Xeno-free generation of human embryonic stem cells (hESCs) is important to prevent potential animal contaminations in culture for advanced cell-based therapeutic applications. Xeno-free production of hESCs is the first step for manufacturing clinical-grade hESC lines.To produce new hESC lines in xeno-free condition.This lab resources report was conducted at Stem Cell Biology Research Center, Yazd, Iran from 2019-2022. 4 new hESC lines from 11 (10 fresh and 1 frozen) donated surplus discarded human embryos were established. In this study, we report the xeno-free derivation of new Yazd hESC lines (Yazd4-7), without using immunosurgery, by culturing intact zona-free blastocysts obtained from discarded embryos onto the YhFF#8 cells as a feeder layer in a microdrop culture system. The pluripotency gene expression profile of the cell lines was assessed by reverse transcription polymerase chain reaction and the expression of specific surface markers was detected using immunofluorescent staining. In vitro differentiation was induced using embryoid body formation and gene expression profile of 3 germ layers and germ cells. Reverse transcriptase polymerase chain reaction was investigated to prove their pluripotent capacity.In sum, we have been able to generate 4 new hESC lines (Yazd4-7) from 11 discarded embryos in xeno-free culture conditions using a micro drop culture system and YhFF#8 as a human source feeder layer.The outcome of this work can be the foundation for the future allogeneic cell-based therapeutic application using clinical grade good manufacturing practice-derived hESC derivatives. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells are 2 major types of human pluripotent stem cells (hPSCs) that do not have any counterpart in vivo (1) and produce an in vitro cell culture artifact from different origins (embryos and somatic cells). These pluripotent stem cells have an unlimited self-renewal capacity in culture and pluripotency potential to differentiate the cells from all 3 germ layers and germ cells (2). These properties have made them good candidates as raw materials for producing allogeneic and autologous clinical-grade cells, tissues, and organs for cell-based therapeutic applications and regenerative medicine .Current progressions in understanding the clinical potential of hPSCs and their derivatives (cellular and extracellular) have raised concerns about the requirements for the derivation of clinical-grade hPSCs in reproducible, fully defined, xeno-free culture conditions (5).In the case of hESCs, for generation of these pluripotent cells in a xeno-free condition should get rid of animal products such as animal-derived antibodies and mouse embryonic fibroblasts (MEFs), which are used in derivation during immunosurgery and expansion procedures. Successful derivation of hESCs in xeno-free conditions is the first step for producing these cells in a good manufacturing practice condition to generate cells and their derivatives in clinical-grade standards, which is critical in progressing stem cell therapies (6-8). To ensure the safety of the hESCs derivatives for the ultimate cell-based therapeutic cures, all potential risks, such as animal pathogens infection transmission by animal-derived materials, must be removed during the procedures . Therefore, all phases of the manufacturing of hESC lines should prevent the usage of animal cells and animal-origin materials while doing derivation, expansion, and differentiation of hESC lines in culture medium and processes. Otherwise, such xeno-contaminated hESCs and their derivatives are typically known as inappropriate raw materials and final products for transplantation due to the hazards of zoonosis infections which may not be rejected by the immune system .One of the sources of animal pathogen infections is MEFs, which have been used as feeder layers during the derivation and expansion of hESCs. Alternatively, several human-originated feeder layers exist such as fetal muscle, fetal skin, adult fallopian tubal epithelial cells, and foreskin fibroblasts . The hESC lines derived using various sources of human feeder layers display similar features to those generated on MEFs, with distinct morphological characteristics being small and roundish with a high ratio of nucleus to cytoplasm while expressing specific markers (15).Moreover, it has been demonstrated that hESC lines cultured on human feeders are more feasible to expand with less spontaneous differentiation than those cultured in feeder-free culture conditions . The other way of animal pathogen transmission during the generation of hESC lines are antibodies developed in animals against human antigens used during immunosurgery procedures for the derivation of hESCs. It has been reported that by using a microdrop culture system, hESC lines can be derived without the need of immunosurgery . In the current study, we are reporting the generation of new Yazd hESC lines (Yazd4-7) from discarded embryos (19) using a microdrop culture system  onto the Yazd human foreskin fibroblasts #8 (20) feeder layer. The cells were characterized using specific markers following derivation and expansion. Their pluripotency capacity was evaluated by applying in vitro differentiation in monolayer culture and embryoid body (EB) formation as the most obvious alternative to the teratoma assay (21).This study aimed to set up the generation of new hESC lines in xeno-free conditions before establishing a good manufacturing practice construction to generate clinical-grade hESC lines and their derivatives for future regenerative medicine applications.This lab resources report was conducted at Stem Cell Biology Research Center, Yazd, Iran from 2019-2022. 4 new hESC lines from a total of 11 (10 fresh and 1 frozen) donated surplus discarded human embryos were established.Chemicals and culture media (with their supplements) were obtained from Sigma Aldrich  and Invitrogen-Gibco (UK) unless otherwise stated.For the derivation of new Yazd hESC lines (Yazd4-7) in a xeno-free condition, Yazd human foreskin fibroblasts batch 8 (20) were used as human feeder following mitotically inactivation with 4 hr of mitomycin-C treatment. Microdrops of feeders were prepared as explained elsewhere .To prevent using animal antibodies for inner cell mass isolation in immunosurgery procedure, whole zona-free blastocyst culture into the microdrop culture system was applied. The zona-pellucida (ZP) was removed by treating the human embryos with pronase  for 3-5 min, depending on the ZP thickness and stiffness. Followed by washing, the ZP-free human embryos were cultured into the microdrops coated with mitotically inactivated YhFF#8 cells as a human feeder layer. Depending on different factors, mainly, the blastocyst quality, between days 3-8 following seeding ZP-free blastocysts in microdrops of YhFF#8, the initial outgrowths were observed. Within some of the initial outgrowths, distinguished clumps of hESCs (18) were observed, then by using pulled glass pipettes they were mechanically cut and pasted into the new microdrops of feeders. Following 2 or 3 passages, the colonies appeared flattened, and hESCs were observed in a unique morphological feature. The new lines were expanded using mechanical passaging in microdrop, and their EB formation capacity was evaluated using nonadherent culture conditions similar to what was explained elsewhere (14). New cell lines were frozen using the vitrification method for producing master and working cell banks for each cell line.The expression of pluripotency markers and genes by the newly derived Yazd hESC lines (Yazd4-7) was assessed using immunofluorescent (IF) staining and reverse transcriptase polymerase chain reaction (RT-PCR). IF and RT-PCR were performed according to the manufacturer's instructions (14). Antibodies used for IF are listed in table I. RT-PCR was done using the cDNA synthesis and primers for different genes (Table II). The chromosomal content of the cells was evaluated using G-banding karyotype as explained elsewhere (14). The pluripotency capacity of the cell lines was evaluated by differentiation in vitro assay (21) using spontaneous differentiation in monolayer culture and EB formation similar to that explained elsewhere (14). Primers that were used to evaluate pluripotency are listed in table II.This study was ethically approved by the Ethics Committee of the Shahid Sadoughi University of Medical Sciences, Yazd, Iran (Codes: IR.SSU.REC.1394.104 and IR.SSU.REC.1394.105). All embryos were coded to ensure anonymity. Moreover, the derivation and characterization procedures of the cell lines were done according to the guidelines and the ethical considerations. Informed written consent was obtained from every infertile couple.From 11 discarded human embryos, 4 new hESC lines (Yazd4-7) were derived on neonatal human foreskin fibroblasts (20) as feeders using microdrop culture conditions . The establishment of each cell line is summarized in figure 1. Master and working cell banks were established for each cell line using vitrification (14). Cell lines were thawed to check their survival and expansion capacity following vitrification.4 new hESC lines were derived in a xeno-free condition using YhFF#8 as a human feeder and microdrop culture instead of immunosurgery. Each cell line was characterized using specific markers and assessment of their gene expression profile during undifferentiated and differentiated conditions to prove their pluripotency. The cells in an undifferentiated state showed distinct morphological characteristics with a high nucleus: cytoplasm ratio with distinct nucleoli (16).NANOG, SOX2, and OCT4/POU5F1  by the cells. Moreover, G-banding karyotyping showed that Yazd4 cells contained normal 46, XY chromosomal content . As it is shown in figure 2L, differentiation in vitroassay (15) by EB formation and gene expression analysis for 3 germ layers .IF results have indicated that undifferentiated Yazd4 hESCs express specific surface markers such as SSEA4 , TRA-1-60 , and TRA-2-49 , while RT-PCR data confirmed the expression of pluripotency genes POU5F1, NANOG,and SOX2 . The karyotype of Yazd5 hESCs was normal 46, XX as revealed by a G-banding . The pluripotency capacity of Yazd5 cells was proven by in vitro differentiation assay to 3 embryonic germ layers  and germ cells (VASA and DAZL) using EB formation after 4, 7, and 14 days .Undifferentiated Yazd5 colonies showed positive expression for surface markers such as SSEA4 , TRA-1-60 , TRA-2-49 , and pluripotency genes such as POU5F1, NANOG, and SOX2 . Yazd6 hESC line was a normal 46, XX cell line as shown by G-banding . Following EB formation after 4, 7, and 14 days Yazd6 cells showed positive expression of 3 embryonic germ layers  and germ cells (VASA and DAZL) and shown in Figure 4L.Similarly, undifferentiated Yazd6 hESC colonies displayed specific characteristics such as SSEA4 , TRA-1-60 , and TRA-2-49  while expressing pluripotency genes POU5F1, NANOG, and SOX2 as pluripotency genes  while displaying normal 46, XY karyotype . Cells within differentiated EBs of Yazd7 have shown the expression of 3 germ layers  and germ cells (VASA and DAZL) as confirmed by RT-PCR .Likewise, Yazd7 hESCs showed positive expression of SSEA4 , TRA-1-60 , and TRA-2-49  markers and 4 new hESC lines (Yazd4-7) were generated using discarded embryos (19) in a xeno-free condition using YhFF#8 as a human feeder and established following characterization with specific markers and genes in undifferentiated status. The pluripotency of the new hESC lines was assessed by in vitrodifferentiation and gene expression profile analysis for 3 germ layers and germ cells (21). Master and working cell banks were established using vitrification method (14).Since the first derivation of hESCs by Thomson and colleagues , scientific societies, and policymakers in health (24) were concerned regarding the safety of hESCs and their derivatives besides their effectiveness during their manufacturing procedures (25). Conventionally hESCs were derived using immunosurgery onto the mitotically inactivated MEFs as feeder layer  due to the presence of the animal antibodies and mouse feeders, there was the risk of animal source pathogens infection transmission into the products (26).The alternative process for immunosurgery is using whole zona-free blastocyst seeding onto the feeders in a microdrop culture system , which was used to derive xeno-free Yazd hESCs (Yazd4-7).On the other hand, different sources of human cells are used as feeder layers to replace MEFs. Human foreskin fibroblasts (hFFs) isolated from infant foreskin are the most popular source of human feeders, which have been used for expansion (14) or derivation of hESCs . Besides the xeno-free concern, hFFs lifespan is longer than MEFs before entering the decline phase in growth and propagation and starting senescence .Some groups produce hFFs in their labs (20), and others buy the commercially available hFFs (27) as human source feeder cells to propagate and derive hESCs. Here, we have used Yazd human foreskin fibroblasts #8 (YhFF#8), produced in our center (20), as a human feeder source. Before using YhFF#8 as a feeder for the derivation procedure, their capacity to support the expansion of the hESCs in an undifferentiated state was tested using already derived hESCs on MEFs .In sum, 4 new hESCs (Yazd4-7) were derived in xeno-free conditions avoiding animal products during the manufacturing procedure to set up the first step of the generation of clinical-grade hESCs for future applications in regenerative medicine. The cell lines were characterized morphologically as they have shown a high nucleus: cytoplasm ratio and dominant nucleoli (16).Furthermore, cells displayed specific markers and pluripotency gene expression profiles, as shown in the results. All the cell lines contained normal chromosomal content. In vitro differentiation assay (21) using EB formation and gene expression assessment for 3 germ layers and germ cells indicated the pluripotency capacity of the xeno-free Yazd hESC lines (Yazd4-7).Moreover, the rate of the hESC lines derivation success from discarded embryos  and testing them as a feeder layer for propagation and expansion of Yazd hESCs , new hESC lines (Yazd4-7) were generated on YhFF#8 feeder layer in a microdrop culture condition with KOSR/HES without immunosurgery as a xeno-free condition. Xeno-free derivation of hESCs, as the first step of clinical-grade manufacturing of hESCs, will lend itself toward producing good manufacturing practice -grade hESCs in its suitable hygiene construction.The authors declare that there is no conflict of interest.


