Hepatitis C virus (HCV) eradication by direct-acting antiviral agents (DAAs) reduces de novo hepatocellular carcinoma incidence in cirrhosis; however, contrasting evidence on higher incidence of hepatocellular carcinoma (HCC) was reported in patients previously treated for HCC. Here, we showed that sofosbuvir and daclatasvir can modulate cell proliferation, invasion capability and gene expression in HCC-derived cell lines, suggesting that off-target effects of these drugs might be responsible for both the increase and reduction of cell proliferation and migration capability. Off-target gene modulation, mainly affecting mitochondrial functions, ribosomal genes and histones, was consistent with matched phenotypic changes and might account either for pro-oncogenic or tumor-suppressive functions of DAAs, that seemed to be dictated by the molecular background.Background and Aims: HCV eradication by direct-acting antiviral agents (DAAs) reduces de novo hepatocellular carcinoma (HCC) incidence in cirrhosis; however, contrasting evidence about beneficial or detrimental effects still exists in patients who have already developed HCC. Methods: we investigated whether sofosbuvir and daclatasvir modulate cell proliferation, invasion capability and gene expression (RNA-seq) in HCC-derived cell lines, hypothesizing possible off-target effects of these drugs. Results observed in HCC cell lines were validated in non-HCC cancer-derived cell lines and a preliminary series of human HCC tissues by qPCR and IHC. Results: DAAs can affect HCC cell proliferation and migration capability by either increasing or reducing them, showing transcriptomic changes consistent with some unexpected drug-associated effects. Off-target gene modulation, mainly affecting ribosomal genes, mitochondrial functions and histones, points to epigenetics and proliferation as relevant events, consistent with matched phenotypic changes. A preliminary validation of in vitro findings was performed in a restricted cohort of HCC patients previously treated with DAAs, with immunohistochemical correlations suggesting DAA-treated HCCs to be more aggressive in terms of migration and epidermal-to-mesenchymal transition. Conclusions: Our findings suggested the possible occurrence of off-target effects ultimately modulating cell proliferation and/or migration and potentially justified previous findings showing some instances of particularly aggressive HCC recurrence as well as reduced incidence of recurrence of HCC following treatment with DAAs. In western countries as well as in Japan, a large proportion of hepatocellular carcinomas (HCCs) are associated with hepatitis C virus (HCV) infection in the cirrhotic stage. It was hardly possible to cure this infection in the cirrhotic phase until the advent of direct-acting antiviral agents (DAAs). The target mechanism of action is inhibition of viral replication by blocking non-structural viral proteins. Therefore, they are classified as NS3-4A inhibitors, NS5A inhibitors and nucleoside or non-nucleoside inhibitors of NS5B, according to the viral protein they block. A significant reduction in first HCC incidence results from HCV eradication by DAAs . HoweverOne hypothesis is that DAAs do not induce neoplasia directly; rather, HCV clearance might reduce the immune response, favoring the development or progression of neoplastic clones, especially in patients with pre-existing dysplastic/neoplastic foci . FaillacOff-target effects of DAAs were previously described. Bachovchin et al.  used higSofosbuvir and daclatasvir have been the most used DAAs in Italian cirrhotic patients. Daclatasvir inhibits NS5A. In clinical practice, it is usually combined with sofosbuvir, which is a nucleoside polymerase inhibitor of the RNA polymerase NS5B. After phosphorylation to nucleoside triphosphate, it competes with nucleotide substrates that are incorporated into the viral RNA chain, resulting in chain termination.In order to screen for molecular mechanisms linking DAAs to tumor behavior, we tested whether possible off-target effects might occur in HCC-derived cell lines, by evaluating sofosbuvir and daclatasvir modulation of cell proliferation, invasion capability and gene expression. The results observed in HCC cell lines were validated in non-HCC cancer-derived cell lines and a restricted series of primary human HCCs. HepG2, Hep3B, SNU449 and SNU475 cell lines were obtained from the American Type Culture Collection  and were maintained according to ATCC instructions. Huh7 cells were from Prof. Alberti’s laboratory, University of Padua , and were cultured with RPMI-1640 . Hep10 cells were purchased from Thermo Fisher Scientific . TFK1 and Huh28 cholangiocarcinoma cell lines were provided by Prof. Svegliati Baroni, University of Ancona , and were grown in RPMI-1640. LnCAP and MCF7 cells, respectively, derived from prostate cancer and breast cancer, were obtained from the ATCC. Media were supplemented with 10% fetal bovine serum (FBS), L-glutamine and penicillin/streptomycin (Life Technologies).Cell proliferation assay, cell cycle analysis, scratch tests, real-time qPCR and immunohistochemistry (IHC) on DAA-treated cells were performed as detailed in the 6 reads per sample with an average deep of coverage of ~60X. After BCL to FASTQ conversion (bcl2fastq—https://www.illumina.com), quality control and adapter trimming , the paired-end reads were mapped on the human reference genome HG19 with TopHat/Bowtie (http://ccb.jhu.edu/software/tophat). The abundance of transcripts was computed by using the Python function htseq-count (https://htseq.readthedocs.io) with the Ensembl72 release as the gene feature annotation. Gene expression profiling was performed by using a suite of R-bioconductor packages (https://bioconductor.org), such as edgeR  and limma .RNA sequencing was used to analyze the gene expression profiles of HCC-derived cell lines following DAA treatments as well as primary HCCs and cirrhotic tissues. Total RNA was extracted from cell lines (treated with DAAs for 24 h) and tissue samples of HCC and adjacent cirrhosis (control and next-generation sequencing (NGS) cohorts) by using TRIzol . Whole transcriptome sequencing was performed on the Illumina HiScanSQ platform by using the paired-end strategy (2 × 80 bases) as previously described , producihttp://bioinformatics.psb.ugent.be/webtools/Venn/).Principal component analysis (PCA) was performed with prcomp function (R package stats) and the 3D projections were visualized using plot3d (R package ggplot2). All the heatmaps were built with the R package pheatmap. A Venn diagram analysis was performed to visualize the overlapping genes of the different clusters using an online software (Two independent patient cohorts were examined: The first one (post-DAA cohort) was composed of 13 patients with first HCC diagnosed and surgically resected within 12 months (range 0–11) from the end of DAA treatment . Formalin-fixed, paraffin-embedded (FFPE) HCC and surrounding tissues were available; thus, these patients were studied by IHC to determine whether the deregulated expression of molecules in DAA-treated HCC cell lines might be confirmed in this subgroup of HCCs too.The second cohort (active HCV cohort) was composed of 39 surgically resected HCC patients with active HCV infection and who were not previously exposed to DAAs. These patients were studied by IHC and RT-qPCR. Of these, 28 patients, who were surgically resected for first HCC in a background of active HCV infection in the pre-DAA era, had already been analyzed by RNA sequencing (RNA-seq), as previously reported . HCC genCharacteristics of these patient cohorts are described in p-value and corrected p-value  were reported and genes significantly modulated were considered with q-value < 0.01. RNA-seq raw data are available upon request. For qPCR studies (human tissues and cell lines) and IHC studies, differences between groups were analyzed using a double-sided Student’s t-test. Pearson’s correlation coefficient was used to investigate relationships between two variables (qPCR gene expression studies). Differences between categorical variables were explored by Chi-square test. Reported p-values were two-sided and considered significant when lower than 0.05 . Statistical calculations were performed using SPSS version 19.0 . Gene function and pathway analysis was performed on the differentially expressed genes using Visualization and Integrated Discovery v6.7 (http://david.abcc.ncifcrf.gov/home.jsp).For both patient samples and cell lines, the statistical methods adopted to evaluate significant differences in gene expression were as follows: differentially expressed genes were evaluated using the eBayes approach  for the HCC versus surrounding cirrhosis comparison (paired test) and treated versus untreated cell lines (unpaired test). Log2 fold change, nominal Our first question was whether a single administration of sofosbuvir and/or daclatasvir plays any effect on HCC cell proliferation. We performed a preliminary screening of five HCC-derived cell lines  by crystal violet staining after exposure to either sofosbuvir and/or daclatasvir. Proliferation was increased, reduced or unaffected at 24 h, depending on the cell line . Any effTo go deeper into the mechanisms sustaining variations of proliferation, we analyzed cell cycle after DAA treatments. Sofosbuvir reduced Huh7 and SNU449 cell proliferation by arresting cells in the G1 phase. A limited increase of proliferation was induced by sofosbuvir in SNU475 cells. Daclatasvir triggered cell proliferation resulting from a reduction of the G1 phase in SNU475 and SNU449 cells, while it had no effect on Huh7 cells. Either drug did not modify the proliferation or cell cycle of HepG2 and Hep3B cells . NeitherThese data showed that DAAs can modify HCC cell proliferation, either by reducing or increasing it, in the different cell lines and pointed to the relevance of the molecular background in dictating DAAs effects.Cancer cells may not proliferate while migrating, and they may stop moving while dividing. Hence, migration and proliferation may be mutually exclusive. Previous studies showed that microvascular invasion and aggressive features characterize HCCs that develop after