There was no similar pattern of phenotypic alterations among eleven resistant cell lines, including expression/activity of crucial regulators, such as MITF, AXL, SOX, and NGFR, which suggests that patient-to-patient variability is richer and more nuanced than previously described. This diversity should be considered during the development of new strategies to circumvent the acquired resistance to targeted therapies.The clinical benefit of MAPK pathway inhibition in BRAF-mutant melanoma patients is limited by the development of acquired resistance. Using drug-naïve cell lines derived from tumor specimens, we established a preclinical model of melanoma resistance to vemurafenib or trametinib to provide insight into resistance mechanisms. Dissecting the mechanisms accompanying the development of resistance, we have shown that (i) most of genetic and non-genetic alterations are triggered in a cell line- and/or drug-specific manner; (ii) several changes previously assigned to the development of resistance are induced as the immediate response to the extent measurable at the bulk levels; (iii) reprogramming observed in cross-resistance experiments and growth factor-dependence restricted by the drug presence indicate that phenotypic plasticity of melanoma cells largely contributes to the sustained resistance. Whole-exome sequencing revealed novel genetic alterations, including a frameshift variant of RBMX found exclusively in phospho-AKT BRAF and alternative splicing of BRAF transcript, loss of cyclin-dependent kinase inhibitor 2A (CDKN2A) and alterations of genes encoding the components of the phosphoinositide-3-kinase–protein kinase B/AKT (PI3K/AKT) signaling pathway [A constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway due to genetic alterations is detected in the majority of melanomas . Addicti pathway ,12,13,14 pathway . Cell pl pathway . Diverse pathway ,18,19,20V600E) or trametinib . The original drug-naïve melanoma cell lines were previously characterized at the level of cell morphology, proliferation rate, activity of multiple signaling pathways and response to changes in the microenvironment [To address the challenges of extensive variability of acquired resistance mechanisms, we have taken the advantage of melanoma cell lines derived from tumor specimens to obtain their counterparts resistant to either vemurafenib , ERK1/2, phospho-MEK1/2 (Ser217/Ser221), p-AKT (Ser473), AKT, AXL, SOX2, EGFR , BRAF, DUSP6, PARP, SOX10, GAPDH , active β-catenin (dephosphorylated on Ser37 and Thr41) from Millipore or β-actin  were used followed by binding of the secondary HRP-conjugated anti-mouse or anti-rabbit antibodies . The membranes were incubated with Pierce® ECL Western Blotting Substrate , and the proteins were visualized by using ChemiDoc Imaging System , or on a medical X-ray film .Preparation of cell lysates, electrophoresis, protein transfer, and immunodetection were performed according to the procedures described previously ,25. BrieAXL: 5’-GGTGGCTGTGAAGATGA-3′ and 5′-CTC AGA TAC TCC ATG CCA CT-3′; IL-8: 5′-AGG TGC AGT TTT GCC AAG GA-3′ and 5′-TTT CTG TGT TGG CGC AGT GT-3′; MMP-2: 5′-AGC GAG TGG ATG CCG CCT TTA A-3′ and 5′-CAT TCC AGG CAT CTG CGA TGA G-3′; SOX2: 5′-GCT AGT CTC CAA GCG ACG AA-3′ and 5′-GCA AGA AGC CTC TCC TTG AA-3′; SOX9: 5′-GAC CCG CAC TTG CAC AAC-3′ and 5′-TCC GCT CTC GTT CAG AAG TC-3′. Relative gene expression was calculated versus a reference gene RPS17, and efficiency correction for real-time PCR was included.Total RNA isolation, cDNA synthesis, quantitative real-time polymerase chain reaction  procedures and data analysis were described elsewhere . In brieAnalysis of 43 phospho-proteins by using Human Phospho-Kinase Array  was performed according to the manufacturer’s protocol. In brief, drug-resistant cell lines and matched drug-naïve cell lines cultured in the same conditions for 19 h were used to prepare cell lysates. Antibody-coated membranes were incubated with Array Buffer followed by overnight incubation with cell lysates. After three washes, the membranes were incubated with biotinylated secondary antibodies and streptavidin conjugated with HRP. Chemiluminescence signal was developed on X-ray film or by using ChemiDoc Imaging System . Pixel densities of dots were analyzed using a transmission-mode scanner and ImageJ free software. Mean pixel densities of the pair of duplicate spots representing each protein of interest and HSP60 protein (a loading control) were calculated. Relative level of each protein was calculated by using the formula: (mean pixel density of the protein)/(mean pixel density of HSP60). Obtained values were used to prepare color coded matrix.LIVE/DEAD Fixable Violet Dead Cell Stain Kit  was used to exclude dead cells from the analysis. Antibodies against NGFR (PE-conjugated) were from