and BRDT, influences gene expression by recruiting transcriptional regulators to specific genomic locationsHere, we describe a detailed study of drug response and combination treatments across a panel of haematological malignancy derived cell lines focusing on BL. We identify a subgroup of BL lines responsive to PI3K and BCR pathway inhibition and delineate numerous cooperative interactions of PI3K/AKT/mTOR pathway and BET inhibition. Strong synergy between BET and cyclin dependent kinase (CDK) inhibition by SNS-032 provides a rational for clinical testing of this combination.24.To identify molecular dependencies and potential therapeutic targets in BL, we measured the effect of 32 drugs in 10 concentrations on the viability of 42 blood cancer cell lines, including 17 BL cell lines, and 6 isogenic BL lines with targeted deletion of p53, SYK, BTK, BLK or CD20 Fig. . In lineBecause of the potential bias introduced by response surrogates, we considered IC50, AUC and individual doses Figs , S1. We r = 0.93), venetoclax (median r = 0.81) or thapsigargin .To understand the relation of effects produced by different agents, we analysed drug-drug correlations across concentrations. This matrix uncovered a number of highly correlated drugs driven by both multiple concentrations of individual compounds as well as drugs targeting overlapping pathways or nodes of the same pathway. For example, strong positive correlations were shown for multiple concentrations of everolimus (median correlation coefficient 25. Dasatinib and saracatinib (Src family), the CHK inhibitor AZD7762, and the DNA-PK inhibitor NU7441, which has also been shown to inhibit PI3K and mTOR26 also showed similar response across cell lines.Idelalisib (PI3K), everolimus (mTOR) and MK-2206 (AKT) effects were highly correlated as the drugs target members of the PI3K pathway. In line with the known role of SYK, PI3K and BTK in BCR signalling, ibrutinib (BTK) and PRT062607 (SYK) Fig.  and idelIn summary, the analysis of drug-drug associations allowed us to provide evidence for the coherence of the screening platform as well as to provide insight into the pathways involved in viability effects of drugs.We clustered cell lines based on the effect on viability for all drugs and concentrations Fig.  and idenThe second response group (Cluster II) showed the strongest response to kinase inhibitors including PI3K inhibitor, as well as a number of other targeted drugs including SRC inhibitors . The dependence of those cell lines on BCR and SYK was validated by knocking out BTK, SYK and CD79A/B using CRISPR/Cas9. The effect of gene knock-out followed the pattern of drug sensitivity and showed that the cell line with highest IC50 values for BTK and SYK inhibition (Z138) was also the least affected by the knockout of CD79A, CD79B and SYK compared to BJAB and BL-60 Fig. . These r29, a subgroup of BCL-2 dependent lymphoma lines (OCI-LY-1 and OCI-LY-8)32, a BL cell line (DOGKIT) known to harbour BCL2 rearrangements33 and the B-NHL line Granta-519, which has been shown to overexpress BCL234  clustered with its parental line  analysis35. We classified combinations as synergistic for CI values below 0.85. Values between 0.85 and 1.15 indicate additive effects, and CI above 1.15 points was assigned antagonistic. The use of the CI analysis has its limitations, e.g. in the context of very toxic combinations or if the drug itself had a very small IC50 value, based on the logarithmic transformation used in the model36. Therefore, we also assessed drug interactions using dose-response curves of relative viabilities . Six of 32 idelalisib combinations were synergistic, while for none of the ibrutinib combination CI were below 0.85 Fig. . The AKT11. Bcl-2 inhibtiors, obatoclax and venetoclax, combined with ibrutinib showed median CI values off 1.12 and 0.96 respectively, which was explained by differential sensitivity to the drugs , chemotherapeutics, the AKT inhibitor (MK-2206), a downstream node of PI3K pathway, and the SYK inhibitor PRT062607 with corresponding median CIs of 0.76 (JQ1), 0.80 (doxorubicin), 0.70 (MK-2206), 0.82 (PRT062607). This suggests a positive influence of combining more than one inhibitor of the PI3K pathway, as well as combining inhibitors of BCR proximal kinase SYK and PI3K. These data also provide a rationale for combinations of idelalisib with doxorubicin, which is used in BL treatment regimens.18.Although we observed few differences between the response patterns to ibrutinib and idelalisib Fig. , we founWe observed multiple strong synergistic effects for the BET inhibitor OTX015. The