DAAs ,18. TherBased on our results in HCC cells, we investigated whether DAAs might also affect cell proliferation and migration of cholangiocarcinoma (CCA) cells. To this aim, TFK1 and Huh28 cells treated with sofosbuvir and daclatasvir were analyzed by crystal violet staining, flow cytometry (FACS) analysis and Incucyte Live-Cell Analysis. Sofosbuvir increased proliferation and accelerated cell cycle progression, displayed by G1-phase reduction, in both TFK1 and, to a lesser extent, Huh28 cells. Daclatasvir increased cell proliferation in both cell lines, although with less prominent variations of cell cycle . A woundWe additionally tested MCF7 (breast cancer-derived) and LnCAP (prostate cancer-derived) cell lines to study the effects of sofosbuvir and daclatasvir on extrahepatic cancer cells. Neither sofosbuvir nor daclatasvir affected MCF7 cell proliferation and migration. Conversely, LnCAP cells displayed an increase in proliferation after sofosbuvir treatment, resulting from G1-phase reduction , while tThese data confirmed the influence of specific cellular backgrounds on DAA-mediated effects on the proliferation and migration of non-HCC cell lines. Thus, we can speculate that off-target effects of DAAs inducing either decreased or increased cell proliferation and migration depend upon the genetic backgrounds of some but not all tumor types and liver lineages, in keeping with the heterogeneous clinical HCC courses.p-value < 0.01, n = 4783), we were able to highlight substantial differences in expression patterns that also showed through the hierarchical clustering analysis -treated control. To account for variations among biological replicates, experiments were done in duplicate. Principal component analysis (PCA) of treated cells showed a clear separation between cell lines, suggesting distinct expression profiles linked to cell type and minor differences due to the treatments A. Lookinanalysis . Assuminanalysis . Gene exn = 321) were identified, including 50 upregulated genes, 256 downregulated genes and 15 genes that were differently regulated in the two cell lines. Functional enrichment analyses showed that co-downregulated genes were significantly enriched in 20 Gene Ontology-Biological Process (GO-BP) terms, with the top 10 reported in Sofosbuvir reduced proliferation in SNU449 and Huh7 cells. Several commonly deregulated genes  downregulated gene, as shown in n = 101), upregulated by daclatasvir and downregulated by sofosbuvir, were identified . Twenty-seven common genes were identified , among wThese findings supported a role of sofosbuvir and daclatasvir as triggers of gene expression changes in HCC cells. Remarkably, these changes were in accordance with corresponding proliferation and migration changes observed in vitro. t-test, p < 0.05; Among these deregulated genes, expression changes of two histones (HIST1H4A and HIST1H3A) were validated by qPCR. Both the histone-coding genes were downregulated in primary HCCs, which suffered post-surgical recurrence within two years after surgery  that were not treated with DAAs. This analysis was expected to indicate a possible relevance in human hepatocarcinogenesis of genes deregulated in vitro by DAAs. We focused on genes with an annotated function in cancer. This procedure led to the identification of 90 genes that were deregulated in vitro and ex vivo. The 37 genes involved in proliferation were oppositely deregulated in HCC tissues and in the two cell lines (Huh7 and SNU449) whose proliferation was reduced by sofosbuvir. Conversely, the 25 genes involved in migration were upregulated in the same cell lines, which showed accelerated migration after sofosbuvir and in HCC tissues as well. The corresponding numbers for daclatasvir (tested in SNU475 and SNU449 with increased proliferation and which displayed different effects on migration) were 15 genes associated with proliferation and 13 genes related to migration F.To further assess the extent to which in vitro findings were representative of HCCs developed after DAA treatment, we analyzed by IHC the expression of Forkhead box protein M1 (FOXM1), Ki67 and vimentin (VIM) in thirteen surgically resected HCCs diagnosed within 12 months after completion of treatment with DAAs (post-DAA cohort) and in an active HCV cohort of 39 patients composed of 28 patients tested by RNA-seq and 11 randomly selected additional patients with clinical characteristics and tumor stage matched with the post-DAA cohort  pathway analysis revealed that sofosbuvir-downregulated genes were enriched in multiple biological processes interfering with mitochondrial function. The downregulation of these gene families might account for the molecular basis of reduced proliferation of Huh7 and SNU449 cells upon treatment with sofosbuvir. Indeed, mitochondrial toxicity is a known side effect of nucleoside reverse transcriptase inhibitors ,24,25.DAA treatment in HCC cell lines modulated ribosomal genes and pseudogenes, outlining the relevant contribution of ribosomal changes in this setting. These factors participate in the translation of viral transcripts , but areThese data do not allow to define the relevance of each molecular event, among those identified here, in a tumorigenic or tumor suppressor perspective. It is conceivable that the molecular background might play a central role in dictating the effective relevance of mitochondrial toxicity, ribosomal alterations and epigenetic or transcriptomic changes in each setting.To assess whether our in vitro findings might reflect the biology of human HCC, we analyzed the overlapping genes regulated by DAAs and those significantly modulated in HCC compared with matched cirrhosis. In line with the opposite effects induced by sofosbuvir and daclatasvir on cell proliferation, most overlapping genes among those regulated by DAAs and those modulated in HCC went in the opposite direction in the case of sofosbuvir, while they were concordant in the case of daclatasvir treatment. Moreover, common genes involved in cell motility were concordant in HCC tissues and in sofosbuvir-treated cells that displayed increased migration capability.A further supportive contribution came from IHC and in vitro data displaying respectively an increase of EMT tissue markers such as vimentin and FOXM1 in DAA-related HCCs and enhanced migration capabilities in specific DAA-treated HCC cell lines. These findings let us to suppose that, in specific contexts, DAAs might elicit pro-tumorigenic molecular changes in HCC cells and thus contribute to aggressive HCC features. At the same time, in vitro evidence also supported reduced proliferation in specific cell lines resulting from DAAs, in line with the benefits reported in some clinical studies. Of course, HCCs available for molecular characterization were presumably those in which DAA predominant effects had been characterized by increased proliferation and migration. Unfortunately, the surgical series of HCCs diagnosed or recurred soon after DAA treatments was very small because these HCCs are often diagnosed beyond surgical criteria. Of note, all cases in which beneficial/tumor suppressive effects of DAAs predominate are not likely to develop HCC and thus cannot be studied. A major limit of the present study was that, while identifying the molecular background as a relevant player mediating the effects of DAAs, specific drivers were not identified. In addition, this study focused on molecular changes occurring in cancer cells, without any evaluation of tumor microenvironment, which is supposed to play a relevant role too. Clinical trials concordantly point to a reduction of HCC development in patients with chronic hepatitis without cirrhotic evolution or HCC, ruling out oncogenic effects of DAAs on normal hepatocytes. The nearly absent constitutive proliferation and migration capability of normal hepatocytes prevented a reliable evaluation of possible “oncogenic” or “anti-oncogenic” changes in these cells. Indeed, the expected effects of DAAs in terms of proliferation and migration reduction cannot be observed because of the ‘quiescent-like’ state of normal hepatocytes. Thus, for both epidemiological and technical reasons, the experiments were performed on HCC-derived cell lines. Interestingly, HepG2 and Hep3B cells, displaying the most “epithelial” features, did not show any phenotypic change after DAA treatment. Conversely, the most “mesenchymal” HCC cells were more affected by DAAs. Even though our data were still too preliminary to be translated into the clinical practice, they appeared to indicate that “mesenchymal” features might be more affected by these drugs. At the same time, we cannot exclude that these drugs themselves were responsible for triggering EMT in specific molecular backgrounds. Thus, the characterization of resected HCC or dysplastic nodules by EMT markers prior to DAA treatment is a possible further line of research that can be proposed, which might help to identify those cases that have to be more strictly surveilled during and after DAA treatment or in whom DAA administration could be at least temporarily withheld. In this perspective, relevant molecular information might be derived from the characterization of extracellular vesicles, which might provide a source of non-invasive biomarkers to be repeatedly assayed.This study added a novel observation on the effects of sofosbuvir and daclatasvir by supporting their modulation of proliferation and migration in some HCC- and CCA-derived cell lines. These phenotypic changes were associated with concurrent variations of gene expression likely resulting from off-target effects. Several gene families modulated in vitro by DAAs were deregulated in primary HCCs too. Thus, while treating HCV infection definitely reduces HCC incidence, a field to investigate further is the possible off-target effects of DAAs, which in some instances may explain HCC risk reduction, while in others might support its increase. Our findings suggested that a molecular characterization of the cured HCC might be performed before DAA treatment, and patients bearing an increased risk of HCC development or recurrence should continue to be strictly surveilled when DAA treatments are to be prescribed.