BD Pharmingen . Appropriate isotype controls were included in each experiment. Flow cytometric data were acquired with FACSVerse  and analyzed using BD FACSuite.ELISA kit Quantikine High Sensitivity Human CXCL8/IL-8  was used to determine IL-8 secretion to the culture medium by melanoma cells. Briefly, culture media were diluted 40x, added to each well of the pre-coated plate and incubated for 2 h at room temperature. After 6 washes, the plate was incubated with IL-8 HS Conjugate for 1 h, and washing steps were repeated. This was followed by an incubation with the Substrate Solution, Amplifier Solution and Stop Solution. The optical density of each well was determined using a microplate reader Infinite M200Pro . Concentrations of IL-8 in the medium samples were obtained by applying a four parameter logistic (4-PL) curve fit, and normalized to cell number.http://www.ncb.nlm.nih.gov), and gene expression values were log2-transformed. The AXL expression profiles were developed from paired tumor specimens collected from patients before treatment and post-relapse with acquired resistance either to vemurafenib or combined treatment, dabrafenib and trametinib.The publicly available microarray data sets  were downloaded from the GEO database  derived from six different tumor specimens were used to develop resistance to vemurafenib (PLX), whereas to obtain melanoma cell lines resistant to trametinib (TRA), in addition to BRAFV600E melanoma cell lines, one Harvey rat sarcoma viral oncogene homolog (HRASQ61R) cell line (DMBC17) was employed. After 4–5 months of continuous exposure to increasing concentrations of PLX and TRA finally reaching 10 µM and 50 nM, respectively, six PLX-  and five TRA-resistant cell lines  were obtained. None of two attempts to obtain 12_TRAR and 33_TRAR was successful. 33_PLXR cell line derived from DMBC33, which originally exerted very high doubling time (107.7 h) [BRAF107.7 h) , althougCCND1 expression. Proliferation rates of resistant cells were high even if all PLXR cell lines had significantly lower transcript levels of cyclin D1 than their drug-naïve counterparts, whereas in TRAR cell lines, CCND1 expression was enhanced (11_TRAR), diminished (29_TRAR and 17_TRAR) or remained at similar level (21_TRAR and 28_TRAR) compared with parental cell lines   was even substantially reduced ), which revealed that the development of resistance to vemurafenib or dabrafenib combined with trametinib was accompanied with increased expression of AXL only in selected cases, and the AXL level either remained unchanged or was decreased in the majority of relapse samples  D,E. At t DMBC11) D. The pr reduced E. Althou samples F.SOX2, SOX9, and SOX10 at the transcript levels was compared between drug-naïve cell lines. SOX10 was quite uniformly expressed in all drug-naïve cell lines relative to the median value , and several substitutions in RAS guanyl-releasing protein 4 (RASGRP4) and son of sevenless homolog 1 (SOS1) were found in 21_PLXR and 28_PLXR cells  or was substantially diminished (11_TRAR and 12_PLXR cells) during the development of resistance  and hepatocyte growth factor (HGF), and lack of them in the culture medium do not affect cell morphology and activity of several signaling pathways in patient-derived melanoma cells . In thisAR cells B, which AR cells C. TRAR cAR cells C and 17_AR cells C. In conAR cells C.low resistant cell lines, 21_PLXR and 28_PLXR . 21_PLXR, 21_TRAR, and 28_PLXR cells were used for comparison. The activity of EGFR downstream signaling pathways remained unaffected in EGFR 28_PLXR D. In con 28_PLXR D. Notabl 28_PLXR D. Furthe 28_PLXR D. Result 28_PLXR E.217/221 (BRAFwt/HRASQ61R). Lack of cleaved PARP indicates that apoptosis was not induced in any of cell lines  and drug-naïve cells after short treatment (immediate response) were performed. Gene expression at the transcript and protein levels as well as activity of corresponding proteins were (re)-analyzed based on results of the present report and previously published data ,27 and sS473 also diverse in drug-naïve cells , remained unchanged (29_TRAR) or was significantly enhanced , whereas during immediate response the percentages of NGFR-positive cells were increased in all melanoma cell lines although to different extent , AXLhigh (invasive), and NGFRhigh . In acquired resistance phase, about 70% of melanomas present the AXLhigh phenotype and only about 23% are enriched with the MITFhigh phenotype. Our present study as well as previously published [high/AXLlow (five cell lines) or MITFlow/AXLhigh (two cell lines). Among eleven resistant cell lines, six vemurafenib-resistant and five trametinib-resistant, none became MITFlow/AXLhigh , MITFlow/AXLlow/NGFRlow , MITFlow/AXLlow/NGFRhigh (11_PLXR), MITFmedium/AXLlow/NGFRhigh , patient-to-patient variability