Compounds interfering with glucose uptake and NAD metabolism are potential candidates for cancer therapy. Tumor cells are sensitive to such compounds in terms of glucose uptake as well as cell proliferation and survival. We have carried out these types of studies to neuroendocrine tumor (NET) cells, i.e., pancreatic Bon-1 and QPG-1 NET cell lines and GLC-2 and GLC-36 small cell lung cancer (SCLC) cell lines. Cells treated with substances that provoke a chemical inhibition of facilitative glucose transporters (GLUT) and nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in NAD metabolism, lead to decreased cell proliferation and increased cell death.(1) Background: the potency of drugs that interfere with glucose metabolism, i.e., glucose transporters (GLUT) and nicotinamide phosphoribosyltransferase (NAMPT) was analyzed in neuroendocrine tumor  and small cell lung cancer  tumor cell lines. (2) Methods: the proliferation and survival rate of tumor cells was significantly affected by the GLUT-inhibitors fasentin and WZB1127, as well as by the NAMPT inhibitors GMX1778 and STF-31. (3) Results: none of the NET cell lines that were treated with NAMPT inhibitors could be rescued with nicotinic acid , although NAPRT expression could be detected in two NET cell lines. We finally analyzed the specificity of GMX1778 and STF-31 in NET cells in glucose uptake experiments. As previously shown for STF-31 in a panel NET-excluding tumor cell lines, both drugs specifically inhibited glucose uptake at higher (50 μM), but not at lower (5 μM) concentrations. (4) Conclusions: our data suggest that GLUT and especially NAMPT inhibitors are potential candidates for the treatment of NET tumors. Neuroendocrine tumors (NETs) derive from neuroendocrine cells that in turn are descendants of the embryonic neural crest or the endodermal layer ,2. InterDependent on the tumor type and stage different treatment strategies are available for NETs, comprising surgery, chemotherapy (as the application of the multi-tyrosine kinase inhibitor sunitinib and the mTOR inhibitor everolimus in metastatic pancreatic NETs) or peptide receptor radionucleotide therapy (PRRT)  . Recentl177Lu-DOTATATE treatment in nude mice that had been xenografted with the human neuroendocrine cell line GOT1 -fluoro-deoxyglucose  was kindly supplied by Advanced Accelerator Applications .Fasentin, WZB117, and STF-31 were from Merck Millipore. GMX1778 was obtained from Selleck Chemicals and nicotinic acid (NA) from Sigma. -FDG (specific activity of 2.5 to 5.0 GBq/µmol) were added in 100 µL per well for 30 min. Afterwards, radioactive FDG containing medium was discarded and cells were rinsed twice with PBS and subsequently solubilized in PBS containing 2% Triton X-100. Glucose uptake was detected by measuring radioactivity using a gamma-counter. Values were matched according to the decay of fluorine-18.For glucose uptake assays 1.5 × 10p-values < 0.05 were considered to be statistically significant.For statistical data analysis, the one-way-ANOVA and the posthoc Tukey’s multiple comparison test were used with GraphPad Prism 6 Software . NET cell lines Bon-1, QPG-1 (pNET cell lines), or GLC-2 and GLC-36 (SCLC cell lines) were cultivated for four days in the presence of increasing concentrations of fasentin, GMX-1778, STF-31, or WZB to analyze cell proliferation and survival. In all cell lines, a strong impact of fasentin A became In this context, we analyzed the expression level of NAMPT and NAPRT in NET cells . WhereasWe next tried to rescue the effect of NAMPT inhibition via the addition of nicotinic acid. Supplementation with NA allows NAD+ generation only when the Preiss–Handler salvage pathway is intact  and, in particular, needs the expression of functional NAPRT . In all 18F]-FDG in the absence of inhibitors to analyze glucose uptake efficiency. After an incubation time of 30 min with [18F]-FDG in serum- and glucose-free medium, the uptake rate varied by a maximum of about factor two between the cell lines, but depicted no statistical difference (not shown). These kinds of variations are also not reflected by differences in the expression level of GLUT1 transcripts as revealed by real time RT-PCR analysis (not shown).We have previously shown that the GLUT-1/NAMPT inhibitors used in the present study interfere with Glucose-uptake in a drug- and cell-line-specific manner . To expaWe next examined if GLUT or NAMPT inhibitors interfered with the cellular uptake of glucose .Fasentin (100 µM) inhibited glucose uptake in the range of 30–40%, with the exception of GLC-2 cells where an inhibition rate around 70% was observed. Whereas 50 µM GMX1778 led to a pronounced inhibition of glucose uptake in the range of 90%, no inhibition took place at a concentration of 5 µM as already shown for other types of tumor cells [2 via the tricarboxylic acid cycle [Tumor cells possess particular metabolic properties adapted to cancer-specific features, especially regarding replicative immortality . Dysreguid cycle . Hence, id cycle ,28. Thisid cycle ,29,30. Bid cycle . Recentlid cycle . Moreoveid cycle  have preid cycle . Therefoid cycle . The firid cycle . The ATPid cycle . Furtherid cycle . Hence, The GLUT inhibitors fasentin and WZB117 showed similar effects on neuroendocrine and small cell lung cell tumor cells: both substances negatively affected the cell numbers only at higher concentrations (50/100 µM). This observation is in good agreement with results of our group on cell lines of different tumor entities. Although the cellular response varied between cell lines after treatment with those GLUT inhibitors, a dramatic effect was shown only at higher concentrations (50/100 µM) ,30. GMX-Cellular glucose uptake is reduced after treatment of Bon-1, GLC-2, GLC-36, and QPG-1 by all four substances. The inhibition profile between each cell line does not differ significantly. The results are comparable with data from our recent study . StimulaTo summarize, GLUT and NAMPT inhibitors negatively affect cell numbers of NET cells. Resistance to NAMPT inhibitors depends on the expression level of NAPRT and thus on the predominant NAD producing Preiss–Handler pathway. Finally, our data suggest that GLUT and especially NAMPT inhibitors are potential candidates for the treatment of NET tumors.