lowest CI scores were found for the CDK2/7/9 inhibitor SNS-032, inhibitors of the PI3K/AKT/mTOR and BCR pathway inhibitors. Median CI scores across 18 cell lines ranged from 0.56 (SNS-032) to 0.81 (doxorubicin). Combinations of OTX015 with thapsigargin and YM155 yielded CIs of 1.24 and 1.46, suggesting that these combinations were antagonistic. We analysed the individual dose-response curves and found a subset of cell lines, which showed cooperative effects for this combination , while in 7 out of 18 lines the interaction between OTX015 and YM155 was antagonistic. Cell lines with lower OTX015 sensitivity showed higher CI values Fig. , suggest5, and OTX015 within a subgroup of BL cell lines. Specifically, the PI3K inhibitors idelalisib and IPI-145 (duvelisib), the allosteric AKT inhibitor MK-2206 and the mTOR inhibitors everolimus and deforolimus  and OTX015 showed a CI50 of 0.46 and 0.35 in BJAB and BL-60 respectively, pointing towards strong synergy. The concave gradient of viability heatmap of the 10 × 10 combination matrix also indicated these cooperative interactions  in parallel to uncovering new targets within a Burkitt lymphoma set (BTK and SYK in a subset of BL).The platform uncovered known vulnerabilities  showed strong combination activity with the highest number of compounds targeting different pathways in nearly all tested models. Synergistic combinations with idelalisib and ibrutinib were found in a subset of cell lines for a smaller set of drugs. Current clinical trials of OTX015 in non-leukaemia haematological malignancies have shown relatively good tolerance of the drug, with low-grade adverse events of anaemia, neutropenia, diarrhoea, fatigue and nausea; high-grade thrombocytopenia was also observed19. Also, it has been suggested that BET inhibition sensitizes PI3K and MYC driven cancer cells to PI3K inhibitor by blocking PI3K pathway reactivation caused by induction and activation of receptor tyrosine kinases (RTKs) through blocking the expression of RTKs45. Synergistic interactions between the PI3K pathway inhibitor and BETi have been previously described in vitro in other entities including haematological malignancies MCL46 and ABC-DLBCL14 as well as in solid malignancies including glioblastoma47, osteosarcoma48 and breast cancer45, which underscores the necessity of evaluation of this combination in clinical trials. Interestingly, dual-activity PI3K-BRD4 inhibitors have been recently developed and are tested in clinical trials for hepatocellular carcinoma49.A high number of cooperative drug interactions with OTX015 were found with multiple drugs inhibiting the PI3K pathway, including the PI3K inhibitors idelalisib and IPI-154, the AKT inhibitor MK-2206 and mTOR inhibitors everolimus and deforolimus. Those synergies are in line with the oncogenic signalling activated by tonic BCR signalling50, which consists, among others, of CDK9 and cyclin T, and plays a crucial role in activation of transcription. Another possibility is that a Brd4-independent mechanism may recruit or retain P-TEFb and lead to elevated MYC transcription22, which in turn can be down-regulated by CDK9 inhibition by dinaciblib in BL cell lines51. However, it has to be noted that dinaciclib is not as target-specific as SNS-032. SNS-032 targets not only CDK9, leading to inhibition of transcription, but also CDK7 and CDK2, whose inhibition can block cell cycle progression53.The current study uncovers a novel synergistic combination in BL, a specific CDK2,7,9 inhibitor SNS-032 with BET inhibitor OTX015. This exact mechanism of this combination activity is currently unclear. The targets of both drugs, cyclin-dependent kinase 9 (CDK9) and Brd4, act as positive regulators of p-TEFbin vitro pharmacological profiling in cell lines is an excellent tool of preclinical evaluation of drug combinations. The dataset provided can function as a training set to predict synergistic drug interactions on a larger scale54 and help plan future evaluation of drug combinations. The possibility of in silico modelling of predicting drug resistance, drug-target interaction and potential synergistic drug combinations on a large scale based on current drug target and pharmacological profiling databases57 has been extensively discussed as a relevant alternative drug discovery. Information from such models could help develope efficient in vitro experimental setups in the context of developing rational combination therapy.This study shows that Cell lines were obtained from G. M. Lenoir , A. Rickinson  or the DSMZ . p53-deficient BL line Séraphine was generated and provided by J. Hüllein . MCL lines  were kindly provided by University of Frankfurt and University of Würzburg. KS. Lee  generated isogenic BL lines. Cell lines for the screen were chosen with a focus on BL, other entities were introduced for comparison.2 with humidification. Cells with a passage number of 3–6 were used for drug screening. Cell lines were authenticated externally at Multiplexion GmbH  by Single Nucleotide Polymorphism (SNP) profiling and by comparison of obtained signatures to reference signatures58.Cells were cultured in RPMI 1640 medium supplemented with 10–20% FBS, 1% Glutamine and 1% Penicillin/Streptomycin at 37 °C, at 5% COFor the single agent drug screen we built a library of 32 drugs, 10 concentrations each using threefold dilution to reach end concentrations from 30 µM to 1.5 nM. The range of concentrations allows for a wide range of viability effects, which is needed to generate IC50 and AUC. For the combination screen we built a library of 32 drugs, 10 concentrations each using threefold dilution to reach concentration ranges 30 µM–1.5 nM, 15 µM–0.76 nM or 10 µM–0.5 nM depending on drug potency. The choice of library drugs aimed at covering a wide range of key oncogenic pathways, especially those active in haematological malignancies. For easier translation of the results into the clinical setting only drugs that are currently in clinical use or in clinical trials were used.We used a parallel approach probing three combination components: ibrutinib at 200 nM, idelalisib at 200 nM and OTX015 at 200 nM with the drug library. Single agent control was generated by combining drug library with solvent solution.59. The remaining drugs were obtained from Sigma-Aldrich , Enzo Life Sciences  and Selleck Chemicals . Compounds were dissolved in DMSO at 0.1–50 mM and stored at −20 °C.SD07 was provided by Prof. A. Mokhir, Erlangen, Germany3–7 × 103 cells per well depending on the growth rate and exposed to the drug library (10 µl) or the drug library combined with 200 nM ibrutinib, idelalisib or OTX015 (10 µl each) for 48 h; the final volume per well was 50 µl. We included DMSO and PBS controls to exclude DMSO toxicity effects. Cells were then incubated with 14 µl CTG reagent. Luminescence was measured after 20 minutes using a TECAN Microplate Reader (Tecan Group) with an integration time of 200 ms. Relative viability was determined by normalizing luminescence units for each well to DMSO control.Cell viability was determined using CellTiterGlo (CTG) ATP assay (Promega). Cells were seeded onto 384-well plates (Griner Bio One) at 1.5 × 102) between the datasets was 0.77  from 0.76 to 0.96 was shown  of two drugs; we determined optimal starting concentrations based on dose-response curves to those drugs: 1 µM for SNS-032, 10 nM for everolimus, 10 µM for MK-2206 and 10 µM for OTX015. Luminescence data from the CTG viability assay was normalized to DMSO controls and then bound between 0% and 100% by a second normalization.61. All sgRNA target sequences were extracted from Human Brunello CRISPR knockout pooled library  except sgRNA target sequences for mCherry and Luciferase (see below). Lentivirus packaging protocol was adapted from elsewhere61. In brief, it was performed in HEK293T/17 cell line  using 2ndsgRNA was cloned into pLKO5.sgRNA.EFS.GFP plasmid vector  followed by cloning protocol from elsewhereCells were infected with lentivirus particles in 6 well cell culture plate and were maintained at appropriate cell culture condition. Cells were analyzed using BD LSRII flow cytometry  to determine the percentage of GFP positive cells. Data obtained from FB LSRII was analyzed using FlowJo v.10 software .Normalisation of the data and basic data analysis were performed with Microsoft Excel. Luminescence data was normalized to a mean of DMSO controls to obtain relative viability in per cent, which in turn was used for further analyses.IC50 curves and bee swarm plot were generated using GraphPadPrism 5 software . For the IC50 curves a non-linear regression with viability data bound between 0 and 100% was used. This model:P values (two-tailed) were calculated using an unpaired parametric t-test.https://www.r-project.org/).Subsequent data analysis was performed in R statistical environment . Correlation matrices were calculated using cor function and plotted using corrplot function  from the ‘corrplot’ package.35. IC50 values for this analysis stem from the drm calculation Combination indices for 10 × 10 matrices and isobologram analyses were performed using CompuSyn software .Combination index (CI) was calculated using the formula:Supplementary informationDataset 1: Supplementary figuresDataset 2: Supplementary tables


