Recent studies have reported that Klotho is expressed in podocytes and protects glomeruli through auto- and paracrine effects. Here, we examined renal expression of Klotho in detail and explored its protective effects in podocyte-specific Klotho knockout mice, and by overexpressing human Klotho in podocytes and hepatocytes. We demonstrate that Klotho is not significantly expressed in podocytes, and transgenic mice with either a targeted deletion or overexpression of Klotho in podocytes lack a glomerular phenotype and have no altered susceptibility to glomerular injury. In contrast, mice with hepatocyte-specific overexpression of Klotho have high circulating levels of soluble Klotho, and when challenged with nephrotoxic serum have less albuminuria and less severe kidney injury compared to wildtype mice. RNA-seq analysis suggests an adaptive response to increased endoplasmic reticulum stress as a putative mechanism of action. To evaluate the clinical relevance of our findings, the results were validated in patients with diabetic nephropathy, and in precision cut kidney slices from human nephrectomies. Together, our data reveal that the glomeruloprotective effects of Klotho is mediated via endocrine actions, which increases its therapeutic potential for patients with glomerular diseases. Transgenic overexpression of αKlotho in hepatocytes results in protection against renal insults, possibly through modulation of the ER stress response by circulating αKlotho. In contrast, αKlotho overexpressed in podocytes is not renoprotective. Klotho knockout mice develop an accelerated aging phenotype characterized by hypervitaminosis D, phosphate retention, growth retardation, soft tissue calcification, infertility, osteoporosis, skin atrophy, and premature death2. The majority of these phenotypes are explained by membrane-bound Klotho forming complexes with fibroblast growth factor (FGF) receptor-1 that bind circulating FGF23 and regulate mineral metabolism4. However, cumulative evidence also points to independent functions of soluble Klotho, which is released from the cell membrane after enzymatic cleavage and can be detected in blood, urine, and cerebrospinal fluid5. Notably, renal Klotho levels are downregulated in patients with acute and chronic kidney disease6 as well as in numerous animal models of kidney injury and fibrosis11. Conversely, Klotho overexpression and treatment with recombinant protein have been demonstrated to be renoprotective in numerous animal models of kidney disease. Although the main effects appear to be on the tubulointerstitium, overexpression in a mouse model of glomerulonephritis alleviated both tubulointerstitial and glomerular damage12. Similar findings were reported in diabetic Ins2Akita mice, in which insertion of a Klotho transgene protected against glomerular injuries13. These two studies are supported by many other reports showing glomeruloprotective effects of soluble Klotho in several kidney disease models14. Considering the critical role of podocytes in the maintenance of glomerular integrity, it is biologically plausible that the beneficial actions of Klotho are mediated, at least partly, through effects on podocytes. In that respect, over the last 5 years, several studies have reported that Klotho is expressed in podocytes and inhibits proteinuria15, diabetes-induced oxidative stress18, podocyte apoptosis19, and glomerular hypertrophy20. Moreover, some single-cell sequencing studies have reported expression of Klotho in podocytes21, supporting a role for Klotho in podocyte biology. These results conflict with earlier studies reporting undetectable expression of Klotho in glomeruli27. As Klotho is a key player in renal physiology and disease, we set out to determine the expression and function of Klotho in murine and human podocytes in vitro, ex vivo, and in vivo. We hypothesized that Klotho is not expressed in podocytes and that its glomeruloprotective effects are mediated through systemic mechanisms rather than via local expression and/or direct effects on podocytes. To address this hypothesis, we assessed murine and human Klotho podocyte expression and generated mouse models with either podocyte-specific Klotho deletion or overexpression, and with systemically increased levels of soluble Klotho. The transgenic mice were challenged using nephrotoxic serum (NTS)-induced glomerulonephritis to examine the potentially protective effects of systemic or podocyte-derived Klotho on the glomerulus.αKlotho (hereafter referred to as Klotho) is a type I membrane-bound protein mainly expressed in renal tubules, parathyroid cells, choroid plexus, and sinoatrial node28, the highest expression of Klotho was found in distal tubular cells, and lower expression was seen in proximal tubular cells. No or extremely low expression was observed in human  26Sortm14(CAG-tdTomato)Hze/J mouse , as previously describedNTS Fig. . HoweverNTS Fig. . PodocytNTS Fig. . FurtherNTS Fig. .To understand whether the reported glomeruloprotective effects of Klotho are mediated via local or systemic effects, we generated a transgenic mouse lines in which human full-length Klotho were overexpressed in either podocytes or hepatocytes. In these transgenic mice, a cassette containing a lox-PolyA-lox construct next to the full-length human KL (hKL) gene fused to a T2A linker-EGFP, under the CAG promoter was introduced into the Rosa26 locus Fig. . The floTo test whether increased expression of Klotho in hepatocytes or in podocytes protect against glomerular injury, we induced NTS glomerulonephritis in Alb-hKL and Pod-hKL mice. All mice developed albuminuria that peaked on day 3 and returned closer to baseline levels on day 7 Fig. . However35, IGF-136, Wnt37, TRPC615, Nrf216, and