Brain tumours are the leading cause of cancer-related death in children, and there is no effective treatment. A growing body of evidence points to deregulated epigenetics as a tumour driver, particularly in paediatric cancers as they have relatively few genomic alterations, and key driver mutations have been identified in histone 3 (H3). Cancer stem cells (CSC) are implicated in tumour development, relapse and therapy resistance and thus particularly important to target. We therefore aimed to identify novel epigenetic treatment targets in CSC derived from H3-mutated high-grade glioma (HGG) through a CRISPR-Cas9 knockout screen.The knockout screen identified more than 100 novel genes essential for the growth of CSC derived from paediatric HGG with H3K27M mutation. We successfully validated 12 of the 13 selected hits by individual knockout in the same two CSC lines, and for the top six hits we included two additional CSC lines derived from H3 wild-type paediatric HGG. Knockout of these genes led to a significant decrease in CSC growth, and altered stem cell and differentiation markers.UBE2N, CHD4, LSM11, KANSL1, KANSL3 and EED) were validated individually thus demonstrating their importance for CSC growth in H3-mutated and wild-type HGG. These genes should be further studied and evaluated as novel treatment targets in paediatric HGG.The screen robustly identified essential genes known in the literature, but also many novel genes essential for CSC growth in paediatric HGG. Six of the novel genes (The online version contains supplementary material available at 10.1186/s13148-023-01498-6. H3F3A, DAXX and ATRX for paediatric HGG) [Cancer is the second most common cause of mortality in children in developed countries , and theric HGG) . Mutatioric HGG) . The mutric HGG) . The WHOric HGG) , 13. DNAric HGG) , 14–16. ric HGG) , which aric HGG) –21.UBE2N, CHD4, LSM11, EED, KANSL1 and KANSL3 are essential for CSC growth and suggest them as therapeutic candidates in paediatric HGG.The Nobel Prize winning method CRISPR  provides unprecedented ease of alterations in the genome –24. Multr = 0.7 and 0.6, respectively, for GU-pBT-7 and GU-pBT-19; Additional file Two CSC lines derived from H3 K27-mutated paediatric HGG were used for a CRISPR knockout screen with an epigenetic/chromatin modifier library , where aPCNA, CDK1 and PLK1, further demonstrating the validity of the screen results. However, these genes have been termed as essential for all cells and studied extensively [CHD4, a chromatin-remodelling factor that is part of the NuRD (nucleosome remodelling and deacetylase) complex, EED, part of the polycomb repressive complex 2 (PRC2) complex, and UBE2N , an ubiquitin-conjugating enzyme that promotes the loss of tumour suppressor p53 [The hit list in our screen reassuringly contained many well-known cancer genes such as ensively , 26, 29,ensively  , a known stem cell marker required for self-renewal of glial stem cells [SOX2 vastly decreased their proliferation  instead of lentivirus, since RNPs are highly efficient, yield faster cleavage as the cell does not need to produce the components and reduce off-target effects , 32. Firem cells , was knoem cells  analysisKANSL3, whereas others, e.g. UBE2N, gradually decreased the cell growth  that yielded the largest effect on cell number in both cell lines were selected for further evaluation. We knocked out the six genes in two additional CSC (both derived from H3 wild-type paediatric HGG) and also in two NSC lines. The gene knockouts had a significant effect on these CSC as well (except for EED), demonstrating a robust effect on cell proliferation/viability in a range of CSC lines regardless of the H3 mutation state  and morphology . The total cell number during the experiment period (one month) was normalised against the negative controls . Twelve of the 13 genes (92%) were successfully validated, as knockout of these genes elicited a large decrease in cell number in both cell lines (significant decrease in both cell lines for 10/13 genes), thus validating the genes’ importance for CSC growth in H3K27M HGG Fig. A. The efOn-target cleavage effect at the intended loci and potential off-target effects at two of the top putative genome locations were evaluated with ICE analysis approximately 10 days after knockout. The on-target cleavage efficiency was > 80%, and no off-target effects were observed . Fifteen additional proteins were predicted as druggable by ligand-based assessment. A web-based search revealed that 35 of the hits had commercially available inhibitors. One of these was directed against the candidate UBE2N (NSC697923 inhibitor), and we therefore tested it on four CSC (two H3 wild-type and two K27 mutated) and two NSC lines. The UBE2N inhibitor promisingly had a large effect on all tested CSC lines, killing all cells at the highest examined concentrations  tolerated it significantly better than all CSC  have previously been labelled as “core essential”, “fitness genes” or “pan-cancer” genes [PLK1 has previously been reported as essential in paediatric midline gliomas [Several of the hits in our screen . The findings suggest that these three genes are essential for CSC, and as such potential drivers of the tumour and represent novel treatment targets in HGG. We also tested the commercially available UBE2N inhibitor, NSC697923, and it efficiently killed all CSC, further verifying the importance of UBE2N in CSC and HGG. The median effective value of the inhibitor (ED50) was significantly lower in the two H3K27M cell lines (GU-pBT-7 and GU-pBT-19). The inhibitor seemed to induce a toxic response in CSC as well as NSC, even though one of the NSC lines was significantly more resistant to the inhibitor than the CSC, particularly the H3K27M-derived CSC. Such rapid decrease in cell growth was not observed in the cells after knockout of UBE2N, where instead a decrease in proliferation and SOX2 together with an increase in GFAP was observed, indicative of cell differentiation. These changes were also seen after knockout of the other candidate genes suggesting that loss of these candidate proteins may induce differentiation of the CSC and contributing to the slowed growth that we observed.Six of our hits were knocked out in NSC, and we noted, in all cases, trends that the NSC tolerated the knockout better than the CSC. The difference was significant for three of the genes  1 complex that regulates gene transcription through, for example, histone acetylation . KANSL1 n cancer  and mutan cancer . UBE2N ilymphoma . Intereslymphoma , 47. Inhlymphoma , and a sbinostat . CHD4, le of AML , and reqe of AML . EED, a  complex . EED washood HGG .The optimisation of our pipeline for individual knockout in paediatric patient-derived CSC was essential to routinely obtain high DNA cleavage efficiencies (> 80%), similar to previous publications using RNP , 55, andUBE2N, CHD4 and LSM11. Our results suggest that the six candidate genes UBE2N, CHD4, LSM11, KANSL1, KANSL3 and EED are vital for CSC growth, and potential therapeutic targets in paediatric HGG.In conclusion, we have performed a CRISPR knockout screen on paediatric H3K27-mutated HGG with CSC lines accurately reflecting the disease. The screen robustly identified the positive controls and essential genes known in the literature, but also several novel genes essential for CSC growth in paediatric HGG. Interestingly, many of the hit genes were predicted as druggable highlighting their therapeutic potential, and we evaluated an UBE2N inhibitor, which successfully killed the CSC. We optimised an individual knockout pipeline (nucleofection of RNP complexes) for the cell lines routinely achieving high DNA cleavage efficiencies (> 80%) and loss of protein while detecting no off-target effects at the top predicted locations. The optimised pipeline was used to successfully validate 12 genes, demonstrating their importance for CSC growth. Six of these genes were knocked out in four CSC lines, derived from H3 wild-type as well as H3K27-mutated HGG, proving their broad effect on cell growth. The decrease in cell growth may partially be caused by differentiation as the expression of the astrocytic marker GFAP increased, while stem cell markers decreased, but this requires further studies. We also knocked out the six candidate genes in NSC, and while their viability was affected by the knockout, it was not as detrimental as for the CSC, especially for the genes No formal sample size estimation was performed as well-characterised cell lines from paediatric high-grade gliomas are very rare. We therefore used two CSC lines, which could tolerate the lentiviral transduction of the knockout library, for the CRISPR knockout screen. To compensate for the relatively small sample size, we used stringent thresholds for hit calling and used four CSC lines in the validation step. Blinding was not performed as, for example, specific pulse programs for nucleofection were required for the different cell lines.P values for the pooled CRISPR knockout screen was calculated according to the MAGeCK software . Validatn = 3 or 4). Welch one-tailed t test was used  to test if the cell growth after gene knockout was significantly different compared to the growth of the negative controls of the cell lines. It was also used to test if the growth after knockout was significantly reduced in CSC compared to NSC on a group level .The number of cell doublings during the one-month culture was summed up for each gRNA and used to calculate the total number of cells. An average of the two gRNA for each gene was calculated and then normalised to the total cell number for the negative controls n = 3 or . Welch ovel Fig. A was sigp value < 0.05 was considered significant.The dose–response curve Fig. B was fitThe study was approved by the regional ethics committee in Gothenburg (Dnr 604–12) and carried out according to relevant guidelines and regulations. The cell lines GU-pBT-7 , GU-pBT-19  and GU-pBT-28  have been characterised and presented previously , 58, andThe foetal NSC were a kind gift from Professor Steven Pollard , and the cells were derived as previously described , 60. TheGU-pBT-7 and GU-pBT-19 were transduced with a lentivirus coding for Cas9, blasticidin resistance and BFP (under an Ef1a promoter) and then selected with blasticidin and sorted with FACS for BFP expression. A guide RNA (gRNA) library with four gRNA/gene targeting 1212 genes was designed with guide sequences from the Brunello library  (AdditioBased on the ranking score (lower ranking means more depleted) of the positive ribosomal gene controls, we put a hit threshold at top 250-ranking with at least three out of four concordant gRNA for both replicates. Next, all positive and negative controls were excluded and false-positive hit genes were removed based on the following criteria: 1) genes with more than 1 gRNA having multiple matches in the genome were removed as multiple cuts induce a negative growth effect on cells , 65 and  genes wiNext, we compared our identified hits to CRISPR screen data from more than 300 traditional cancer cell lines . We exclOR1A2 at two different locations in the gene . 20 µl of this cell solution, typically containing 60 000–200 000 cells depending on the cell line, was used for one reaction and combined with 5 µl of the pre-made RNP complex and 0.5–1 µl Alt-R® Electroporation enhancer (IDT). The cell/RNP solution was nucleofected with the 4D-Nucleofector® X unit (Lonza) with an optimised pulse program for each cell line see Fig. A. Warm mhttps://ice.synthego.com). We also used this method to examine potential off-targets at two of the top putative off-target locations for the gRNA  using algorithms by Hsu et al. [On-target effect of the knockout was performed ~ 10 days after knockout by ICE  analysisu et al. .CSC were fixated with 4% paraformaldehyde, and immunocytochemistry and EdU imaging were performed as previously described , 67 usinBriefly, cells were seeded in 384 well plates (Corning) and a UBE2N inhibitor , dissolved in DMSO, was added to the cells one day after seeding. The cells were cultured with the inhibitor for four days and imaged with the Incucyte. At the end point, cells were analysed with the CellTiter-Glo® 2.0 cell viability assay on a Glomax Discoverer  according to the manufacturer’s instructions.Additional file 1. Supplementary figures and captions.Additional file 2: Supplementary table 1. Underlying data for figure 1.Additional file 3: Supplementary table 2. MaGeCK analysis of the CRISPR knockout screen.Additional file 4: Supplementary tables 3–6. ED50 analysis of UBE2N inhibitor (S3), gRNA sequences used in CRISPR knockout library (S4) and for validation (S5), and PCR primers (S6).