Prediction of response to specific cancer treatments is complicated by significant heterogeneity between tumors in terms of mutational profiles, gene expression, and clinical measures. Here we focus on the response of Estrogen Receptor (ER)+ post-menopausal breast cancer tumors to aromatase inhibitors (AI). We use a network smoothing algorithm to learn novel features that integrate several types of high throughput data and new cell line experiments. These features greatly improve the ability to predict response to AI when compared to prior methods. For a subset of the patients, for which we obtained more detailed clinical information, we can further predict response to a specific AI drug. Breast cancer is the second most common type of cancer in women, with an incidence rate of over 250,000 cases per year, and breast cancer cases show significant heterogeneity in clinical and omic measures. Estrogen receptor positive (ER+) tumors typically grow in response to estrogen, and in post menopausal women, estrogen is only produced in peripheral tissues via the aromatase enzyme. Inhibition of aromatase is often an effective treatment for ER+ tumors, but aromatase inhibitor therapy is not effective for all tumors, and causes of this heterogeneity in response are largely not known. In this work, we present a feature construction and classification method to predict response to aromatase inhibitor therapy. We use network smoothing techniques to combine tumor omic data into predictive features, which we use as input to standard machine learning algorithms. We train predictive models using clinical data, including high-quality clinical data from UPMC patients, and show that our method outperforms previous approaches in predicting response to aromatase inhibitor therapy. A number of recent large efforts have focused on collecting genomic data from tumors. While these datasets led to several successful studies and insights, in many cases the clinical data available for patients enrolled in these studies is incomplete. This makes it hard to use such datasets for predicting tumor specific outcomes and tailoring treatments to individuals.To develop accurate methods for for predicting treatment responses we need both, a comprehensive genomic dataset profiling the individuals being studied and accurate complimentary clinical information. To date, methods that used the former (detailed genomic data) usually were unable to use the latter for a significant number of individuals while methods that only relied on clinical information are limited in their ability to distinguish between tumor responses .de novo pathway identification via identification of mutually exclusive mutations , we compute a function F(v) ∀v ∈ V that is both smooth over the network and accounts for the prior knowledge about each node.As has been shown in the past, protein interaction networks provide a useful way to overcome data sparsity and noise when predicting cancer responses . Here weet al.  to combiW′, computed via Laplacian normalization of the edge weight matrix W: we first construct a diagonal matrix Δ with Δ = ∑jW, and compute W′ = Δ−1/2WΔ−1/2. Given a prior knowledge vector Y, we then compute the smoothed vector F using the iterative procedure described by Zhou et al.  quantifies gene j’s proximity to both somatic mutations in tumor i and the gene set S represented by the smoothed vector T. We compute DP,T similarly, replacing MP with DP in Given one of the smoothed “target” vectors described above, denoted as i:We use these matrices to compute features for response to treatment in tumor e.g. targets of a specific drug).Row-wise mean, representing the total network proximity between a tumor’s somatic mutations  and genes in a predefined set , and the aromatase inhibitor Arimidex. Each of these two drugs were tested on a single cell line, and we create LINCS features for each tumor by combining that tumor’s continuous log-fold differential expression with the expression change induced by that drug in the appropriate cell line. We compute two features for each  pair:Correlation between that tumor’s differential expression and the cell line differential expression induced by administering that drug.Dot product between that tumor’s differential expression and the cell line differential expression induced by administering that drug.magnitude of change in expression between a tumor and the cell line in question.While the two features above are conceptually similar, we note that in addition to the direction of agreement, the dot