VEGF38. However, its putative receptors have not been definitely identified. Herein, we employed an unbiased approach to elucidate the mechanism by which soluble Klotho exerts protection of the glomeruli. We performed RNA sequencing of glomeruli- and tubulointerstitial fractions of WT and Alb-hKL mice 7 days after injection of NTS. From the generated datasets, 2725 upregulated and 3573 downregulated genes were identified in the glomerular fraction of Alb-hKL mice compared to WT controls, while only 111 upregulated and 27 downregulated genes were found in the tubulointerstitial fraction  and CHOP (encoded by the DDIT3 gene) were confirmed by immunohistochemistry s48 Fig. . Higher ons Fig. . Additiotry Fig. .Fig. 6LoPodocyte dysfunction is a well-established mechanism behind the progression of proteinuria and kidney diseases. Accumulating evidence strongly supports that Klotho has a protective role against various forms of glomerular injury. Although many pathways through which these protective effects are mediated have been proposed, the mechanism(s) of action remain unclear. Importantly, there are conflicting data on whether Klotho is expressed in podocytes or not. In this study, we addressed these topics through various approaches. First, we analyzed the expression of Klotho at transcript and protein levels in human and mouse kidneys. Using several different methods, we were unable to detect Klotho in human or mouse podocytes, at baseline or after injury. Second, to rule out potential effects of extremely low Klotho expression in podocytes, we generated podocyte-specific Klotho knockout mice and demonstrate that complete deletion of Klotho from podocytes does not induce a glomerular phenotype, nor does it affect the outcome after glomerular injury. Third, we generated a transgenic mouse line allowing conditional overexpression of human full-length Klotho. Notably, mice overexpressing Klotho in podocytes were not protected against glomerular injury. However, increased circulating concentrations of soluble Klotho derived from hepatocytes protected against glomerular injury and helped to maintain glomerular integrity and function. Functional studies in mouse and human tissue identified modulation of ER stress/UPR pathways as a potential mechanism of action.2, renal Klotho expression was described in cortical tubules, with no expression in glomerular cells. This expression pattern was later confirmed by numerous studies in various species, on transcript as well as on protein level51. However, more recently several studies have reported Klotho expression also in the glomeruli, most frequently in podocytes20. Furthermore, Klotho has been reported to protect the podocytes from injury through various mechanisms. Expression of Klotho in podocytes is of importance to understand whether its effects on podocytes are direct or indirect, as it is a large protein (approximately 130 kDa) unlikely to pass the glomerular filtration barrier. Hence, soluble Klotho released from extraglomerular cells would not be able to reach the podocytes and exert its protective effects directly. Herein we wanted to elucidate (i) whether podocytes express Klotho, and (ii) how the reported glomeruloprotective effects of Klotho are mediated.At its discovery in 1997Using two previously validated antibodies and in situ hybridization we failed to detect Klotho expression in podocytes in FFPE or frozen sections from mouse and human kidneys. Similarly, we could not detect Klotho in isolated podocytes by qPCR or Western blot. The specificity of the methods was confirmed using kidneys from tissue-specific and global Klotho knockout mice. Our results from these experiments indicate that Klotho is absent or expressed only at extremely low levels in podocytes. There are different possible explanations for the observed discrepancies regarding expression of Klotho in podocytes, such as the use of non-specific antibodies or contamination by tubular cells that can lead to the detection of a “false positive” signal.31. Therefore, we wanted to explore if the potential low-grade expression of Klotho in podocytes could still have a biological relevant role. Characterization of mice with a targeted deletion of Klotho in podocytes (Pod-KO) revealed no discernable abnormalities in glomerular structure or function. Furthermore, Pod-KO mice challenged with NTS-induced glomerulonephritis developed a similar degree of glomerular injury and proteinuria as WT mice, as evidenced by quantification of albuminuria and histological examination. Together, these data suggest that the protective effects of Klotho against glomerular injury observed in previous studies cannot be attributed to the local expression of Klotho in podocytes but is rather due to the effects of Klotho derived from other cell types. To address this hypothesis, we generated two novel transgenic mouse lines overexpressing full-length human Klotho in podocytes (Pod-hKL) or in hepatocytes (Alb-hKL), which do not express Klotho endogenously. The lack of protection afforded by overexpression of Klotho in podocytes when mice were challenged with NTS-induced glomerulonephritis confirms that locally expressed Klotho is unlikely to have a glomeruloprotective role. In contrast, mice overexpressing Klotho in hepatocytes (Alb-hKL) had significantly less glomerular injury after NTS challenge, supporting the notion that the beneficial role of Klotho in the context of glomerular injury is mediated via systemic effects from its soluble form.Previous studies have demonstrated that Klotho can play a vital role also in tissues where its expression is close to undetectable, such as in osteocytes52. Similarly, in a patient with a translocation causing