Ustilago maydis is one of the dominant fungal diseases in plants. The resistance mechanism to U. maydis infection involving alterations in the cell wall is poorly studied. In this study, the resistant single segment substitution line (SSSL) R445 and its susceptible recurrent parent line Ye478 of maize were infected with U. maydis, and the changes in cell wall components and structure were studied at 0, 2, 4, 8, and 12 days postinfection. In R445 and Ye478, the contents of cellulose, hemicellulose, pectin, and lignin increased by varying degrees, and pectin methylesterase (PME) activity increased. The changes in hemicellulose and pectin in the cell wall after U. maydis infection were analyzed via immunolabeling using monoclonal antibodies against hemicellulsic xylans and high/low-methylated pectin. U. maydis infection altered methyl esterification of pectin, and the degree of methyl esterification was correlated with the resistance of maize to U. maydis. Furthermore, the relationship between methyl esterification of pectin and host resistance was validated using 15 maize inbred lines with different resistance levels. The results revealed that cell wall components, particularly pectin, were important factors affecting the colonization and propagation of U. maydis in maize, and methyl esterification of pectin played a role in the resistance of maize to U. maydis infection.Common smut caused by  Ustilago maydis (DC) Corda. High temperature and low moisture conditions are favorable for its development. U. maydis is a parasitic, biotrophic fungus and a plant pathogen, mainly infecting gramineous plants. Its infection can significantly decrease the yield of corn, wheat, barley, sorghum, and other staple crops  were inoculated with U. maydis at the seedling stage. This experiment included three independent biological replicates under controlled conditions. For each line, an average 100 plants were scored according to the symptoms. Interestingly, the susceptible maize lines  originated from five different heterotic groups; all five partially susceptible lines  originated from at least three groups, whereas all five resistant lines  originated from four different groups , partially susceptible (Dan598 and Cheng351), and resistant (444 and Ji1037) lines displayed significantly different demethylation of pectin during disease development, an LSD test showed that there was significant otherness between the susceptible and resistant groups at the 0.05 level , namely, Dong6002, P007, Dan598, Cheng351, 444, and Ji1037, for the qualitative analysis of pectin at 8 dpi using low/high-methylated pectin immunolabeling antibodies JIM5 and LM20. Immunolabeling with the JIM5 antibody was weaker in all lines after infection than in the control. Moreover, partially susceptible and resistant lines exhibited relatively stronger immunolabeling than susceptible lines after the infection compared with the control. Immunolabeling with the LM20 antibody exhibited stronger signals in the susceptible and partially susceptible lines but a weaker signal in the resistant lines after the infection. In contrast, partially susceptible and resistant lines exhibited weaker immunolabeling than susceptible lines before culation . TherefoArabidopsis, and rice were subjected to conjoint analysis using MegaX and Tbtools software. Seven genes with similar structures and functions were selected for qRT-PCR according to the comprehensive analysis and our previous transcriptome data  subunits CESA 4, CESA 7, and CE-SA 8 enhanced resistance to Ralstonia solanacearum and Plectosphaerella cucumerina [HvCslD2) was silenced through RNA interference (RNAi), which resulted in decreased cellulose content in the epidermal cell wall; its mastoid structure was more easily penetrated by powdery mildew pathogens [U. maydis (Arabidopsis to pathogens [Atxyl1) was silenced, after which, remarkable changes in xyloglucan composition were observed, along with alterations in the growth pattern [U. maydis infection progressed, more proliferative cells were formed, eventually promoting hemicellulose accumulation  was significantly upregulated during the infection, which suppressed sheath blight disease [2+ bonds or the so-called “egg-box” model structure to form gel and improve plant resistance, provides a degradation target for other pectin-degrading enzymes to reduce the resistance by effecting the cell wall structure and hardness, or acts as an activated substance to be recognized by the downstream defense response [Fusarium acuta (F. oxysporum) and Phytophthora soya (P. sojae) with a defense mechanism via SA resistance pathway. Lionetti [AtPMEIs is strictly regulated by JA and ET signaling during Botrytis infection in Arabidopsis, which upregulates the defense-related genes to limit PME activity, resulting in higher pectin methylation and abnormal functioning of the CWDEs secreted by pathogens [U. maydis infection, the contents of jasmonic acid-isoleucine (JA-ILE), 1-aminocyclopropane-1-carboxylic acid (ACC), and SA were extremely significantly increased in Ye478, confirming that hormones are required for cross-linking with PMEIs to fine-tune the biosynthetic homeostasis of pectin polysaccharides [The activity of PME is regulated by pectin methylesterase inhibitors (PMEIs). It is reported that PMEIs have originated from PME for the spatio-temporal coordination of the expression model to regulate methyl esterification degree of HG ,40. More disease . Interesresponse ,44. Furtresponse . Bano [4response  analyzedathogens . In our U. maydis infection, the low-methylated pectin antibody JIM5 and the high-methylated pectin antibody LM20 were used for immunolabeling in Ye478 and R445. The result revealed that low-methylated pectin was mainly distributed in the vascular bundle sheath, primary leaf vein, mesophyll cells, and epidermis  infection in tobacco. Therefore, the reduced expression of PME slows the progression of viral infections [Arabidopsis led to increased resistance to B. cinerea and P. carotovorum via overexpression of pectin methylesterase inhibitors AtPMEI-1 or AtPMEI-2. Actually, PME is the enzyme modulating the degree and pattern of methyl esterification of HG, which is delicately tuned to adjust the HG properties according to plant growth and development [GAE1 and GAE6  were repressed by a P. syringae pv maculicola infection in A. thaliana, the gae1 and gae6 double mutation significantly altered the OG signaling pathway and led to restricted soluble pectin release during pathogenic attack [CaPMEI1 in Arabidopsis could lead to a higher methyl esterification level that enhanced resistance to biotic and abiotic stresses [The activity of PME gradually increased as the dpi extension; however, the increased PME activity could promote the pectin methyl esterification level. This demonstrated that the degree of pectin methyl esterification significantly changed during fections . Lionettfections  reportedelopment . Generalelopment ,54,55,56elopment  reportedelopment . Similarc attack . Overexpstresses . These rU. maydis infection . In the treatment group, the leaves were infected with U. maydis (labeled as Ye478-Inf and R445-Inf). We selected 15 maize inbred lines with different resistant levels, i.e., susceptible, partially susceptible, and resistant to assess the role of the cell wall in U. maydis infection (The susceptible inbred line Ye478 was used as the recipient genetic background. The resistant chromosome segment substitution line (SSSL) R445 was used for the evaluation of resistance phenotype and the determination of cell wall components and immune antibody markers. The R445 line contained only one segment of donor chromosome derived from Qi319 marked by umc1528 and umc1489 introgressed into Ye478 . In the nfection . LSD  on a rotary shaker (200 rpm) at 28°C, followed by centrifugation at 5,000 rpm for 5 min. The cell pellet was resuspended in ddH2O to obtain cell suspension with OD600 = 1 [D600 = 1 .U. maydis as follows: 2 mL of the cell suspension was injected into the whorls at the six-leaf stage using a syringe as described previously [U. maydis strain SG200 are preserved at the Specialty Corn Institute, College of Agronomy, Shenyang Agricultural University, China.Three-week-old maize seedlings were inoculated with U. maydis or ddH2O. Cellulose, hemicelluloses, lignin, and PME were isolated using a Cellulose (CLL) Content Assay Kit , Themicellulose Content Assay Kit , and Lignin Content Assay Kit  , respectively, and plant pectin methylesterases (PME) ELISA Kit  as per the manufacturer’s protocol and quantified using Thermo Scientific Microplate Reader .The leaves collected at 0, 2, 4, 8, and 12 dpi were inoculated with The specific CWDEs including α-L-arabinofuranosidase, β-galactosidase, β-glucosidase, and β-1,4-glucosidase were extracted from the leaf samples of Ye478 and R445 lines collected at 0, 2, 4, 8, and 12 dpi using an α-L-Arabinofuranosidase (α-L-Af) Activity Assay kit , β-galactosidase (β-GAL) Activity Assay Kit , β-glucosidase (β-GC) Activity Assay Kit , and 1,4-β-D-Glucan Cellobilhydrolase (C1) Activity Assay Kit  , respectively, and quantified using an automatic microplate reader .U. maydis-infected plant leaves, the focal tissue below the infection site was collected and fixed in FAA solution. The fungal and plant cell walls were co-stained using 4’,6-diamidino-2-phenylindole  and WGA-AF 488  as described by Gao [To visualize the hyphae in d by Gao  with somU. maydis were treated as described previously with increased dehydration time [w/v) toluidine blue. The progress and characteristics of U. maydis infection in the treatment and control groups of Ye478 and R445 were assessed using a Photometrics SenSys CCD camera and a stereomicroscope .The maize leaves infected with ion time  for para® 250 Flash III, 3DHISTECH Ltd., Budapest, Hungary) was used to obtain a panoramic photo, and fluorescence signal intensity was regulated using Caseviewer (V2.3) software . The photographed images were analyzed using Image J  software, and the average fluorescence intensity was calculated as the average fluorescent point pixel value of each target site.Pectin, hemicelluloses, and cell wall glycoproteins were qualitatively analyzed via immunohistochemical staining. The monoclonal antibodies, namely, LM10 (for xylan), JIM5 (for low-methylated pectin), and LM20 (for high-methylated pectin) were used for immunolabeling. Cellulose was visualized via staining with 1% Calcofluor White  as previously described by Tang . Immunof2O 7.0 μL. The 2−ΔΔCt method was used to process the data. The method was modified by Zou [Total RNA was extracted from the leaves collected at 0, 2, 4, 8, and 12 dpi using an RNA extraction kit  as per the manufacturer’s protocol. The quantity and quality of RNA was confirmed using a NanoDrop 2000  according to A260/A280 and electropherograms . ComplemThe primers used are described in U. maydis infection, both susceptible and resistant lines exhibited an increased content of cellulose, hemicellulose, pectin, and lignin as revealed via qualitative and quantitative analyses. However, the activity of PME increased. As it was competitively symbiotic, U. maydis also produced CWDEs to degrade cellulose, hemicellulose, and pectin while the fungal biomass was significantly increased at 8 dpi and 12 dpi. The degree of pectin methyl esterification affected the rheological properties of the cell wall and affected the disease resistance of plants. The PME activity significantly increased in the susceptible lines, followed by partially susceptible and resistant lines. These results revealed that the resistance to U. maydis infection is correlated with the degree of pectin methyl esterification. This study enhanced the understanding of the interaction between the cell wall and pathogenic basidiomycetes during plant defense mechanisms.In summary, after