Melissa officinalis L. (MO), known as lemon balm, an aromatic plant that spontaneously grows in the South and Western areas of Romania, being traditionally recommended as anxiolytic, antispasmodic, or as digestive remedy. Our aim was to investigate the phytochemical profiling and the antiangiogenic and chemopreventive bioactivity of MO from Banat region, on breast cancer. Two ethanolic extracts of MO (MOE96 and MOE70) and one methanolic extract (MOM80) were subjected to polyphenol and triterpene profiling by HPLC-MS, and the antioxidant capacity was evaluated. The antiangiogenic potential was investigated using the chorioallantoic membrane assay (CAM). The MTT-2-5-diphenyltetrazolium bromide) assay was used to investigate the cytotoxic effects on MCF-7 and MDA-MB-231breast cancer cells, as well as on MCF-10A normal breast epithelial cells, while apoptosis was performed by DAPI staining. Rosmarinic acid (RA) and ursolic acid (UA) were revealed as dominant phytocompounds. The highest concentration in phytochemicals were found in MOM80; MOE96 was more concentrated in UA, while MOE70 extracted more RA. MOE96 inhibited cancer progression and angiogenesis in the in ovo CAM model using MDA-MB-231 cells, inhibiting breast cancer progression and angiogenesis for the MDA-MB-231 breast cancer cell line; no secondary tumoral areas were registered, indicative for a preventive effect against breast tumor cell invasiveness. The highest cell inhibitory activity was also exhibited by MOE96, in particular against the estrogen receptor positive MCF7 breast cancer cell line, with no cytotoxic effect on healthy cells. The estrogen receptor positive MCF7 cell line proved to be more sensitive to the extract antiproliferative activity than the triple negative MDA-MB-231 breast cancer cell line. Nevertheless, the chemopreventive potential of MOE96 extract is phenotype-dependent and is rather related to the apoptosis and antiangiogenic effects suggesting a multitargeted mechanism of action due to its multiple compound composition next to a concentration ratio of RA : UA in favor of UA.Breast cancer is the most frequently diagnosed malignant pathology, representing the primary cause of cancer death in women. Natural products are an appealing strategy to limit the progression of the disease. Targeting angiogenesis in breast cancer may positively impact on poor prognosis of breast cancer. As source of natural compounds, we investigated the leaves of  Breast cancer is an important worldwide public health problem, being the most frequently diagnosed cancer and the first cause of cancer death in women . In receMelissa officinalis L. (MO), a perennial aromatic plant belonging to the Lamiaceae family, also called lemon balm due to its lemon scent. The chemical composition consists in low amounts of essential oils, triterpenes, and important concentrations of polyphenols (caffeic acid derivatives as rosmarinic acid and flavonoids) , where A0 = absorbance of blank sample and As = absorbance of tested samples.The colorimetric microculture tetrazolium assay (MTT) was used to study the viability of MCF-7, MDA-MB-231, and MCF-10A cells in accordance with Mosmann . The celμg/mL), RA and UA standard compounds (50 and 100 μM), and DMSO used as negative control. DAPI staining was performed as previously described [Detection of the state of MCF-7 and MDA-MB-231 cells' nuclei condensation was assessed by the means of DAPI, a nuclear stain able to emit blue fluorescence of the live cell nuclei and bright blue fluorescence of apoptotic cell nuclei, upon UV excitation. The cells were seeded on coverslips in 6-well plates until the optimal confluence was reached, followed by 24 h treatment with MO extracts .All experiments were performed in triplicate. Results are presented as mean ± standard deviation (SD). One-way ANOVA analysis followed by Tukey's post hoc test was performed to detect statistical differences among the tested groups μg of phytocompound per gram dry weight (d.w.). Also, the SIM MS spectra in the negative ion mode of the polyphenolic profile of MOE70 sample are depicted in All samples were subjected to LC-MS analysis which enabled the identification and quantification under identical solution and instrumental conditions of two major classes of secondary metabolites in MO: polyphenols and pentacyclic triterpenic compounds . All ideμg/g d.w.) and almost 25 times higher than 96% ethanol maceration (86637 vs. 3515 μg/g d.w.)  was found to be the most abundant polyphenolic compound. These findings are consistent with many literature reports , 10, 38,/g d.w.) .The use of methanol generally favored the extraction of all polyphenolic compounds to an average increase in concentration of 500% when compared to ethanolic extraction, but in some cases , the difference can reach double the average. Methanol combined with 30 minutes sonication was previously found to be the most favorable method for polyphenolic compounds extraction , but theNext, the phytochemical characterization of MO extracts continued with the screening and quantification of all samples for pentacyclic triterpenic compounds; detected and quantified compounds in MOE70, MOE96, and MOM80 are listed in μg/g d.w.) and almost double than the 96% ethanol maceration (11234 vs. 6103 μg/g d.w.)  . Similarμg/ml), next to the two pure compounds, RA and UA (50 μM). Firstly, we assessed the general tolerability and the effect on the normal developing chorioallantoic membrane, 24 hours following treatment . When comparing the six groups of samples—one control and five treatment groups—experimental data showed that the MOE70 extract was best tolerated with no modification induced on the normal angiogenic process. When compared to MOM80, MOE96 showed a slightly higher vessel density, though none of the samples induced toxicity on the vascular architecture and functionality. In case of the two standard compounds used in 50 μM concentration, ursolic acid induced a slight reduction of the vascular density, while rosmarinic acid did not influence the normal CAM vascular plexus. Collectively, these data indicated the lack of significant toxicity of pure compounds and total extracts as well, when applied on normal tissues.As far as we know, this is the first evaluation of lemon balm ethanolic or methanolic extracts on the CAM assay, regarding their potential effects on the angiogenesis process involved in breast cancer progression. The suppression of the angiogenesis process is known as an antitumor and antimetastatic mechanism . We invereatment . From ouSecondly, we assessed the effects induced by the tested samples onto the developing CAM after the inoculation of MCF-7 and MDA-MB-231 breast cancer cells. The follow-up was performed 24 hours posttreatment of CAMs already inoculated with tumor cells and 96 hours posttreatment as well . Secondai.e., MOM80 and MOE70) induced more important reduction in all angiogenic parameters, except the number of complexes, for the MDA-MB-231 cell line  in a dose related manner [Previous evaluations regarding the possible angiogenic implications of lemon balm extracts were performed only d manner , while hd manner . The antd manner . CAM-basd manner .μg/mL) next to standard compounds RA  and UA  and compared to ascorbic acid  as control (50 for the AOA in DPPH assay: IC50 for UA∼5 mg/mL > IC50 for AA∼15 μg/mL > IC50 for RA = 5.5 μg/mL [The antioxidant activity (AOA) was evaluated for the three types of MO extracts (50  control . All thr.5 μg/mL –50. Henc.5 μg/mL . The lemμM (corresponding to 9–36 μg/mL·RA and 11.4–45.6 μg/mL·UA), exhibited an opposite effect on the cell viability of the two breast cancer cell lines; on both breast cancer cells, ursolic acid induced a significant decrease of cell viability  while RA showed a complete lack of inhibitory activity or even stimulated cell proliferation, in particular on the MDA-MB-231 cell line. The inhibitory potency of ursolic acid was similar on the two tested cancer cell lines.The three MO extracts and standard compounds, RA and UA, were evaluated in terms of their effect on cell viability on two breast carcinoma cell lines, MCF-7 and MDA-MB-231, the former representing an ER+ type of breast cancer and the latter a triple negative breast cancer. The effect of the tested samples on MCF-7 and MDA-MB-231, respectively, after 24 h stimulation is shown in Figures 50 values previously reported for UA on the two breast cancer cell lines are controversial, ranging from approximately 1 μM to 500 μM [μM) on two phenotypically distinct breast cancer cell lines (MCF-7 and MDA-MB-231) was attributed to the changes induced by UA to the glycolytic pathway leading to cytotoxic autophagy as well as apoptotic cell death [Although considered as a valuable potential chemotherapeutic agent in terms of arresting cancer progression, the ICo 500 μM , 51. Thell death . Anotherll death .μg/mL, RA increased cell viability of breast cancer cells, in particular estrogen-dependent cells (MCF-7) [50 values ranging from as low as 2 μM to 200 μM [With regard to rosmarinic acid tested as pure compound, our results are consistent with other previously published studies showing that at 50  (MCF-7) . Howevero 200 μM .