product also represents the We use the following categorical variables from the general TCGA clinical data:Tumor pathological stageNode pathological stageMetastasis pathological stageOverall pathological stageHistological typeICD-10 typeICD-O-3 histologyHER2 immunohistochemistry level resultPost-surgery margin statusWe expand each categorical variable listed above into 0/1 indicator columns for use in classification methods. We additionally extract the estrogen receptor status of each tumor, used for selecting additional patients based on prior clinical knowledge.With the above features, we perform cross-validation experiments to assess our ability to predict response to aromatase inhibitor treatment. We examine all patients who were given any of the aforementioned drugs: 279 patients were given anastrozole, 51 were given exemestane, and 80 were given letrozole. An additional 180 samples were not considered for aromatase inhibitor therapy due to having ER– tumors, which are known not to respond to AI therapy. In this general aromatase inhibitor response prediction task, we assign a patient a “non-response” label if they were removed from any such drug for clinical reasons, or if the patient died during drug treatment. We also include prior clinical knowledge in this “all aromatase inhibitor” analysis; we integrate this prior knowledge by also computing the above features for the 180 patients who were not given an aromatase inhibitor, but who had estrogen receptor negative (ER–) tumors. These tumors are known not to respond to this type of treatment, so we assign these samples “non-response” labels. We use the features discussed above to learn various types of classifiers including logistic regression (with both L1 and L2 regularization), Random Forest and Probabilistic SVMs. For each of these methods and each setting we perform leave-one-out cross-validation.We performed cell line experiments to compare breast cancer cell line growth with and without the addition of estrogen. We initially grow cell cultures for 5 days, with estrogen present, simulating the initial growth of breast cancer in a patient. We then separately grow cultures with a continued supply of serum estrogen, or with replacement cell medium that lacks estrogen—the environment without estrogen simulates the introduction of an aromatase inhibitor. We measure cell line growth with and without serum estrogen after the initial growth period, and from these cell counts we compute measures of how much each cell line responded to the presence of estrogen. We performed this experiment with 12 replicates of each cell line, 6 with and 6 without estrogen after the initial growth period, and used a mixture of ER+ and ER– cell lines , using a VICTOR X4 plate reader . Bars represent the mean of six biological replicates ± SD. 17β-Estradiol (E2) was obtained from Sigma-Aldrich .MCF-7, BT474, BT483, CAMA1, Uacc812, ZR75-1 ZR75-30 and T47D breast cancer cell lines were purchased from American Type Culture Collection [ATCC], Manassas, VA, USA. SUM44PE was purchased from Asterand Bioscience, Detroit, MI, USA, and 600MPE cells were a gift by Dr. Rachel Schiff. For the estrogen removal experiments, the cells were kept for 5 days in IMEM supplemented with 10% charcoal stripped serum (CSS) with 1nM E2, and then plated into 96-well plates with or without 1 nM estradiol. An exception are Sum44PE cells that were kept in IMEM with 2% CSS. After 5 days, cell numbers were measured using Cell-titer Glo  according to the manufacturer’s instructions. Luminescence was measured with GloMaxp = γpc+(1 − γ)pp, with pc denoting predictions from cell lines and pp denoting predictions from tumors. The validation set predictions then combines the tumor and cell line predictions via the hyper-parameter γ tuned by cross-validation .We separated a random 10% of patients to use for training weights between tumor and cell line classifiers, and used the remaining 90% of patients for cross-validation analysis. In each cross-validation fold, we fit classifiers to the corresponding training set of patients, and then used those classifiers to produce non-response predictions of the 10% of patients initially set aside. We then computed predictions for those 10% of patients using classifiers trained on cell lines, and chose the optimal convex combination of tumor and cell line predictions in the training set, producing final prediction S1 TextDescriptive statistics of the UPMC patient cohort, details of comparisons with other methods, and results of additional analyses.(PDF)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.