Populus GT43 clades B and C (GT43BC) corresponding to Arabidopsis clades IRX9 and IRX14, respectively, involved in xylan backbone biosynthesis, GATL1.1 corresponding to AtGALT1 involved in xylan reducing end sequence biosynthesis, or ASPR1 encoding an atypical aspartate protease. Their productivity, wood quality traits, and saccharification efficiency were analyzed. The only lines differing significantly from the wild type with respect to growth and biotic stress resistance were the ASPR1 lines, whose stems were roughly 10% shorter and narrower and leaves showed increased arthropod damage. GT43BC lines exhibited no growth advantage in the field despite their superior growth in greenhouse experiments. Wood from the ASPR1 and GT43BC lines had slightly reduced density due to thinner cell walls and, in the case of ASPR1, larger cell diameters. The xylan was less extractable by alkali but more hydrolysable by acid, had increased glucuronosylation, and its content was reduced in all three types of transgenic lines. The hemicellulose size distribution in the GALT1.1 and ASPR1 lines was skewed towards higher molecular mass compared to the wild type. These results provide experimental evidence that GATL1.1 functions in xylan biosynthesis and suggest that ASPR1 may regulate this process. In saccharification without pretreatment, lines of all three constructs provided 8-11% higher average glucose yields than wild-type plants. In saccharification with acid pretreatment, the GT43BC construct provided a 10% yield increase on average. The best transgenic lines of each construct are thus predicted to modestly outperform the wild type in terms of glucose yields per hectare. The field evaluation of transgenic xylan-reduced aspen represents an important step towards more productive feedstocks for biorefineries.Xylan that comprises roughly 25% of hardwood biomass is undesirable in biorefinery applications involving saccharification and fermentation. Efforts to reduce xylan levels have therefore been made in many species, usually resulting in improved saccharification. However, such modified plants have not yet been tested under field conditions. Here we evaluate the field performance of transgenic hybrid aspen lines with reduced xylan levels and assess their usefulness as short-rotation feedstocks for biorefineries. Three types of transgenic lines were tested in four-year field tests with RNAi constructs targeting either  MutatioGAUT12-B ; the cop/GATL1-B ; and theis GT47C . These P mutants  and are d growth , whereaswn trees . The effbidopsis  and genebidopsis . HoweverGT43 family genes and GATL1.1 based on a four-year field experiment in Southern Sweden. We also report a novel xylan defect in aspen lines with suppressed expression of ASPARTIC PROTEASE 1 (ASPR1) which was not previously implicated in xylan biosynthesis but was tested here since the gene was highly induced during secondary wall formation in developing wood  clone T89 was used to make transgenic lines , as described previously . A permiMelampsora spp.), venturia (Venturia spp.), chlorosis, necrosis, and hypersensitive responses (HR). Chlorophyll content was determined with a CCM-200 plus instrument  on six fully developed leaves per tree for 50% of the tallest trees in each line, as previously described  measurements were conducted at RISE Research Institutes of Sweden AB using previously reported procedures . Briefly2.4https://plantcv.readthedocs.io/en/stable/tutorials/vis_tutorial/. The major (a) and minor (b) axis of the ellipses fitted over the contour of each tree image are obtained, as well as the area of the tree bounding convex hull (chA), the tree bounding box length (bbL) and width (bbW), and the surface area of the contour of each tree (treeA), i.e. the number of pixels corresponding to the tree. Other parameters are calculated as follows:To assess differences in branching and growth morphology, trees representing the studied genotypes (N = 14) were photographed in the field before and during bud burst in May and early June of 2017. Images were analyzed using scripts written in the object-oriented programming language Python  using thThe number of main (primary) and secondary branches was counted manually from the images.2.5Dry stem segments were cut into pieces roughly 5 cm long and ground in an SM 300 Cutting Mill with a 2 mm sieve . The rough powder was then sieved using an AS 200 vibratory sieve shaker (Retsch) and divided into the following particle size fractions:< 50, 50–100, 100–500, and > 500 μm. Wood powder of each size fraction from sets of three trees was then pooled. Four such pooled samples  were used for each transgenic line; eight were used for the WT.Alcohol-insoluble residue (AIR) was obtained from 10 mg of each pooled biological replicate of the 50–100 μm particle size fraction and treated with α-amylase and α-amyloglucosidase  as described by 2SO4 at room temperature for 3 h, then diluted with 1375 μL of deionized water and incubated at 100°C for 3 h. The resulting hydrolysates were diluted with 0.9 mL of MilliQ water and filtered through a 0.2 mm syringe filter (Chromacol 17-SF-02-N) into a high-performance anion-exchange chromatography - pulsed amperometric detection (HPAEC-PAD) vial for sugar analysis.All four biological replicates of the 50-100 µm sized particle fractions were pooled to obtain one pool representing 12 trees per line for transgenic trees and two pools of 12 trees each for the WT. Each pool was then analyzed in technical duplicates to determine the sugar composition by a two-step sulfuric acid hydrolysis method . Aliquot2.6Hemicelluloses were purified by alkaline extraction  by incubO-MeGlcA and GlcA) with concentrations of 0.005 - 0.1 g/L.The sugar composition of the alkaline extracts was determined after acidic methanolysis  using 1 The molar mass of the alkaline extracts was determined by size exclusion chromatography coupled to refractive index and UV-detectors . The freeze-dried extracts were dissolved in dimethyl sulfoxide  to a concentration of 2 mg/mL with 0.5% w/w LiBr  at 60 °C overnight and filtered through 0.45 μm PTFE syringe filters (VWR). Separation was performed using GRAM Analytical columns of 100 and 10 000 Å  at a flow rate of 0.5 mL/min at 60 °C. The columns were calibrated using pullulan standards between 345 and 708 000 Da .Bacillus subtilis (-1) for 16 h at 37 °C. After incubation, the enzyme was inactivated at 95°C and the mixture was filtered. Oligosaccharides were analyzed by liquid chromatography system  coupled with electron spray tandem mass spectrometer  as described by For oligosaccharide mass profiling (OLIMP), the alkali-extracted hemicelluloses were digested using GH30 endo β-(1-4) glucuronoxylanase from subtilis , kindly 2.7Analytical enzymatic saccharification experiments were performed as described previously  using 5033.13.1.1Young trees belonging to the transgenic lines and WT were planted in the field during the summer of 2014. The mortality up to the summer of 2018 never exceeded one out of 28 trees per line (transgenic lines) or 56 trees (WT) and the survival of the modified trees therefore did not deviate from the survival of WTs . The summer of 2018 was exceptionally dry in Southern Sweden  were damaged by hares, some of them multiple times. This damage tended to occur at specific locations in the field but no significant differences were observed between the lines  relative to the WT in the ASPR1 construct (particularly the ASPR1_2A line) showed growth retardation.Most of the transgenic lines thus exhibited growth similar to the WT and had normal morphology; only the 3.1.2ASPR1_2A line than in the WT and other lines  than in the wild type, and the GT43BC lines also exhibited reduced fiber coarseness (by 5%)  and a slight increase in cellulose microfibril angle . Overall, these changes were very modest; the SilviScan wood traits were only slightly affected in the ASPR1 and GT43BC lines and not at all in the GATL1.1 lines.Wood of transgenic and WT trees was analyzed using a SilviScan instrument. The annual ring width initially decreased in the 3.3ASPR1 lines were slightly reduced and their phenolic content was increased , while GT43BC lines had increased levels of Man, mGlcA and Glc  , which was a minor component.When the biomass sugar composition was analyzed using two-step hydrolysis with sulfuric acid, approx. 50% of the biomass weight was recovered, with Glc and Xyl being main sugars  yielding approx. 5% of biomass recovered as sugars, or to enzymatic saccharification after acid pretreatment (pretreated) with the recovery above 50% of biomass weight Figure 6ASPR1 and GATL1.1 lines gave increased yields of Ara and Gal relative to the WT. Total yields of Glc in saccharification with acid pretreatment were increased from biomass of GT43BC construct lines (by 9% on average) even though Glc production rates did not differ significantly from WT for any transgenic line. The total yields of sugars other than Ara and Gal (which were released during acid hydrolysis) were unaffected by acid pretreatment.Biomass of transgenic and WT trees gave similar yields of Xyl, Man and Glc in acid pretreatment liquid  released by GH30 indicated a reduced frequency of widely-spaced glucurosylation and an increased abundance of more tightly spaced glucuronosylation . These tGT43BC hybrid aspen  of xylan , and a pid aspen . Howeverid aspen . In accoynthesis . Our ana4.2ASPR1 belongs to a large family of atypical aspartic proteases and nucellins. Its clade includes eight genes in P. trichocarpa and ten in Arabidopsis  as well as a higher chlorophyll index and more extensive chewing damage  were increased in these lines, this indicated that the genetic change that resulted in decreased xylan content in these lines lead to an altered cell wall architecture and higher accessibility of saccharification enzymes to substrates. In the case of GT43BC lines, the increased accessibility was also observed after acid pretreatment, which is of particular interest considering the presence of the pretreatment step in current technologies. Because these lines exhibit normal growth but some have slightly lower wood density than WT plants, these higher Glc yields should result in modestly improved total Glc gains per hectare.Considering the potential of the studied lines as biorefinery feedstocks, we found that all constructs improved Glc yields in saccharification without pretreatment (The original contributions presented in the study are included in the article/MD-M overviewed field trial setting up and phenotyping in 2017. PS carried out wood grinding and chemical analyses with help of RV and EH and supervision of FV and EM. ED carried out statistical analyses, data plotting and prepared the figures. MG and LJ were responsible for pretreatment and saccharification analyses. ZY and GS were responsible for SilviScan analyses. UJ was responsible for field management, operation and phenotyping. FA and BA were responsible for biotic stress estimation in 2017 and leaf phenotyping. MH produced and selected transgenic lines for field testing, EM coordinated the study, ensured the funding and wrote the paper with contributions from all the authors. All authors contributed to the article and approved the submitted version.