μg/mL, on the two breast cancer cell lines, respectively, the first observation to be noted is that all three extracts exhibited an inhibitory activity against the two cancer cell lines in a dose-dependent manner, in particular MOE70 and MOE96; MOM80 showed a weak inhibitory activity, only when used in the highest tested concentration, 100 μg/mL, the lower concentrations exhibiting either a lack of activity (50 μg/mL) or even inducing cell proliferation (25 μg/mL). A slightly stronger inhibitory activity was registered for both MOE70 and MOE96 against the ER+ MCF-7 cell line. Their effect on cell viability can be perfectly correlated with their concentrations in the pure compounds, RA and UA. MOM80 contains the highest concentration in both compounds . The excess of RA as compared to UA is maintained in MOE70 which contains 8627.84 μg/g d.w. RA and 3577 μg/g d.w. UA; however, in this case, the RA : UA concentration ratio becomes 2.4 : 1, indicating that UA may strongly exert its inhibitory effect. Indeed, for MOE70, the lowest concentration did not affect cell viability or even induce a slight stimulatory effect, while higher concentrations significantly decreased cell viability in a dose-dependent manner. As one can see, the median dose, 50 μg/mL, inhibited in particular the estrogen-dependent cell line MCF-7 while the highest dose of MOE70, 100 μg/mL, had a similar effect against both breast cancer cell lines. For MOE96, the RA : UA concentration ratio is reversed, becoming 1 : 2 (3515.6 μg/g d.w. RA and 6103.47 μg/g d.w. UA); this change is reflected in its effect on cell viability. All of the three concentrations show an inhibitory effect on both cell lines, also in a dose-dependent manner, with a slightly higher potency against the MCF-7 cell line; in this case, the inhibitory activity of ursolic acid prevailed to the proliferative activity of RA.When evaluating the three types of lemon balm extracts, tested in concentration of 25–100 μM, its concentration in the used extracts was much lower: for example, 100 μg/mL MOE96 extract contains 0.35 μg/mL·RA and 0.61 μg/mL UA, corresponding to a concentration of 0.97 μM and 1.34 μM, respectively. Therefore, we may state that we achieved 30% cell inhibition for both types of breast cancer when using a very small concentration of 1.34 μM active UA (in the form of MOE96 extract), even in the presence of a significant amount of pro-proliferative RA (0.97 μM), while a much higher UA concentration (25 μM) achieved cell inhibition of only approximately 50%; this phenomenon may be the result of a multitargeted activity that might occur due to UA and other compounds such as oleanolic or betulinic acid also present in the extract in smaller amounts, as indicated by the HPLC analysis. The synergistic activity exhibited by oleanolic and ursolic acids was previously documented [in vitro studies on two human melanoma cell lines (A375 and A2058). RA is also capable of inducing synergistic effects in spite of its lack of cytotoxicity; in 2017, Bahri et al. reported a significant decrease of the IC50 value for the combination of RA and carnosic acid compared to carnosic acid alone, while RA alone had no cytotoxic effect on human lung fibroblasts [If we compare the cell inhibition induced by pure UA versus MOE96, a superficial look might indicate that the use of UA is clearly preferable due to its much stronger cytotoxic effect; however, the two respective concentrations in UA must be taken into account when comparing the two biological activities. While pure UA was applied in a concentration range of 25–100 cumented , 55 by iroblasts . Their dAnother possibility may consist in the sensitization of the two breast cancer cell lines to the cytotoxic activity of UA by the presence of the large plethora of polyphenolic compounds revealed by the HPLC assessment. The use of polyphenols as sensitizers was reported by Singh et al. on MCF-7 and MDA-MB-231 cell lines; the combination of low doses of polyphenols with cytotoxic compounds augmented the apoptotic activity of the active drug while providing the advantage of reduced side effects .Melissa officinalis L.; the results varied in a large range, as a great variability lays in the extraction procedure, next to the geographical source of plant material. Some studies reported that aqueous Soxhlet extracts of MO from Turkey may affect breast cancer cell viability, with IC50 values of 18 μg/mL against MCF-7 and 19 μg/mL against MDA-MB-231 [50 of 196 μg/mL for the triple negative cells MDA-MB-231 [50 values for the ethanolic extracts (122 μg/mL), compared to the methanolic ones (181 μg/mL), despite their higher content in polyphenolic compounds. The same study indicated that hydroalcoholic extracts had intermediate effects, while the aqueous decoctions were less active [Several other research groups studied the potential chemopreventive effect of the abundantly available and frequently used medicinal plant, A-MB-231 . A more s active . Our resκB-) related gene that leads to a higher aggressiveness and cell resistance [The cell inhibitory activity is in particular worth noticing on the ER− cancer cell line MDA-MB-231; while the ER+ cell lines such as MCF-7 are sensitive to treatment with hormone and other targeted therapies, triple negative breast cancers such as MDA-MB-231 do not express any receptors and can be therefore subject only to cytotoxic chemotherapy . MDA-MB-sistance ; these fWhen confronting the results attained by the pure compounds and MO extracts on cell viability and the antioxidant capacity, an inverse relationship can be noticed. MOE96 (the extract with the highest concentration in UA) and UA exert the strongest effect on the reduction of cell viability, while exhibiting the lowest AOA, thus implying that extract effect may be due to the UA contained.μM, affected MCF-10A cell population, displaying a cell viability percentage of 70.86%. Simultaneously, the best tolerated extract in all applied concentrations was MOE96, followed by MOM80 and MOE70. When corroborating the in vitro results obtained on both cancer and healthy cell lines, we may conclude that in spite of the strong antiproliferative activity exerted by UA against the two breast cancer cell lines, the compound cannot be considered as the optimal solution for cancer treatment due to its lack of selectivity. At the same time, MOE96 extract exhibited a significant cytotoxic activity against both breast cancer cell lines while lacking any negative effect on the normal MCF-10A cell line, thus showing a high selectivity degree. MOM80 also lacked cytotoxic effect on the normal cell line but its in vitro antitumor activity was also negligible. The second most active antiproliferative extract, MOE70, exhibited a moderate cytotoxic effect against normal cells, in particular in the highest concentration, 100 μM, displaying a cell viability percentage of 82.77%. The samples were also screened in regard to their cytotoxic potential on another normal cell model, using immortalized human keratinocytes—HaCaT cells. Results were similar to the ones obtained on MCF-10A cell, mentioning that the HaCaT cells seemed more sensitive, especially after treatment with the highest dose, 100 μM, of UA (data not shown). This cytotoxic activity of UA against HaCaT cells was previously documented by Harmand et al. who reported a decrease of cell viability in a time and dose-dependent manner through caspase-3-mediated apoptosis and cell cycle arrest [When assessing the cytotoxic activity of synthetic or natural compounds, an important issue is their selectivity, namely, their ability to exert an antiproliferative effect against cancer cells while leaving healthy cells unharmed. In this regard, we tested the biological activity of pure compounds and extracts also on breast epithelial MCF-10A cells . One cane arrest . CollectTo investigate if MO extracts and standard compounds (RA and UA) have the potential to induce apoptosis of breast adenocarcinoma MCF-7 and MDA-MB-231 cells, DAPI staining was employed. The results are presented in Figures DAPI nuclear staining revealed different responses in terms of apoptosis after the treatment with the tested samples, dependent of the cell line genotype (ER−/ER+). Typical signs of apoptosis (chromatin condensation and nuclear membrane blebbing)  were marMCF-7 cells incubated with MO extracts exhibited an increase of apoptotic markers as compared to the effect observed in the case of MDA-MB-231 cells. Among the MO extracts, the most significant apoptotic effects were induced by MOE96 followed by MOE70 on MCF-7 cell line, observing the loss of round-shape nucleus in several cells .μM UA, a much higher concentration than that present in the tested extracts.The pure compounds were less active against tumor formation and acted as antiangiogenic compounds especially 96 hours postinoculation with MDA-MB-231 cells. RA reduced all angiogenic measured parameters 96 hours posttreatment for the MDA-MB-231 cells. UA affected strongly the process of tumor angiogenesis, reducing the number of blood vessel complexes for both cancer types. Still, the total extracts, in particular MOE96, exhibited more important reduction of the angiogenesis process compared to RA on both cell lines after short and long stimulation, while reducing the size of the blood vessels comparable to the effect of 50 in vitro, with a slightly stronger effect towards the estrogen receptor positive type MCF7 cell line, consistent with the proapoptotic assessment.The 96% ethanolic extract MOE96 induced the lowest cell viability of breast cancer cells When compared to the effect of pure RA and UA, MOE96 exhibited a potent cytotoxic effect at a much lower concentration in active principles. MOE96, with a concentration ratio RA : UA of 1 : 2 induced a stronger inhibitory effect on both cell lines, in a dose-dependent manner, with a slightly higher potency against the MCF-7 cell line. MOE96 extract containing the equivalent of 1.34 μM antiproliferative UA and 0.97 μM of the proproliferative RA decreased breast cancer cell viability with about 30% in both cell lines, while a much higher UA concentration (25 μM) was needed in order to reduce tumor cell viability with approximately 50%.Among the MO extracts, the most significant proapoptotic effect was induced by MOE96 followed by MOE70 on MCF-7 cell line, observing the loss of round-shape nucleus in several cells. The ER− cancer cell line MDA-MB-231 seemed to be less affected by exposure to MO extracts for 24 h, yet some apoptotic signs can be observed especially in the case of MOE70 and MOE96.A multitargeted effect of the multiple components found in the composition of the MOE96 total extract together with a concentration ratio of RA : UA in favor of UA could be suggested as an explanation for the cytotoxic activity against breast cancer cells, but not affecting healthy cells. In addition, the study revealed that the reduction of breast cancer progression is not manifested through an antioxidant mechanism but rather through the proapoptotic activity and antiangiogenic effect of the extract with the concentration ratio RA : UA in favor of UA and presumably potentiated by the association with other polyphenols and pentacyclic triterpenes.Melissa officinalis L.), a highly available and used medicinal plant in the Western area of Romania, were previously reported with promising antitumor effects. Our work contributed to this research topic by evaluating breast cancer antiangiogenic effect and chemoprevention induced by methanolic and ethanolic lemon balm leaves extracts. Although the highest contents in both polyphenols and triterpenes were detected in the 80% methanolic extract, with rosmarinic acid and ursolic acid as the most concentrated compounds, the highest cell inhibitory activity was exhibited by the 96% ethanolic extract, in particular against the estrogen receptor positive MCF7 breast cancer cell line, with no cytotoxic effect on healthy cells. The estrogen receptor positive MCF7 cell line proved to be more sensitive to the extract's antiproliferative activity than the triple negative MDA-MB-231 breast cancer cell line. The in ovo CAM assessment also revealed the 96% ethanolic extract as the most potent chemopreventive agent, inhibiting breast cancer progression and angiogenesis for the MDA-MB-231 breast cancer cell line. The antioxidant potential was directly correlated with the concentration of rosmarinic acid. Nevertheless, the chemopreventive potential of MOE96 extract is phenotype-dependent and is rather related to the apoptosis and antiangiogenic effects suggesting a multitargeted mechanism of action due to its multiple compound composition next to a concentration ratio of RA : UA in favor of UA.Various types of extracts from lemon balm (