might be richer and more nuanced than previously suggested. Especially interesting is MITFlow/AXLlow/NGFRlow phenotype of 28_PLXR cell line. This is vemurafenib-resistant cell line, in which dedifferentiation, shown as inhibition of expression of MITF and MITF-dependent pigmentation-related genes, is irreversible [FGFR, FMS-like tyrosine kinase 3 (FLT-3) and ERBB4, which is exclusively observed in this cell line. Moreover, enhanced activity of STAT3 followed by increased expression of SOX2, which were observed only in 28_PLXR cells could be also considered as the mechanism supporting cell survival. It has been demonstrated that SOX2 as a part of STAT3-SOX2-CD24 axis rescued melanoma cells against acute exposure to vemurafenib [BRAF or NRAS) was present [SOX2 expression in 29_TRAR and 17_TRAR cells were less pronounced probably because of an elevated MITF level and high frequency of differentiated cells [The plasticity of melanoma as enabler of drug resistance was a subject of most recent reviews ,37,53. Aublished , which btudy and ). Thus, tudy and ,34,35. Ttudy and ,34,35, ahibition ,60, but hibition . Our stuhibition ,63, it chibition . Therefoversible . In the versible ,66,67, dNGFRhigh _PLXR, MIversible . In 28_Purafenib . SOX2 wa present . Recipro present , which wed cells . SOX2 leed cells , vemurafed cells ,75,76,77CCND1 expression, including upregulation, was found in resistant cells. The stop-gained variants of CDKN2A were acquired by two vemurafenib-resistant cell lines, 21_PLXR and 28_PLXR, and a possibly damaging D148N variant of E2F transcription factor 3 (E2F3) was additionally found in 21_PLXR cells  [Proliferation curves of resistant cells and their drug-naïve counterparts were similar. While immediate response to PLX and TRA was associated with reduced cyclin D1 transcript levels in all drug-naïve cell lines , variablXR cells , but the of BRAF  and CCNDF [CCND1 , respect4 (CDK4) . Interes4 (CDK4) . In the 4 (CDK4) , IL-8 thF57C substitution exerted phospho-MEK2low/phospho-ERK1/2high signature [P124L variant [P124S or MEK1P124L variants faced rapid progression upon administration of BRAF inhibitor [Reactivation of the MAPK (ERK1/2) pathway ,81,82 orignature , (ii) re variant , and  TRAR cells neither expressed SOX2 protein at the detectable level nor activated JNK as shown by the phospho-protein array. In addition, mutually exclusive expression of MITF and EGFR was reported [low [EGFR expression in both 21_TRAR and 28_TRAR cells. Moreover, as deprivation of exogenous EGF was enough to trigger inhibition of EGFR signaling, production of EGFR-activating ligands in resistant melanoma cells was insufficient. Addiction to EGFR activation was, however, advantageous to melanoma cells only in the presence of drug since the cessation of trametinib treatment led to a growth factor-independent maintenance of TRA-resistant EGFR-expressing cells. This was an immediate effect, which was presumably due to a high plasticity of melanoma cells, and not as a result of depletion of EGFRhigh subpopulation as reported in BRAF inhibitor-resistant cells [EGFR is expressed in epithelial cells and its overexpression is found in cancers of epithelial origin [EGFR [EGFR expression is more frequent in metastatic melanoma [A high plasticity of resistant melanoma cells was also detected as the rapid regrowth and reprogramming of EGF-dependent EGFRurafenib ,104,105,of SOX10 ,103 follof SOX10 , and SOXof SOX10 . It is ureported ,102, andted [low , howeverted [low . In addited [low . Thus, al origin , whereasin [EGFR . Positivin [EGFR . EGFR exmelanoma  and has melanoma . In addimelanoma . Short tmelanoma ,115. OurThis in vitro study was performed to characterize melanoma cells that developed resistance to vemurafenib or trametinib after 4–5 months of treatment. A high variability of molecular and cellular changes was observed, as there was no similar pattern of genetic/non-genetic alterations among eleven investigated resistant cell lines, including phenotypic changes in expression/activity of crucial melanoma regulators e.g., MITF, AXL, NGFR, AKT, ERK1/2, and SOX2 . Moreove