CMYC, TP53, GPX1, and TXRND1 expression levels were assessed using qPCR. Our results showed that parthenolide promoted a metabolic activity decrease in all cell lines in a time-, dose-, and cell-line-dependent manner. The mechanism induced by parthenolide was demonstrated to be cell line dependent. Nonetheless, parthenolide promoted cell death by apoptosis with significant ROS increase (peroxides and superoxide anion) and GSH decrease combined with a ΔΨmit reduction across all studied cell lines. Despite the need to further understand parthenolide mechanisms, parthenolide should be considered as a possible new therapeutic approach for B- and T-lymphoid malignancies.Lymphoid malignancies are a group of highly heterogeneous diseases frequently associated with constitutive activation of the nuclear factor kappa B (NF-κB) signaling pathway. Parthenolide is a natural compound used to treat migraines and arthritis and found to act as a potent NF-κB signaling inhibitor. This study evaluated in vitro parthenolide efficacy in lymphoid neoplasms. We assessed parthenolide metabolic activity in NCI-H929 (MM), Farage (GCB-DLBCL), Raji (BL), 697 and KOPN-8 (B-ALL), and CEM and MOLT-4 (T-ALL), by resazurin assay. Cell death, cell cycle, mitochondrial membrane potential (ΔΨ Lymphoid neoplasms comprehend a highly heterogeneous group of diseases currently ranked between the 6th and 7th most common cancers worldwide . These hBCL-2, BCL-xL, CCND1, CMYC, and TP53. This transcription factor family is responsible for regulating various biological processes, such as survival, proliferation, and inflammation [The nuclear factor kappaB (NF-κB) transcription factor family comprises five mammalian family members, particularly RelA p65), cREL, RELB, NF-κB1 (p105 and its precursor p50), and NF-κB2 (p100 and its precursor p52). NF-κB dimers are generally sequestered in cytoplasm by their inhibitor IκB family in an inactive complex. Phosphorylation of IκB by IκB kinase (IKK) on the cytosolic domain and subsequent ubiquitination and proteasome degradation of IκB lead to the activation of NF-κB. The activated NF-κB then translocates to the nucleus and induces the transcription of its many target genes, such as 5, cREL, ammation ,8.Aberrant activation of NF-κB has already been observed in different types of cancer, and lymphoid malignancies in particular rely on the constitutive activation of NF-κB for cell survival and proliferation . Furtherfeverfew (Tanacetum parthenium), a traditional medicinal plant that has been conventionally used in the treatment of fever, migraines, and arthritis. Parthenolide has also been highly studied for its anticancer activities in multiple cancer types, including hepatocellular carcinoma, breast cancer, and hematological malignancies [Parthenolide is a naturally occurring sesquiterpene lactone isolated from gnancies ,14,15,16gnancies . A seriegnancies ,19,20,21gnancies ,23,24,25Our study explored the antitumor effect of parthenolide in several lymphoid neoplastic cell lines, unveiling its role in apoptotic mechanisms and oxidative stress dysregulation.50) ranging between 1 µM and 10 µM.To evaluate the potential effect of parthenolide on metabolic activity, seven lymphoid neoplastic cell lines were incubated in the absence and presence of increasing concentrations of parthenolide, ranging between 0.1 µM and 100 µM. Resazurin assay was performed at 24 h, 48 h, and 72 h. Our results showed that parthenolide reduced the metabolic activity in all cell lines in a dose-, time- and cell line-dependent manner, as represented in 50 observed for each cell line were 1.5 µM for Farage, 2.5 µM for 697 and KOPN-8, 5 µM for H929 and CEM, 7.5 µM for MOLT-4, and 10 µM for the Raji cell line.To further understand the mechanisms associated with parthenolide in these cell lines, subsequent studies were performed using two concentrations. One concentration was common to all seven cell lines and corresponded with 1 µM; other concentrations closer to the IC2/M phase was observed in both the Raji  and the MOLT-4 cell lines . No differences in cell cycle distribution were observed for the other four cell lines: H929, Farage, 697, and CEM.Our first approach to evaluate the mechanistic effect of parthenolide was to assess if the decrease in metabolic activity was induced by a cytostatic effect. To evaluate this, cell cycle distribution was assessed using flow cytometry (FC) and a propidium iodide (PI)/RNAse kit. As previously mentioned, two concentrations of parthenolide were used for each cell line for a 72 h incubation. Parthenolide induced cell cycle arrest in a cell line-dependent manner . Cell arp < 0.001; Farage: 53.0 ± 2.1, p < 0.001; Raji: 57.3 ± 0.7, p < 0.001; 697: 53.3 ± 2.4, p < 0.001, KOPN-8: 37.3 ± 1.3, p < 0.001; CEM: 49.7 ± 1.8, p < 0.001; MOLT-4: 48.0 ± 2.1, p < 0.001). In agreement with these results, morphological analysis performed in these higher concentrations showed typical features of cell death mediated by apoptosis, confirming apoptosis activation by parthenolide. These apoptotic features included nuclear fragmentation and blebbing in the parthenolide-treated cells , CEM  and MOLT-4  cell lines.To evaluate whether parthenolide also induced a cytotoxic effect, cell death analysis was first assessed using flow cytometry with anexin V (AV) and 7-Aminoactinomycin D (7-AAD) double staining. Our results a showed ed cells b. Moreovp < 0.001) increase and MOLT-4 with a 12.8-fold  increase in caspase-3 expression levels. The H929 and Raji cell lines presented the lowest increase in caspase-3 levels, 2.2-  and 3.9-fold , respectively.To further confirm apoptosis induction, we assessed the activation of caspase-3, an effector caspase d, and FAp = 0.047; Raji—control: 16.0 ± 0.6, PRT 10 µM: 22.0 ± 2.3, p = 0.044; MOLT-4—control: 14.0 ± 0.6, PRT 7.5 µM: 30.0 ± 1.5, p < 0.001), highlighting a possible involvement of the extrinsic apoptotic pathway.FAS-L expression levels were also increased in response to parthenolide in all cell lines, except for H929 and CEM cell lines e. The otmit) using a JC-1 probe using flow cytometry. Cellular apoptosis is associated with a higher JC-1 monomer (M)/aggregate (A) ratio corresponding to a lower mitochondrial membrane potential. Parthenolide induced a dose-dependent increase in the M/A JC-1 ratio in all the lymphoid neoplastic cell lines, except for the Raji cell line, demonstrating that the intrinsic apoptotic pathway is also involved in parthenolide response (p < 0.001), KOPN-8 , CEM , and MOLT-4 , i.e., the four acute lymphoblastic leukemia cell lines.We then evaluated the mitochondrial membrane potential (ΔΨresponse f. This i2-DA) and dihydroethidium (DHE) fluorescent probes, respectively, and the intracellular levels of reduced glutathione (GSH), a major antioxidant, using a mercury orange (MO) probe. As demonstrated in p = 0.030), 697 , KOPN-8 , CEM , and MOLT-4 . Similar results were obtained for superoxide anion levels .As parthenolide is also often associated with oxidative stress imbalance, we then assessed the intracellular levels of peroxides and superoxide anion using 2,7-dichlorodihydrofluorescein diacetate (DCFHp < 0.001). These results further prove that parthenolide effects are mainly mediated by the intrinsic apoptotic pathway.As an antioxidant defense, GSH levels were significantly reduced in all cell lines when incubated with parthenolide, particularly with the higher doses. This decrease was more pronounced in the Raji cell line  incubated with the high concentration of parthenolide and a 2.0-fold reduction in the MOLT-4 cell line (p < 0.001), when compared to the control cells. For the other three cell lines , no differences were observed in pNF-κB expression after parthenolide treatment.To assess the inhibition of IKK by PRT, the expression of the active form of NF-kB (p65 subunit) was also evaluated. Parthenolide induced a dose-dependent decrease in pNF-κB expression levels in the four ALL cell lines: 697, KOPN-8, CEM, and MOLT-4 . Among tCMYC and TP53, and two oxidative stress-related genes, GPX1 and TXNRD1, after 72 h incubation with parthenolide at higher doses. As represented in CMYC expression levels in all cell lines, except in the KOPN-8 cell line. However, only the MOLT-4 cell line presented over 50% reduction in CMYC expression  and KOPN-8, which presented increased levels  and CEM  cell lines presenting statistical differences when compared to untreated cells.The oxidative stress-related genes, d levels c. For TX  and buthionine sulfoximine (BSO), a ROS inhibitor and a GSH inhibitor, respectively [Apoptosis induction by parthenolide has been extensively described in several different cancer-associated studies. Recently, a proteomic study conducted by Cui et al. identified several proteins associated with apoptosis promotion in thyroid cancer cells . Despiteectively . ROS indectively ,29. A moectively .As previously stated, parthenolide mechanisms in our study seemed highly dependent on tumor type. Besides the effects related to apoptosis and oxidative stress which have already been discussed, in the H929 cell line, a multiple myeloma cell model, parthenolide did not alter the phosphorylated NF-κB p65 expression levels. Constitutive activation of NF-κB signaling contributes to MM pathogenesis and is usually related to oncogenic mutations and inflammation . However2/M cell cycle arrest after parthenolide incubation, as well as in the Raji cell line. These are the two less sensitive cell lines, which might indicate a dose-dependent effect of parthenolide. Another important study in ALL using in vitro and in vivo studies demonstrated the potential of parthenolide to inhibit different subpopulations of cells, particularly leukemia-initiating cells, that highly contribute to disease progression and relapse [Parthenolide demonstrated apoptosis-associated oxidative stress in B- and T-ALL models and a significant decrease in NF-κB p65 phosphorylation. The first study using parthenolide in B-ALL demonstrated that it induces growth arrest and stress response, specifically in t ALL cell lines . Nonethe relapse . More re relapse .Although it presents a high antitumor potential, parthenolide was shown to be highly lipophilic and to have low solubility, which limits its