Opioid receptor activation was shown to enhance the efficacy of anti‐neoplastic drugs in several human cancer cell lines. In these cell lines, doxorubicin increased the number of opioid receptors and methadone concurrently enhanced cellular doxorubicin uptake. Triggered through lay press and media, animal owners started to challenge veterinary oncologists with questions about methadone use in anti‐cancer therapy. Especially in veterinary medicine, where side effects of chemotherapy are tolerated to a lesser extent and hence smaller doses are given, agents potentiating chemotherapeutic agents would be an optimal approach to treatment. Canine transitional cell carcinoma cells , canine osteosarcoma cells  and canine hemangiosarcoma cells  were incubated with different combinations of methadone, buprenorphine and doxorubicin, in order to test inhibition of cell proliferation. Opioid receptor density was assessed with fluorescence‐activated cell sorting in drug native and doxorubicin pretreated cells. In TCC and OSA cell lines opioid receptor density increased after doxorubicin pretreatment. In combination treatment, however, we did not find significant potentiation of doxorubicin's inhibitory effect on proliferation in these cell lines. Neither was there a significant increase of the effect of doxorubicin when the opioids were added 24 hr before doxorubicin. Hence, we could not confirm the hypothesis that opioids increase the anti‐proliferative effect of the anti‐neoplastic drug doxorubicin in any of these canine tumour cell lines. The lack of effect on a cellular level does not warrant a clinical approach to use opioids together with doxorubicin in dogs with cancer. Triggered through lay press and media, animal owners started to challenge veterinary oncologists with questions about methadone use in anti‐cancer therapy. However, we could not confirm the hypothesis that opioids increase the anti‐proliferative effect of the anti‐neoplastic drug doxorubicin in any of these canine tumor cell lines. The lack of effect on a cellular level does not warrant a clinical approach to use opioids together with doxorubicin in dogs with cancer. Recently, the opioid methadone was described to potentiate the pro‐apoptotic effect of doxorubicin, a commonly used anti‐neoplastic agent in human leukaemia and glioblastoma cells  in vitro Friesen, Bacher, Hormann, Roscher, and Miltner ; FriesenWhen these findings were spread publicly through lay press and media, cancer patients started to confront physicians and pharmacists, willing to use methadone in their cancer treatment , osteosarcoma (OSA) and hemangiosarcoma (HSA)) by flow cytometric analysis before and after doxorubicin exposure. Next, we tested if the concurrent use of opioids in canine tumour cell lines in vitro enhances effects of doxorubicin, measured with proliferation assay. The resulting data will be used for future decisions on whether opioids should be investigated deeper with an aim of a possible use in a clinical setting in dog cancer patients with specific neoplastic conditions receiving doxorubicin.2First, presence and expression of opioid receptors on canine transitional cell carcinoma, canine osteosarcoma and canine cells hemangiosarcoma before and after the treatment with doxorubicin was investigated through flow cytometry. Then, different dosages of doxorubicin, methadone and buprenorphine were tested on all three cell lines. Afterwards, the selected doses were used for different drug combinations, which were examined by means of a cell proliferation assay.2.1®) was obtained from Streuli Pharma AG  and buprenorphine hydrochloride (Temgesic®) was obtained from Indivior Schweiz AG . Both are aqueous solutions in disposable ampoules. For each experiment a new ampoule was opened.Methadone hydrochloride (Methadon Streuli® RD/‐Solution) was obtained from Pfizer AG . Each vial was used three to six times and stored at 4°C for a maximum of 3 weeks. Adriblastin is an aqueous solution.Doxorubicin hydrochloride  and was dissolved in dimethylsulfoxide.2.2™) containing 10% fetal bovine serum (FBS) (Gibco™), 100 units/ml of penicillin (Gibco™), 100 μg/ml of streptomycin (Gibco™) and 10 mM of 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid buffer (Gibco™) and incubated at 5% CO2 and 37°C. The three cell lines are described as TCC , OSA  and hemangiosarcoma  in figures and text.Canine transitional cell carcinoma cells (K9TCC) was obtained from Prof. Knapp, Purdue , canine osteosarcoma cell (Abrams) were obtained from Prof. Rebhurn,  and canine hemangiosarcoma cells (DAL‐4) were obtained from Kerafast Inc. . The K9TCC and Abrams cells were grown in Dulbecco's modified eagle's medium (Gibco™) containing 10% FBS (Gibco™), 100 μg/ml of primocin, 0.05 mg/ml of endothelial cell growth supplement, 0.1 mg/ml of heparin (Sigma‐Aldrich®), 10 mM of HEPES buffer (Gibco™) and incubated at 37°C in 5% CO2 humidified incubator. All cell lines were free of mycoplasma.The DAL‐4 cells were grown in Ham F‐12 . The cells were incubated for 30 min at RT in darkness. After incubation the cells were washed, centrifuged and resuspended in cold PBS/1% FBS. CytoFLEX S flow cytometer  was used for the flow cytometer analysis. The flow cytometry results were analysed by FlowJo 10.6.1 software.2.4The proliferation assay was performed with the cell counting kit CCK‐8 according to the manufacturer's protocol . CCK‐8 reagent is a ready for use solution, which allows determination of the number of viable cells in proliferation assays. Five hundred cells for OSA and TCC cells, and 2000 cells for HSA cells per well were seeded into 96‐well plates. For initial dose finding, cells were treated with 10, 5 and 1 μg/ml of methadone, 0.2, 0.5, 1 μg/ml of buprenorphine and 0.5, 0.1, 0.05, 0.015, 0.01, 0.005 μg/ml of doxorubicin. The cells were incubated up to 72 hr and the proliferation was measured at time points of 0, 24, 48 and 72 hr. At each time point, 10 μl of the CCK‐8 solution was given to the corresponding wells and the absorbance was measured 4 hr later at 450 and 600 nm using microplate reader Epoche 2 (BioTek).For the combination studies, methadone or doxorubicin was added to cells for 24 hr prior to adding the combination compound doxorubicin and methadone, respectively. The concentrations and order of the compound addition for each cell line are indicated in the figure legend. The cell proliferation was measured 72 hr after adding the first compound.2.5t‐tests with the Welch–Satterthwaite approximation were used . Two stars (**) were used for p values below 0.01 and three stars (***) were used for p values falling below 0.001.Statistical analyses were performed using R  along with the multcomp package (version 1.4–10) for post‐hoc tests. For the detection of opioid receptors by fluorescence‐activated cell sorting, two‐tailed unpaired Student 33.1First, we investigated the presence and expression of opioid receptors on canine transitional cell carcinoma, canine osteosarcoma and canine hemangiosarcoma cells through flow cytometry. Flow cytometry revealed receptors on all three tested cell lines. In all tested cell lines, 35%–80% of the cells expressed opioid receptors in untreated cells Figure . After i3.2We tested anti‐proliferative effects of increasing doses of methadone or buprenorphine and doxorubicin on the three cell lines in order to determine the optimal concentration for the combinatorial experiments. Methadone and buprenorphine did not inhibit cell proliferation of all cell lines tested up to concentration of 10 μg/ml and 1 μg/ml, respectively Figure a and b. 3.3When the cells were pretreated with doxorubicin and methadone was added 24 hr later, we did not find a potentiation of doxorubicin's inhibitory effect on proliferation in TCC, OSA or HSA cell lines Figure . Pretrea4The opioid methadone recently gained much attention as an anti‐neoplastic compound, possibly potentiating in vitro and in vivo efficacy of doxorubicin (Friesen, Roscher, Alt, & Miltner, In this study the number of opioid receptors was moderate and increased after doxorubicin exposure in the canine TCC and OSA, but not in the HSA cell line. Cell lines with high opioid receptor density such as human leukaemia cells were found to respond stronger to methadone or combination treatment than cells with only moderate opioid receptor expression (Friesen et al., We could, however, not confirm the hypothesis that opioids increase the potency of doxorubicin in any of the three investigated cancer cell lines of dogs. Comparably, no anti‐neoplastic effect was observed in several established and primary human glioblastoma cell lines using combinations of methadone with temozolomide (Brawanski et al., We selected the herein used three dog cell lines of low, moderate and high sensitivity to doxorubicin treatment. As clinical diseases in dogs, hemangiosarcoma and osteosarcoma are treated with doxorubicin as a single agent or in combination with other anti‐neoplastic drugs (Kent, Strom, London, & Seguin, Buprenorphine was used as a second opioid to test. The oral bioavailability of methadone in dogs is described to be below detection, owing to the first pass effect (Kukanich, Kukanich, & Rodriguez, The cell lines used in this study express moderate amounts of opioid receptors, and its expression increased to over 90% after 72 hr of incubation with doxorubicin in the transitional carcinoma and the osteosarcoma cell line. Nevertheless, no positive or negative effect on cell proliferation could be observed after co‐treatment with opioids. Friesen et al. , 2014 suHowever, along with the findings of our colleagues from human medicine (Brawanski et al., 5The lack of effect on a cellular level does not warrant a clinical approach to use opioids together with doxorubicin in dogs with cancer. In case of further pursuit of such combined approach it should be proactively considered to use an orally bioavailable variant such as buprenorphine.Claudia Cueni: Conceptualization; Formal analysis; Investigation; Writing‐original draft. Katarzyna J Nytko: Formal analysis; Investigation; Supervision; Writing‐review & editing. Pauline Thumser‐Henner: Investigation; Writing‐review & editing. Mathias S. Weyland: Formal analysis. Carla Rohrer Bley: Conceptualization; Project administration; Supervision; Writing‐original draft; Writing‐review & editing. Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Fig S4Click here for additional data file.