Muir-Torre syndrome (MTS) a genetic disorder characterized by predisposition to cutaneous neoplasms. Lipedematous scalp is characterized by the presentation of a thick, sponge-like scalp due to the altered and increased deposition of adipose tissue. We present a case of Muir-Torre-associated sebaceous carcinoma of the scalp consistent with a lipedematous scalp. Sebaceous carcinomas are rare among the general population and are known to be characteristic of Muir Torre Syndrome (MTS). MTS is a genetic disorder that is characterized by a predisposition to sebaceous skin cancers, keratoacanthomas, as well as visceral cancers, most commonly gastrointestinal and genitourinary cancers; it is considered a variant of hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome. Muir originally described MTS in 1967 with Torre reporting a patient in 1968 ,3. The sOriginally described by Combleet in 1935, the term ‘lipedematous scalp’ was not introduced until Lee described it in 1994 in an attempt to separate it from lipedematous alopecia . When a A healthy 37 year old African American female with no significant past medical history presented to the plastic surgery service for treatment of a recently diagnosed sebaceous carcinoma of the scalp. She denied any personal or family history of previous skin cancers or other malignancies. On first exam, the thickness of her scalp was not initially appreciated. However, during the surgical procedure, her scalp was noted to have a ‘sponge-like’ consistency and was noted to be thicker than normal tissue. An increased egress of fluid or increased bleeding was not noted during the operation. Due to the concern for possible satellite metastasis, a biopsy of her scalp was taken from the area inferior to the excision site  that code for proteins that function to correct errors that occur during DNA replication [MYH gene which encodes base excision repair [MTS is a rare genetic condition that predisposes to sebaceous skin conditions and visceral cancers, most frequently large bowel, endometrium, genitourinary tract, breast, and ovarian. The condition most commonly arises in an autosomal dominant inheritance pattern with a mutation in a DNA mismatch repair (MMR) gene – principally n repair .th and 7th decades of life, and women are more commonly affected than men, comprising about 70% of patients diagnosed with sebaceous carcinomas [The most common sebaceous neoplasms in MTS are sebaceous adenomas, sebaceous epitheliomas, and sebaceous carcinomas with varying reports of frequency. Sebaceous adenomas as the initial presentation for MTS have reported associations from 25% to 60%. Sebaceous carcinomas in patients with MTS have reported frequencies of 36–100%, while sebaceous epithelioma associations range from 31% to 86%. Sebaceous neoplasms are rare among the general population and are the most defining characteristic of MTS. Specifically, sebaceous carcinoma represents less than 1% of all cutaneous malignancies and 17% of all sebaceous neoplasms in the general population ,11. Althrcinomas . Twenty-rcinomas . Extraocrcinomas ,5,12.Clinical presentations of sebaceous carcinomas are varied, and it is known for masquerading as common benign conditions. In a study of 31 patients with extraocular sebaceous carcinoma, the most common presentations were blepharoconjunctivitis (32%), eyelid masses (29%), or eyelid thickening (26%), with regional lymphadenopathy accounting for 3% of initial presentations . ExtraocSebaceous carcinomas have the ability to invade and produce metastases; therefore, it is often recommended to excise with 5–6 mm margins. Lymph node investigation is sometimes recommended for larger and more invasive tumors due to the 3–28% risk for local metastasis . ColonosAnother condition seen in our patient was the presence of a lipedematous scalp (LS). LS as well as lipedematous alopecia (LA) are rare conditions that are, as stated previously, most often seen in African-American women. Due to the increased proportion of women affected, hormonal factors have been hypothesized to have some level of influence . TheorieLipedematous scalp is characterized by a thickened subcutaneous and adipose layer in the scalp that presents clinically as a soft, almost sponge-like consistency to the scalp with thickened areas mainly in the scalp vertex and occiput. The lipedematous scalp is measurably thicker ∼10–16mm) than the normal scalp (∼5–6mm) as can be seen in our biopsy specimens above in mm than tSebaceous carcinomas are rare and, when present, should encourage the investigation into possible connections with syndromes such as Muir-Torre Syndrome. MTS is associated with an increased risk for other malignancies, especially GI/GU, and warrants a thorough investigation to rule out the presence of any other tumors. A lipedematous scalp is a very rare finding that has only recently been reported and does not appear to have any connection with sebaceous carcinoma or MTS. We therefore present this unique case that has two rare skin related conditions, sebaceous carcinoma associated with MTS and a lipedematous scalp.Sebaceous carcinomas are associated with patients that have Miur-Torre syndrome.Muir-Torre syndrome places patients at risk for malignancy, especially GI/GU malignancies and they require more surveillance than the general population.A lipedematous scalp may be one way of presentation of sebaceous carcinoma, especially in regards to Muir-Torre patients.


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.