bioavailability and solubility in blood plasma. For this reason, our and other research groups have been focusing on the development of parthenolide derivatives and of new transport nanoparticles that will possibly increase parthenolide efficacy. For instance, a recently published study in acute myeloid leukemia developed a poly lactide co-glycolide (PLGA)-antiCD44-parthenolide nanoparticle that improved bioavailability and selectively targeted leukemic cells .2 and incubated at an optimal density. To ensure mycoplasma-free cultures, cells were periodically tested using PCR.A panel of seven hematological cell lines was used: NCI-H929 (MM), Farage (GCB-DLBCL), Raji (BL), 697 and KOPN-8 (B-ALL), and CEM and MOLT-4 (T-ALL). All cell lines were maintained in Roswell Park Memorial Institute 1640 , containing 2 mM of L-glutamine, 100 U/mL of penicillin, 100 g/mL of streptomycin (Corning), supplemented with 10% fetal bovine serum (FBS) (Corning). The NCI-H929 cell line was supplemented with 20% FBS, 1 mM sodium pyruvate (Corning), and 50 µM mercaptoethanol (Corning). Cells were maintained at 37 °C in a humidified atmosphere containing 5% COThe metabolic activity of cells incubated with parthenolide was evaluated using resazurin assay. This compound was purchased from APExBIO  and dissolved in dimethyl sulfoxide (DMSO). All cell lines were incubated for 72 h in the absence and presence of increasing concentrations of parthenolide, ranging between 0.1 and 100 µM. To avoid DMSO cytotoxicity and to ensure consistency between conditions, a 100-fold concentration was used. The cells were plated on a 48-well plate for 72 h. Every 24 h, resazurin was added to a final concentration of 10 mg/mL and then incubated at 37 °C for at least 2 h. The absorbance at 570 nm and 600 nm was measured using a microplate spectrophotometer , and the metabolic activity was calculated as a percentage of control.6 of untreated and treated cells were collected and washed with PBS for 5 min at 1000× g. The pellet was resuspended in 200 µL of 70% ethanol solution during vortex agitation and incubated for 30 min at 4 °C. Then, cells were washed with PBS and resuspended in 500 µL of PI/RNase solution. Flow cytometry analysis was performed in a FACSCalibur flow cytometer (Becton Dickinson). Results were expressed in the percentage of cells in each cell cycle phase  according to PI fluorescence intensity. A sub-G1 peak was also identified as apoptotic cells. The cell cycle distribution was analyzed using ModFitLT software V2.0 .The cell cycle distribution was evaluated using FC and a propidium iodide (PI)/RNase cell cycle analysis kit , as previously described . BrieflyTo evaluate cell death induced by parthenolide, we used optical microscopy after May–Grünwald–Giemsa staining to study morphological features and flow cytometry (FC), using annexin-V (AV) and 7-Aminoactinomycin D (7-AAD) double staining and a JC-1 probe. FC was also used to evaluate the expression levels of a few apoptotic proteins (FAS ligand (FAS-L) and activated caspase-3) to clarify the mechanisms involved. Cells untreated and treated with two different concentrations of parthenolide were incubated for 72 h and collected for the following measurements.6 cells were washed with PBS, centrifuged at 500× g for 5 min, resuspended in 100 µL of annexin V binding buffer, and incubated with 2.5 µL of annexin V-APC  and 5 µL of 7-AAD (Biolegend) for 15 min in the dark at room temperature (RT). Then, cells were diluted in 300 µL of annexin V binding buffer and analyzed in a FACSCalibur flow cytometer . At least 25,000 events were acquired using CellQuest software v3.3 (Becton Dickinson) and analyzed using Paint-a-Gate v3.0 (Becton Dickinson). The results were expressed as a percentage of viable cells (AV−/7-AAD−), initial apoptotic (AV+/7-AAD−), late apoptotic/necrotic (AV+/7-AAD+), and necrotic cells (AV−/7-AAD+).Cell death was first evaluated using AV/7-AAD double staining by FC, as described by Lapa, 2020 . Briefly6 cells was collected and seeded in glass slides. Then, smears were stained for 3 min with May–Grünwald solution  and for 15 min with Giemsa solution (Sigma-Aldrich). After rinsing with distilled water, cell morphology was analyzed using light microscopy with a Nikon Eclipse 80i microscope equipped with a Nikon digital camera DXm 1200F .Optical microscopy was used to assess the morphological features associated with apoptosis and necrosis. A quantity of 1 × 106 cells was incubated with a monoclonal antibody anti-activated caspase 3-fluorescein isothiocyanate , and FAS-L  antibodies according to the manufacturer’s protocol. For intracellular staining of activated caspase 3, cells were fixed with 100 μL of a fix solution  for 15 min and then washed by centrifugation at 300× g for 5 min. Cells were then permeabilized by incubating for 15 min with 100 μL of a permeabilization solution (IntraCell) and the respective antibody. For membrane staining of FASL, cells were incubated with a FAS-L antibody and incubated for 15 min. After washing, cells were analyzed using FC. At least 25,000 events were acquired using CellQuest software (Becton Dickinson) and analyzed using Paint-a-Gate (Becton Dickinson). The results are presented as mean fluorescence intensity (MFI) arbitrary units and represent MFI detected in the cells, which is proportional to the protein concentration in each cell.The activated caspase 3 and FAS-L expression levels were assessed using FC with fluorescently labeled monoclonal antibodies. A quantity of 1 × 10mit) was evaluated using FC with the fluorescent probe JC-1 . After 72 h of incubation in the absence and presence of parthenolide, 1 × 106 cells were washed with PBS by centrifugation for 5 min at 300× g and incubated at 37 °C for 15 min with 5 µL of JC-1. Then, the cells were washed in PBS and resuspended in 300 µL of PBS. Cells were analyzed using FC, and the results were presented as a JC-1 monomer/aggregate ratio calculated as the mean fluorescence intensity (MFI) fraction observed for each form.Mitochondrial membrane potential  and dihydroethidium , respectively. A quantity of 1 × 106 untreated and parthenolide-treated cells was incubated with 5 µM of DCFH2-DA for 45 min at 37 °C or 5 µM of DHE for 15 min in the dark at RT.Oxidative stress levels were assessed through the redox imbalance between the ROS levels and antioxidant defenses, namely reduced glutathione (GSH), as previously described . Intrace6 cells were incubated with 40 µM of MO for 15 min in the dark at RT. Cells were washed twice with cold PBS by centrifugation at 300× g for 5 min and resuspended in the same buffer. Flow cytometry was then performed using a FACSCalibur flow cytometer (Becton Dickinson).Mercury orange (MO) dye (Sigma-Aldrich) was used to measure the GSH content. Briefly, 1 × 10At least 25,000 events were acquired using CellQuest software (Becton Dickinson) and analyzed using Paint-a-Gate (Becton Dickinson). The results were presented using the probe’s mean fluorescence intensity (MFI) in each condition.6 cells were fixed with 100 μL of fix solution (IntraCell) for 15 min and then washed by centrifugation at 300× g for 5 min. Cells were then permeabilized by incubating for 15 min with 100 μL of a permeabilization solution (IntraCell) and a phospho-NF-κB p65 (Ser536) antibody . After a washing step, at least 25,000 events were acquired using CellQuest software (Becton Dickinson) and analyzed using Paint-a-Gate (Becton Dickinson). The results are presented as mean fluorescence intensity (MFI) arbitrary units and represent MFI detected in the cells, which is proportional to the protein concentration in each cell.The expression levels of phosphorylated NF-κB p65 were assessed using FC, after intracellular staining performed as described in CMYC F 5′-GTCAAGAGGCGAACACACAAC-3′, CMYC R 5′-TTGGACGGACAGGATGTATGC-3′, TP53 F 5′-CAGCACATGACGGAGGTTGT-3′, TP53 R 5′-TCATCCAAATACTCCACACGC-3′, GPX1 F 5′-AGAACGCCAAGAACGAAGA-3′, GPX1 R 5′-TTCACCTCGCACTTCTCG-3′, TXNRD1 F 5′-TGCGTGTCCTGTGCTTAC-3′, TXNRD1 R 5′-TGCTGCCTGCCTTCTATTC-3′, HPRT F 5′-CCCTGGCGTCGTGATTAGTG-3′, and HPRT R 5′-TCGAGCAAGACGTTCAGTCC-3′. Standard curves were created for all studied genes using a serially diluted control sample to assess the reaction efficiency. For each experiment, a non-template control (NTC) was included. The specificity of qPCR reactions was confirmed using the melting curve analysis. Samples were normalized to the endogenous gene (HPRT), and the relative expression values were calculated using the 2−ΔΔCt formula (fold change).Gene expression levels were determined as previously described . Total Rt-test, analysis of variance, Dunnett’s test, and the Tukey test were used to compare the different groups. A significance level of p < 0.050 was considered statistically significant. Results were expressed in mean ± SEM of the number of independent experiments indicated in the figure legends.Statistical analysis was performed using the GraphPad Prism 9 software version 9.5.1 for Windows . A normality test was performed with a Kolmogorov–Smirnov test, and adequate analysis was completed. Student’s Parthenolide is a very promising natural compound to target lymphoid malignancies. Here, we demonstrated that parthenolide might be considered a possible new therapeutic approach for the treatment of B- and T-lymphoid neoplastic diseases. Importantly, parthenolide significantly induced apoptosis through oxidative stress, with increased reactive oxygen species accompanied by a decrease in reduced glutathione levels. Apoptosis induction was also associated with lower mitochondrial membrane potential, increased activated caspase-3, and FAS-ligand expression, thus confirming an apoptosis-mediated effect. Moreover, the levels of phosphorylated NF-κB were inhibited by parthenolide treatment. Despite these promising results, the mechanism of apoptosis activation by parthenolide is highly dependent on the tumor and cell type. Due to the high heterogeneity of lymphoid malignancies, further in vitro and in vivo studies are needed to further unravel the parthenolide mechanisms in these malignancies. Moreover, the research of new delivery systems might also provide new and improved options for parthenolide application in lymphoid malignancies.