Glioblastomas display a high level of intratumoral heterogeneity with regard to both genetic and histological features. Within single tumors, subclones have been shown to communicate with each other to affect overall tumor growth. The aim of this study was to broaden the understanding of interclonal communication in glioblastoma.We have used the U-343 model, consisting of U-343 MG, U-343 MGa, U-343 MGa 31L, and U-343 MGa Cl2:6, a set of distinct glioblastoma cell lines that have been derived from the same tumor. We characterized these with regard to temozolomide sensitivity, protein secretome, gene expression, DNA copy number, and cancer cell phenotypic traits. Furthermore, we performed coculture and conditioned media-based experiments to model cell-to-cell signaling in a setting of intratumoral heterogeneity.Temozolomide treatment of a coculture composed of all 4 U-343 cell lines presents a tumor relapse model where the least sensitive population, U-343 MGa 31L, outlives the others. Interestingly, the U-343 cell lines were shown to have distinct gene expression signatures and phenotypes although they were derived from a single tumor. The DNA copy number analysis revealed both common and unique alterations, indicating the evolutionary relationship between the cells. Moreover, these cells were found to communicate and affect each other’s proliferation, both via contact-dependent and -independent interactions, where NOTCH1, TGFBI, and ADAMTS1 signaling effects were involved, respectively.These results provide insight into how complex the signaling events may prove to be in a setting of intratumoral heterogeneity in glioblastoma and provide a map for future studies. An intratumoral heterogeneous model, U-343, is used.Chemotherapy-resistant clones exist in the tumors. They will grow up and cause tumor relapse after treatment.There are interactions between heterogeneous clones, which are potential therapeutic targets.This study shows that genetically distinct cell populations from a single glioblastoma tumor have different drug sensitivities and phenotypes related to astrocytic versus mesenchymal features. Also, the glioblastoma subtype phenotype was affected in a coculture setting. Furthermore, NOTCH, TGFBI, and ADAMTS1 signaling are indicated as modulators of intratumoral communication in glioblastomas. In summary, we show that the U-343 cell line panel serves as a useful model to study intratumoral heterogeneity.1 Current treatment consists of surgery followed by radiotherapy and adjuvant temozolomide. Single glioblastomas have been shown to consist of an intermixture of cancer cell populations representing the previously described subclasses: classical, proneural, and mesenchymal.2–4 Recent findings in several cancer types support the view that tumors may contain several subpopulations of cells with different genotypic and phenotypic properties, including different response to therapy.5–10Glioblastoma is the most aggressive brain tumor in adults with a median survival of 15 months.PDGFRA) in combination with either epidermal growth factor receptor (EGFR) or MET (hepatocyte growth factor receptor) amplification.12 Furthermore, multiple studies have shown that intratumoral genetic heterogeneity is frequently occurring in glioblastoma, where different cancer cell subpopulations may communicate and depend on each other, like in a social network.14Within the same glioblastoma, different cancer cell populations have been described with distinct genetic alterations including amplification of platelet-derived growth factor receptor alpha  but not glial fibrillary acidic protein (GFAP), and conversely the U-343 MGa cultures express GFAP but not FN1  and cultured as previously described. not FN1  and B.18 Antibodies and primers are provided in Immunofluorescence, western blotting, and real-time PCR were performed as previously described.RNA and genomic DNA were isolated from the U-343 cells. RNA was used for RNA-sequencing (RNA-seq). RNA-seq data have been deposited at the EBI ArrayExpress database (accession number E-MTAB-8620). DNA was used for somatic copy number analysis.19NOTCH1 knockout was performed by CRISPR/Cas9 based gene editing.20Green Florescent Protein (GFP)-expressing cells and gene suppression cells by short hairpin ribonucleic acid (shRNA) were generated by lentiviral transduction as previously described.The invasion capacity of U-343 cells was measured by the Matrigel invasion assay. Cell proliferation was assessed by fluorescence measurements with Tecan microplate reader (Tecan) or by MTT assay.Temozolomide sensitivity in U-343 cell lines was tested by the addition of temozolomide at different concentrations and assessed by MTT assay. In the coculture experiments, the 4 U-343 cell lines were mixed (25% of each) and cultured in the presence of Dimethyl sulfoxide (DMSO) or 200 µM temozolomide for 5 and 10 days.Conditioned media was added in a 1:1 ratio mix with unconditioned media to U-343 cell lines and cultured for 7 days. In coculture experiments, non-GFP-labeled cells were seeded on the bottom for 48 h, followed by addition of GFP-labeled cells on top, and cultured for 5 days.21 Cells were incubated with methionine-free DMEM supplemented with either methionine or L-azidohomoalanine . Collected conditioned media was concentrated and secreted proteins were bound to Click-iT beads by click chemistry. Bound proteins were subsequently digested and identified by mass spectrometry.To detect secreted proteins we used Click-iT Protein Enrichment Kit according to a previously described Click-iT protocol.t-test and the correlation between parameters was assessed by Pearson’s tests.Bar graphs generated in Excel with error bars show average ± standard deviation. The identification of significant differences in average between groups was performed by Student’s 15–17 Here, the identity of the cell lines was confirmed by immunofluorescence staining showing expression of FN1 in U-343 MG and GFAP in the 3 U-343 MGa cultures and by short tandem repeat (STR) analysis  overlapped with “U-343 MG genes” and conversely, 18% (178/1004), 7% (95/1354), and 17% (156/942) of GFAP-correlated genes (r > 0.3) overlapped with “U-343 MGa genes,” “U-343 MGa 31L genes,” and “U-343 MGa Cl2:6 genes,” respectively , glioblastoma subset analysis, and Matrigel invasion assays. We identified genes higher expressed in each cell line compared to the average expression in the other 3 cell lines (>2-fold). These genes were denominated: “U-343 MG genes” (1093 genes), “U-343 MGa genes” (1004 genes), “U-343 MGa 31L genes” (1354 genes), and “U-343 MGa Cl2:6 genes” (942 genes) . To analll lines . To furtignature . Furtherectively . A GSEA ectively . Of partectively . At a clectively . The genll lines . Thus, tll lines .FN1 and GFAP correlated genes, a glioblastoma mesenchymal subtype signature, and the Matrigel invasion capacity indicate that U-343 MG has a more mesenchymal phenotype compared to the U-343 MGa cultures. Overall, these findings indicate that cultured cells from a single glioblastoma tumor may display different gene expression signatures and connected phenotypic traits.In summary, the patterns of EGFR and CDK6, and AKT1, respectively.22 Furthermore, they shared focal gains at 3q26.1, 4q13.3, and 16p11.2 and focal losses at 1p32.2, 1p36.22, 2q24.3, 7p21.1, 8q24.23, 9p21.3, 10q11.22, and 22q12.1. The U-343 MGa cell lines shared loss of 6q, 10p, 18q, and 22q. The U-343 MGa cultures also shared focal gain at 18q12.1 and loss at 7p14.1. Beyond these shared alterations, the U-343 cell lines displayed multiple unique alterations , a known regulator of cell proliferation often lost in glioblastoma,23 was found to have a lower gene copy number and gene expression in U-343 MGa 31L.To determine the genetic relationship between the different U-343 cell lines, we performed DNA copy number analysis  by compaerations . Cell lierations . InteresBy hierarchical clustering, the clonal relationship was investigated based on copy number alterations . The patCDH2 (N-cadherin), SNAI2 (SLUG), and FN1 and decreased expression levels of markers for neural stem and progenitor cells including SOX2, OLIG2, GFAP, PTPRZ1, and CDH1 (E-cadherin)   and B. Tadherin) . In summ24 We thus hypothesized that NOTCH signaling may be involved in cell-to-cell communication between U-343 MG and U-343 MGa Cl2:6. Specifically, JAG1 and JAG2 were highly expressed in U-343 MG, whereas NOTCH1 was highly expressed in U-343 MGa Cl2:6 , followed by U-343 MGa Cl2:6 and U-343 MGa 31L (R2 = 0.43), and U-343 MGa and U-343 MGa 31L (R2 = 0.25). U-343 MG displayed a noncorrelated secretome signature when compared to the U-343 MGa cell lines, where U-343 MGa Cl2:6 only had an R2 of 0.13, closely followed by U-343 MGa and U-343 MGa 31L with R2 values of 0.13 (Due to the identified effects via secreted factors we sought to identify proteins secreted from the U-343 cell lines by a secretome protein enrichment with click sugars (SPECS) analysis. of 0.13 .To compare protein and gene expression levels, we analyzed the concordance between the secretome and RNA-seq data for the U-343 cell lines . Four grTGFBI in U-343 MG by 2 different short hairpin ribonucleic acids (shRNAs), named #1 and #2, and assessed the effect of conditioned media on the growth of U-343 MGa cultures. Western blot analysis confirmed TGFBI suppression by 2 shRNAs (TGFBI suppression (shTGFBI #1 and #2) increased the growth of U-343 MGa 31L to 1.18 and 1.21 as compared to control, respectively  and ADAM metallopeptidase with thrombospondin type 1 motif, 1 (ADAMTS1). We suppressed 2 shRNAs . Conditiectively . Furtherectively . Finallyectively . Since Uectively , we testreatment . Suppres MGa 31L .Taken together, these findings demonstrate that the secretome signature may differ between cells in tumors with intratumoral heterogeneity that are involved in cell-to-cell communication. TGFBI and ADAMTS1 are 2 of these secreted proteins that affect cell proliferation in a coculture setting.2 Several mechanistical studies have recently also highlighted consequences of intratumoral heterogeneity in glioblastoma, where for example subpopulations of neoplastic cells, based on EGFR mutation status, within the same tumor were shown to promote the growth by IL6 signaling.14 In a tumor evolutionary perspective, Ozawa et al.25 have placed glioblastoma gene expression-based subtypes within a framework where a proneural-like precursor is suggested to evolve and give rise to the other glioblastoma subgroups. In connection to this, different expression-based subsets, including stem like or mesenchymal, have been connected to tumor formation capacity related phenotypes.26–28 Furthermore, response to treatment has been connected with glioblastoma subtype transition.29 Together, this highlights the complexity and importance of a deeper understanding of intratumoral heterogeneity.Intratumoral heterogeneity in glioblastoma is an issue that has been studied for a long time and has recently been fueled by novel findings based on single-cell omics.31 In future studies, it will be of importance to investigate if the observed phenotypic differences between the cell lines have a genetic underlying cause since such has recently been described,32 and loss of NF1 has been shown to play a role as a driver of mesenchymal subtype transition.25 But it should also be noted that cells have been described to appear in a spectrum of subtype states, which suggests a more variable situation than simple binomial states.29Here, we used the U-343 model, where the cell lines have been derived from a single glioblastoma, to identify how heterogeneous clones may evolve and phenotypically differ, and how interclonal signaling may exert co-evolutionary consequences. Our results from the U-343 model show that a common confounding clone may have existed that gave rise to phenotypically and genotypically divergent cells. Although it is not unlikely that a field of interacting differentially mutated cells would have been in place already at the initial stage of tumor development.10 Reinartz et al.33 have reported that the subclones from individual tumors exhibited heterogeneity in drug resistance. As we have shown here, U-343 cell lines differ in temozolomide sensitivity. The resistant clone, U-343 MGa 31L, expanded and became predominant after treatment, which is in line with other studies where preexisting clonal temozolomide insensitive populations have been shown to occur.33The heterogeneity caused by tumor evolutionary divergence further raises the question of a need for combinatorial therapy approaches to target different populations. Theoretical models have shown that combinatorial approaches yield better results in diminishing the occurrence of drug resistance.FN1 and SNAI2. This demonstrates that a phenotype transition effect may exist between subsets of cells in a setting of intratumoral heterogeneity.Furthermore, communication between the U-343 cells also occurred via direct cell-to-cell contacts. We demonstrate that the mesenchymal-like cell line U-343 MG stimulates the growth of the non-mesenchymal-like U-343 MGa Cl2:6 cells, and in addition makes them more mesenchymal like with regard to expression of 34 In the coculture experiments, we observed an increased cell proliferation of U-343 MGa Cl2:6 when cocultured with U-343 MG. Ablation of NOTCH1 by CRISPR/Cas9 or shRNA in U-343MGa Cl2:6 lead to increased proliferation of U-343 MGa Cl2:6 in the coculture setting. This suggests that there is an underlying stimulatory effect sent from U-343 MG to U-343 MGa Cl2:6 cells, which is overruled by NOTCH1. But upon removal of the NOTCH1 protection, this effect becomes unleashed. Hence, this growth-promoting function by the U-343 MG cells needs to be further investigated.Here we studied the NOTCH signaling pathway since it has been shown to be involved in glioblastoma initiation.14 In this study, we demonstrated that the different U-343 cell lines indeed affected the proliferation of each other via secreted factors. We used a secretome protein enrichment with click sugars (SPECS), a method developed for secretome analysis, to identify secreted proteins by mass spectrometry without the interference of serum proteins in cell culture.35 In comparison with RNA-seq data, we found a high correlation between identified secreted proteins and the corresponding transcript levels. Candidate proteins that may contribute to the inhibitory effect of U-343 MG on U-343 MGa 31L cells were identified, and TGFBI and ADAMTS1 were selected for testing as potential effectors. In extension, the SPECS results also offer opportunities for studies of further secreted proteins. TGFBI has previously been shown highly expressed in mesenchymal subtype glioblastoma and associated with poor prognosis.36 Few reports are available on ADAMTS1 in glioblastoma, but in other cancer types it has been shown to affect several cancer phenotypes, including proliferation.37 Of note, it is also of importance to investigate the effects of the different secretomes on neighboring non-neoplastic cells, which has not been addressed in this work. For example, secreted factors from glioma cells that have transitioned to a mesenchymal subtype by loss of NF1 have increased capacity to attract microglia.4Intratumoral cell communication has been shown to be important for overall tumor growth by interclonal stimulation.In summary, our study highlights the importance of intratumoral heterogeneity in glioblastoma. We show that genetically distinct tumor cell populations from a single glioblastoma can differ in drug sensitivity and with regard to astrocytic versus mesenchymal features and cell proliferation rates. Furthermore, these heterogeneous cells communicate via both direct cell-to-cell contacts and secreted proteins. An additional analysis of membrane-attached proteins and secreted factors may provide further insights into how glioblastoma cells can intercommunicate and if such factors constitute novel therapeutic targets.vdaa061_suppl_Supplementary_TablesClick here for additional data file.vdaa061_suppl_Supplementary_Material_and_MethodsClick here for additional data file.vdaa061_suppl_Supplementary_Figure_S1Click here for additional data file.vdaa061_suppl_Supplementary_Figure_S2Click here for additional data file.vdaa061_suppl_Supplementary_Figure_S3Click here for additional data file.vdaa061_suppl_Supplementary_Figure_S4Click here for additional data file.vdaa061_suppl_Supplementary_Figure_S5Click here for additional data file.


Here, we used proliferation assay to characterize the plasma sensitivity of fourteen breast cancer cell lines. These assays showed that all tested cell lines were sensitive to NTP. In addition, a good correlation was found comparing cell sensitivity to NTP and radiation therapy (RT), where cells that were sensitive to RT were also sensitive to plasma. Moreover, in some breast cancer cell lines, NTP and RT have a synergistic effect. Adding a dose of PARP-inhibitor olaparib to NTP treatment always increases the efficacy of the treatment. Olaparib also exhibits a synergistic effect with NTP, especially in triple negative breast cancer cells. Results presented here help elucidate the position of plasma use as a potential breast cancer treatment.Cancer therapy has evolved to a more targeted approach and often involves drug combinations to achieve better response rates. Non-thermal plasma (NTP), a technology rapidly expanding its application in the medical field, is a near room temperature ionized gas capable of producing reactive species, and can induce cancer cell death both in vitro and  Each year, more than two million women are diagnosed with breast cancer, the most frequent cancer in women . DependiA new perspective in cancer treatment has come with the use of non-thermal plasma (NTP). Briefly, NTP consists of a partially ionized gas which allows to convert electrical energy into chemical and thermal energy. Usually obtained by applying an electric field to a flowing gas , NTP is BRCA1/2 status of the cell lines.As most studies on plasma oncology examined only a few cell lines to demonstrate the anticancer capacity of NTP, here we use a relatively high-throughput approach to characterize the sensitivity of fourteen human breast cancer cell lines to NTP. Hence, the aim of this work is twofold. First, to determine if the sensitivity of breast cancer cells to NTP is dependent on the molecular subtype. Second, to compare and evaluate the potential combination of NTP with RT and PARP (Poly (ADP-ribose) polymerase) inhibitor olaparib, two other therapies used in breast cancer. By comparing the plasma response of these cell lines to RT sensitivity, we observe a direct correlation between the efficacies of these two DNA damaging agents. Moreover, combining RT and plasma can result in a synergistic effect in a subset of cell lines. In addition, pretreatment with olaparib increased the efficacy of NTP in all tested cell lines. A synergistic effect was also measured for this combination, independent of the Precision oncology involves multidisciplinary approaches to define patient subgroups and adapt the use of various treatment modalities, either alone or in combinations, according to the disease’s sensitivities and patient’s needs. NTP can become a new member of the arsenal to treat patients affected by breast and other cancers. As NTP yield minor side effects ,22, locain vivo, plasma jets are most often used . Ho. Ho24]. γ modes in all cell lines. Cytotoxicity of the jet mode is similar to the cytotoxic effects of RT and in some cases greater than the response to 10 Gy . Additionally, the most radioresistant cell lines, HCC1428 and MDA-MB-175-VII, were sensitive to the jet mode using a sufficient dose of 120 s (p < 0.001).For comparison with plasma treatments, RT was used as a standardized reference for cytotoxic sensitivity of the different cell lines. A 4 Gy reference dose of RT tends to be more cytotoxic than both the Ω and 2 slightly increased the antiproliferative capacity of the treatment, while for other cell lines, the antiproliferative capacity remained unchanged. On the other hand, with the γ mode, addition of O2 to the treatment tends to improve the antiproliferative capacity of plasma for all cell lines. However, the sensitivity to the addition of O2 varies significantly between different cell lines. Comparison of the antiproliferative effect of O2 is illustrated in Another feature of the device is the possibility to inject a secondary gas directly into the plasma effluent or afterglow (via the hollow high-voltage electrode). γ modes is enhanced by the injection of O2 in the high-voltage electrode. However, from the use of GR values, it is possible to quantify the overall sensitivity of the different cell lines. As shown by 2 in the high-voltage electrode is almost inexistent in the Ω mode (p > 0.5 with t-test) but is significant in the γ mode (p < 0.001 with t-test). Even if the injection of O2 in our NTP device is performed downstream (in the effluent or the flowing afterglow region) rather than in the plasma itself, the antiproliferative capacity can benefit from the injection of O2. This feature is in agreement with other studies reporting on the anti-cancer effect of O2 addition to plasma [Results shown in o plasma ,29 but h50  and to RT is shown in 50 B. For ot < 0.005 B. This sWe then determined if sensitivity to plasma treatment can be correlated to a few different clinical aspects of breast cancer. We grouped the cell lines into their different receptor status subtypes . In It is known that NTP can produce RONS , which cγ mode with O2, but both displayed lower antiproliferative capacity than 4 Gy alone . Among the cell lines used, only HCC1569 and MDA-MB-361 contained a BRCA2 mutation. In those cell lines, only 40% (p < 0.001) and 20% (p > 0.05) of growth inhibition was observed, respectively, with olaparib alone. Indeed, the dose used was not enough to reach the synthetic lethality expected in BRCA mutants. Olaparib treatment alone resulted in growth inhibition ranging from 0% to 40% in other cell lines.As shown in In all cell lines, combination with olaparib tends to improve the cytotoxic effect of plasma. Interestingly, CI < 1 for seven out of eleven cell lines, demonstrating a synergistic effect of olaparib and plasma B. This wDNA damage can be visualized through the activation of DNA repair pathways. We used the detection of DNA-damage-associated foci phosphorylation of H2AX (γH2AX) to characterize the effect