Cancer is one of the most dangerous diseases in humans, and no permanent therapy has been developed yet. Breast cancer (BC) is one of the most common cancers in women. The purpose of this review was to clarify how natural products may play a role in the prevention, treatment and progression of BC. For all the compounds examined, in vitro and in vivo studies, as well as clinical studies on BC, are described.In this review, we summarize the most used natural products as useful adjuvants in BC by clarifying how these products may play a critical role in the prevention, treatment and progression of this disease. BC is the leading cancer, in terms of incidence, that affects women. The epidemiology and pathophysiology of BC were widely reported. Inflammation and cancer are known to influence each other in several tumors. In the case of BC, the inflammatory component precedes the development of the neoplasm through a slowly increasing and prolonged inflammation that also favors its growth. BC therapy involves a multidisciplinary approach comprising surgery, radiotherapy and chemotherapy. There are numerous observations that showed that the effects of some natural substances, which, in integration with the classic protocols, can be used not only for prevention or integration in order to prevent recurrences and induce a state of chemoquiescence but also as chemo- and radiosensitizers during classic therapy. The onset, maintenance and progression of BC are influenced by multiple pathophysiological aspects and signaling pathways ,2. Neopl++, triggers the response of NF-kB. ROS are responsible for spontaneous mutations, where they can cause DNA breakage of 1 or 2 strands, deletions, insertions, loss of a base, substitution of a base and a deficiency of SOD in the mitochondria and cytoplasm of many solid neoplasms. The possibility of neoplastic cells exploiting all the resources contributes to growth and cachexia and also derives from the increase in a factor, namely, HIF-1, which increases if stimulated by growth factors [+, are necessary for the process of bone invasion and cell survival. During the neoplastic transformation, the cells of breast tissue lose all the mechanism regulators of cell cycle control, such as P53, retinoblastoma protein (pRB) and P27. Moreover, two genes homologous to P53, namely, P63 and P73, may interact with P53. Cancer stem cells (CSCs) were proposed as one of the determining factors contributing to tumor heterogeneity [The pro-inflammatory NF-kB is implicated in tissue inflammatory status, specifically in BC activation and progression through the stimulation of the cell proliferation, pro-survival and metastatic pathways of angiogenesis. Commonly used therapeutic agents, such as radiotherapy and chemotherapy, can also lead to the activation of NF-kB. This effect can contribute to the resistance of neoplastic cells to the agents themselves and the use of NF-kB inhibitors as sensitizers for therapy could improve the therapeutic target . NF-kB i factors . MMPs ar factors . An abno factors . This deogeneity . They arogeneity ,32. Withogeneity . A microogeneity . During ogeneity . CTLs arogeneity . There aogeneity . The latogeneity . MicroRNogeneity . An overogeneity . MicroRNogeneity . MicroRNogeneity . PreclinPolyphenols are antioxidant substances capable of protecting cells from damage caused by free radicals . PolypheCurcuma longa and is obtained via solvent extraction from the dried and ground rhizome of the Curcuma longa plant. Curcumin and its active metabolites are capable of directly binding to DNA and RNA, thereby sensitizing chemotherapeutics through the modulation of protein kinase C, telomerase, NF-kB and histone deacetylase (HDAC). Curcumin induces cell death and restores sensitivity to tamoxifen in antiestrogen-resistant BC cell lines [Curcumin , which ill lines . From thll lines ,53. Curcll lines ,55. Morell lines ,57. It hll lines  and confll lines ,60. It ill lines . Curcumill lines , enhancell lines , inhibitll lines ,65, inhill lines  and Hedgll lines ,68, and ll lines .EGCG is an important polyphenolic component originating from green tea extract. It possesses various biological functions, including anti-cancer and anti-inflammatory properties . EGCG waI3C is a natural organic substance derived from the degradation of glucobrassicin glucosinolate, which is present in almost all cruciferous plants. Diindolylmethane is the biologically active derivative that spontaneously forms from I3C due to the action of gastric juices . I3C is Artemisia annus). It was found to have biological activity against a variety of cancers [Artesunate is a compound obtained from the plant derivative of Artemisia  belonging to the Zingiberaceae family, which has a biological activity profile similar to that of curcumin [2 are the most appropriate solvents for the extraction of 6-gingerol, whereas microwave-assisted extraction is the best extraction method [Dracocephalum heterophyllum [+ [6-Gingerol is a natural analog of curcumin derived from the root of ginger  [Xanthohumol is a prenylated flavonoid derived from p plant) . Its prop plant) . The invp plant) . Xanthohp plant) . It was p plant) . It alsop plant)  and intep plant) . Xanthohp plant) . Moreovep plant) ,121. Xanp plant) ,123,124.p plant) . None ofp plant) . The dosp plant) .Citrus bioflavonoids are flavonoids present in all types of citrus fruits, including oranges, grapefruits, lemons and limes, and constitute the pigments responsible for the typical yellow-orange color of such fruits. Citrus fruits contain high concentrations of several classes of phenols, including numerous hydroxycinnamates, flavonoid glycosides and polymethoxylated flavones. The latter group of compounds occurred without glycosidic bonds and was shown to inhibit the proliferation of several cancer cell lines, including that of BC . The antBurseraceae family. It is studied for its anti-cancer, anti-inflammatory and antibacterial activities [Commiphora myrrh, Commifora molmol and Commiphora wightii. They are all studied for their anticancerogenic potential [Commiphora molmol (oleo-resin) was studied in vivo at 150 and 500 mg/kg in mice bearing Ehrlich’s ascites and normal carcinoma cells; the viability of these cells revealed a cytotoxic and antitumor activity similar to that of the drug cyclophosphamide, as evaluated in terms of the changes of liver cells and bone marrow on the basis of the frequency of micronuclei and polychromatic and normochromatic cells. It also possesses chemosensitizing activity by inhibiting glycoprotein-P, which is an ABC pump responsible for transporting various substances across the plasma membrane, mitochondrial membrane, endoplasmic reticulum and peroxisomes. It inhibits MMP-P through the inhibition of NF-kB and AP1, which are responsible for its activation. Moreover, it inhibits proliferation by arresting the cell cycle in the S phase [Commiphora myrrh was recently studied in vitro against HeLa BC cell lines, showing that myrrh triterpenes showed cytotoxicity against these cell lines [Commiphora myrrh in triple-negative BC (TNBC) were recently investigated by studying its component Z-guggulsterone. It inhibits the proliferation of the TNBC cell lines MDA-MB-468 and BT-549 in vitro. In vivo studies demonstrated that it inhibits TNBC progression via the p53/cyclin B1 (CCNB1)/polo-like Kinase 1 (PLK1) pathway, inducing cell cycle arrest and apoptosis [Myrrh is an aromatic gum resin extracted from a tree or shrub of the genus Commiphora in the tivities ,137,138.tivities . Three sotential ,141. Spe S phase . The sig S phase ; in thisll lines . The effpoptosis . In a repoptosis . To our poptosis . Myrrh cpoptosis .Sulforaphane (1-isothiocyanate-4-(methylsulfinyl)butane) is a chemical compound belonging to the class of isothiocyanates (ITCs) and naturally present as a metabolite of the phytochemical glucoraphanin in plants belonging to the cruciferous group . Numerous in vitro, in vivo and clinical studies were reported on its anticancer activity . SeveralVitamin D exists in two forms: vitamin D2 or ergocalciferol, of plant origin, and vitamin D3 or cholecalciferol, derived from cholesterol and produced directly by the organism. It is a prohormone and needs to be hydroxylated at the liver and kidney levels. It is mainly synthesized during sun exposure, while dietary intake provides about 10–15% of it. Elevated blood levels of 5-hydroxyvitamin D (25OHD), or vitamin D, are directly correlated with better overall survival in patients with BC, providing compelling observational evidence of an inverse correlation between vitamin D levels, mortality and BC progression risk . VitaminMushrooms belong to the astounding dominion of Fungi and are known as a macrofungus. Significant health benefits of mushrooms were reported worldwide, including anticancer, antioxidant and radical scavenging effects. Mushrooms primarily contain low- and high-molecular-weight polysaccharides, fatty acids, lectins and glucans that are responsible for their therapeutic action . Their aAcetylsalicylic acid (ASA), better known as aspirin, is a chemical modification of salicin, which was originally derived from the bark of the willow tree. When salicin is dissolved in water, it is acidic, and a saturated solution of it has a pH of 2.4, which is why it was renamed salicylic acid. The use of low doses of acetylsalicylic acid is associated with a significant increase in survival in neoplastic disease ,167,168.Galega officinalis L. Metformin reduces the expression of genes that encode for IGF-1 and the expression of IGF-1R receptors [Metformin is a hypoglycemic drug belonging to the group of biguanides that are derived from the plant eceptors . It inhieceptors ,172,173.eceptors . It can eceptors ,176, as eceptors ,178. It eceptors ,180, as eceptors ,182.In this section, the recent clinical studies ) on BC with the involvement of the natural products described are summarized .BC is the most frequent cancer among women. Numerous mechanisms are related to the onset and progression of BC. In BC, inflammation and cancer influence each other, and the inflammatory component precedes the development of the neoplasm, but slowly increasing and prolonged inflammation related to the neoplastic phenomenon also favors its growth. Peritumoral inflammation plays an important role in carcinogenesis, and HSPs play an active part in malignant transformation and progression by allowing the expression of new malignant traits through an accumulation of altered oncoproteins. In this context, natural products play a crucial role. Curcumin; EGCG; I3C; artesunate; ginger; flavonoids and bioflavonoids, such as xanthohumol and citrus bioflavonoids; myrrh; sulforaphane; vitamin D; medicinal mushrooms; acetylsalicylic acid; and metformin were analyzed in detail in order to underline their importance in the onset, progression and/or treatment of BC. The clinical studies on these compounds in BCs were detailed. The integration with these products can be implemented both during therapy in the attack phase, and subsequently to classic protocols. It was shown that some natural substances, through their integration into the classic protocol, produce a synergistic effect with the latter through the stabilization of altered signaling pathways, inhibition of inflammatory factors and increase in immune capabilities. Moreover, some aspects regarding the safety and toxicological involvement of these compounds were analyzed. It must be considered that during the chemotherapy and radiotherapy phase, the dosages taken as integration should be congruent with those that can be taken in the follow-up phase. Some more clinical studies are needed in order to assess the importance of these compounds.