of our combination at the molecular level.p < 0.01 for HCC1428). In addition, pretreatment with olaparib further increased the number and intensity of foci following a treatment by jet mode and RT (p < 0.05). Interestingly, in some conditions, a strong pan-nuclear staining can be observed in a portion of the cells. This pan-nuclear phosphorylation of H2AX signal may suggest cells potentially going through apoptosis following treatment [As expected, in reatment ,45. AccoThese results indicate that the effect of plasma on cancer cells can be improved by a combination with a DNA repair inhibitor such as olaparib. This reinforces the notion that the ability of NTP to induce cytotoxic effects occurs through DNA damage (whether SSB or DSB), similar to radiation.50 (treatment condition resulting in 50% relative viability) [50 is confounded by its slow cell division rate. The GR method is commonly used in a variety of open access databases compiling the drug sensitivity of different cell lines. The Library of Integrated Network-based Cellular Signatures (LINCS) [In this study, we compared the sensitivity to NTP with RT, a conventionally used modality in breast cancer treatment, on a large set of common breast cancer cell lines. A constraint to large-scale comparison of NTP sensitivity across cell lines is the heterogenous proliferation rates of different cell lines. Our method to quantify cell response to NTP was based on the normalized cell number 6 days post-treatment. Therefore, systematic variations in cell division time will affect relative response metrics such as ICability) . To over (LINCS)  and Phar (LINCS)  are two γ modes (in comparison to the jet mode) to reach a similar antiproliferative capacity turned out to be respected for all cell lines. As previously reported [2 to rare gas NTP increases the cytotoxicity of plasma. However, in the present case, O2 is not injected in the plasma per se, it is rather injected in the effluent region (in the Ω mode) or in the flowing afterglow (in the γ mode). As shown in 2 is significant in the γ mode. This suggests that injection of O2 in the flowing afterglow is sufficient to enhance the production of RONS, a fact in agreement with the enhancement of the O  atomic emission line in 2 in the Ω mode could be attributed to the low energy present in the plasma effluent. However, as 2, the mitigate effect of O2 could indicate that the 777.5 nm atomic line is not a good indicator of plasma antiproliferation capacity (at least with downstream O2 injection) or it could be simply due to the NTP dose that is too low to significantly impact cell proliferation. This will be the subject of further investigations.Beside the use of fourteen cell lines, three NTPs, described previously , have bereported ,29, addiWe previously reported that plasma can induce DNA damage in MDA-MB-231 cell line and drive the cells to undergo mitotic catastrophe . Here, i50 for the jet mode and RT, the doses required for combination could be better defined for each cell line in future experiments. Results from the current study supports the fact that the application of NTP in combination with RT may be complementary and a viable clinical strategy for further exploration.Using the same experimental method to investigate the sensitivity of cell lines to NTP and RT allowed to establish a strong correlation between both modalities’ antiproliferative capacity. This correlation highlights the similarity in the mechanisms of action of these modalities. In addition, using the same procedure, the combination of RT and NTP have also been tested. In agreement with previous work by Lin et al.  in otherOlaparib is approved by the US Food and Drug Administration (FDA) as a therapy for select breast, ovarian and prostate cancers, for which plasma treatment is also being evaluated in vitro ,33,54,55In our experiment, we chose to target PARP, which is mainly involved in SSB repair. Unresolved SSB will generally evolve in DSB, and direct induction of both types of DNA damage by RONS is probably the main mechanism of genotoxic stress by plasma treatment . In the In characterizing the relative sensitivity of a large set of publicly available breast cancer cell lines, this study built a platform to further explore the cellular mechanism of action of plasma. As these cell lines’ genomes have been sequenced ,61,62 an2 according to ATCC recommendations. Cells were used at a low passage number (lower than 25) upon reception from ATCC in order to maintain their parental phenotype and genotype. Genomic characterization of all investigated cell lines is publicly available [Breast cancer cell lines Panel 1  was purchased from the American Type Culture Collection . Cells were grown following ATCC recommendations , with or without insulin, with 1% penicillin-streptomycin (Pen-strep). Cells were incubated at 37 °C with or without 5% COvailable . This inγ mode or 4 min of Ω mode [The convertible plasma device used for the treatment of cells in suspension was described elsewhere . In brief Ω mode .−1 grating blazed at 300 nm with a resolution about 1.5 nm, Ocean Optics, Dunedin, FL, USA). The optical emission spectroscopy system was corrected by its complete response curve using an Intellical lamp  above 400 nm and a 900 W tungsten lamp  below.The optical emission of the discharge was collected by an optical fibre for emission spectroscopy . OpticalCell suspensions were prepared in DMEM containing pyruvate supplemented with 10% FBS and 1% Pen-strep. A fixed volume of 400 µL was dispensed in 1.5 mL microtubes for plasma or radiation treatment. Cell concentration was adjusted according to the different cell lines (ranging from 50 000 to 200 000 cells/mL). Plasma treatment was performed by positioning the nozzle of the device inside the tube at a constant distance of 5 mm from the surface of the media. Cells seeded in a 48-well plate were fixed at day 6 after treatment with a crystal violet solution . Fixed cells were then stained with DRAQ5 in PBS, washed and scanned with the Odyssey imaging system . A dilution curve of various cell numbers stained by DRAQ5 was prepared for each cell line in each experiment. DRAQ5 signal in each well was compared to the dilution curves to quantify the cell numbers in each well .x(c) the cell number of the treated sample at concentration c, x0 the cell number at the time t = 0 s , ctlx the cell number of the control sample at the same time as x(c), according to the following equationDose-response curves were calculated using the GR inhibition metrics . This mex0 value were inferred from the number of cells plated, fixed at 18 h and stained with DRAQ5. GR50 for jet and RT were obtained for different doses in order to obtain dose-response curves.To use the GR metrics with our proliferation assay, TM Quantitative Peroxide Assay Kit  was used according to the manufacturer’s instructions. The optical density at 595 nm was measured with the Spark multimode microplate reader .For hydrogen peroxide detection, cell suspensions were centrifuged after plasma treatment, and the supernatant was collected and processed immediately. Pierce−1) just before imaging.Live cells were followed using IncuCyte S3 Live-cell Imaging System , using a 10× objective. During the incubation period, cells were maintained in 48-well plates with 250 μL of medium per well. Only 20% of this medium was carried from the treated microtube as described in For the combination with PARP-inhibitor olaparib , cells were trypsinized, diluted to the appropriate concentration and pretreated with 2 μM of olaparib for 2 h before plasma treatment (or radiation). Following plasma treatment, 50 μL of the treated cells were transferred to a 48-well plate containing 200 μL of fresh media. Cells pretreated with olaparib were also maintained in media containing 2 μM of olaparib during the six days of incubation. For the combination with radiation, the plasma treatment was performed first and then cells were exposed to 4 Gy of radiation, within 30 min. The Chou-Talalay method  for drugAfter treatment, cells were plated in 8-well chamber slides and fixed 24 h later with 10% formalin for 5 min. Cells were permeabilized with 0.25% Triton for 10 min, incubated in blocking solution  for 1 h and with the primary antibody overnight at 4 °C. Cells were washed and incubated with the secondary antibody for 1 h at room temperature and then washed again. Prolong containing DAPI was used for slide mounting and images were obtained using a Zeiss Observer Z1 microscope . Primary antibody used was phospho-H2AX (Ser139)  and secondary antibody was Alexa fluor-488 (1:750).t-test were used to compare different treatments with the control, to compare the effects of NTP between different breast cancer subtypes and to compare the influence of O2 on the antiproliferative effect of NTP in the γ mode. Pearson’s correlation test was performed to determine the correlation of the antiproliferative effect of NTP and RT. Data analysis, microscopy image analysis and statistical analysis were performed using homemade codes with Mathematica 10 .Student’s γ modes were compared when a small concentration of O2 is injected via the high-voltage electrode. Unlike previous works were O2 is injected within the plasma itself, our plasma device allows the injection of O2 downstream of the plasma. In the γ mode, O2 is in contact with the flowing afterglow and a significant increase of the antiproliferative capacity of the plasma occurs. This effect was observed on all cell lines, and highlights the potential benefit on cancer treatment of the precise control of gas composition via downstream injection.In this study, we were able to classify a large panel of breast cancer cell lines according to their sensitivity to NTP in three different discharge modes. Cell lines that are more sensitive to a discharge mode are also found to be more sensitive to other discharge modes. In addition, the Ω and 50 to compare cell lines classified in the aforementioned subgroups, no subgroup of cell lines could be identified as strongly sensitive or resistant to NTP. Hence this highlights the potential clinical benefit to a majority of patient, should NTP be used intraoperatively to treat the tumour bed after tumour resection.One of the aims of this work was to determine if a particular subtype of breast cancer could be particularly sensitive to NTP. Classification was selected with respect to their molecular subtype  and to their receptor status . Using the GRComparing the sensitivity of cancer cell lines to NTP with their sensitivity to RT, a good correlation was found: cell lines more sensitive to RT are also more sensitive to plasma. Combination of NTP and RT was also found synergistic on a subpopulation of cell lines. These results suggest that adding a relatively low dose of NTP to a patient’s therapeutic plan could allow to reduce the dose of RT required, therefore minimizing side effects without compromising efficacy. In addition, we demonstrated for the first time that NTP can synergistically be combined with olaparib, a PARP inhibitor. Olaparib being more and more used in the clinic, its role as a NTP sensitizer is a very important feature towards the eventual position NTP could hold in the arsenal of breast cancer treatment.