increased Klotho expression serum FGF23 was markedly increased, and serum phosphate was low due to renal phosphate wasting53. The reason why there was no increase in Fgf23 or altered phosphate handling in our transgenic mice is unclear, but it could speculatively be due to lack of cross reactivity between human soluble Klotho and mouse Fgf23 and/or Fgfr1. Unfortunately, other studies overexpressing human Klotho did not report on serum Fgf23, phosphate, or calcium levels54. The close interdependence of FGF23 and Klotho often makes it difficult to discern FGF23-mediated effects from the effects mediated by soluble Klotho alone. In our study, the very mild increase in Fgf23 levels and the lack of hypophosphatemia suggests that the phenotypic differences in response to glomerular injury is indeed caused by soluble Klotho, and not by elevated Fgf23 and/or lower phosphate.Despite a rather extreme elevation in blood concentrations of Klotho in the Alb-hKL mice , Fgf23 concentrations were only increased by 2-fold with no changes in serum or urine phosphate or calcium compared to WT mice. This is in stark contrast with results from previous studies of mice overexpressing soluble mouse Klotho by injection of adeno-associated virus, which resulted in up to a 450-fold increase in serum Fgf23 and marked hyperphosphaturia leading to hypophosphatemia8, in healthy mice or in mice with glomerular injury and marked proteinuria, and (ii) the renoprotective effects of soluble Klotho appear to be mediated from the blood/systemic side, and not from the urinary side.Of note, despite the extreme elevation in serum concentrations of Klotho there was no increase in urinary Klotho in Alb-hKL mice. Conversely, Pod-hKL mice had higher urinary Klotho concentrations, but unchanged serum concentrations. From these observations we conclude that (i) there is no substantial passage of Klotho over the glomerular filtration barrier, nor an active transcellular transport in the tubuli that is proportionate to the serum concentrations56, and specific inhibition of CHOP was recently shown to reduce glomerular and tubulointerstitial damage in an animal model of diabetic nephropathy57. Moreover, our data suggest that soluble Klotho attenuates ER stress systemically, rather than specifically in the glomeruli. In support of this hypothesis, previous studies have suggested that Klotho modulates ER stress also in tubular epithelial cells and monocytes60. The exact mechanism by which soluble Klotho modulates ER stress and UPR, and the putative intermediate effector(s) remain unknown and is a topic for further studies. Importantly, pathways related to Wnt, VEGF, and insulin signaling were also modified by Klotho overexpression and are potentially part of the renoprotection . All animals, unless specified otherwise, were on C57BL6/J background, housed in standard, single-ventilated cages with 12 h light–12 h dark cycle and had ad libitum access to water and chow. The house temperature was maintained as 20 ± 2 °C and the relative humidity was kept as 50 ± 5%.25 26Sortm1(CAG-KLOTHO-T2A-EGFP)/J mice were crossed with a albumin-cre or podocin-cre line to activate Klotho expression specifically in hepatocytes or podocytes, respectively. Breeding and genotyping were done according to standard procedures.Mice with a podocyte-specific Klotho deletion were generated using Cre-LoxP recombination as described previouslyAnimals between 8 and 13 weeks old were pre-immunized subcutaneously with 400 µl/kg of sheep IgG Freund’s complete adjuvant . Four days later, glomerulonephritis was induced by intravenous injection of 130 μl of nephrotoxic serum (NTS) purchased from Probetex (PTX-001S) and different batches were used for Pod-KO and Pod-/Alb-hKL mice. Experimental mice were maintained under standard animal house conditions in a conventional animal facility for 7 days. After that period, the animals were placed under isoflurane USP (Baxter) anesthesia to proceed to the organ collection. Histopathology of animals was evaluated by scoring at least 30 glomeruli/mice as “normal” or “abnormal” . To assess the extent of tubular cast formation, the whole cross-section was evaluated. The score was determined as follows: 0, no casts; 1, >0–20%; 2, 20–40%, 3, 40–60%; 4, 60–80%; 5, >80% of the tubules are affected.61. Briefly, podocytes were grown under permissive conditions at 33 °C in RPMI 1640 media containing 10% FBS, 10 U/ml interferon-γ. The cells were treated with 5 μg/ml Tunicamycin  and/or 0.05 μg/ml recombinant human Klotho  for 16 h prior to lysis using RIPA lysis buffer (ThermoFisher Scientific) containing protease and phosphatase inhibitors (Roche).Mouse podocytes were cultured as previously describedIn situ hybridization was carried out on paraffin-embedded sections using RNAscope™ (Advanced Cell Diagnostics) according to manufacturer’s instruction. Kidneys from 8-week-old 129S2/SvPasCrl, CD1(ICR), and C57Bl6/J males were stained using probes against the sequence 879-1844 and 49-1754 of the mouse Klotho and Nephrin genes, respectively. For sections from human kidney probes against the sequence 797-1768 of human Klotho gene was used.63. Mouse podocytes from tdTomato mice were isolated as described previously33. Total RNA was extracted using RNeasy mini kit (Qiagen). First-strand cDNA synthesis was carried out using the iScript cDNA Synthesis Kit (Bio-Rad). For real-time qPCR analysis, theCFX96Real-Time PCR Detection System and iQ SYBR Green Supermix (Bio-Rad) were used. The relative gene expression was calculated with the 2−ΔΔCq method normalizing the gene of interest to housekeeping gene in the same sample. Data are presented as relative fold-change. The used primers were hu28S-F: 5′ttgaaaatccgggggagag3′, hu28S-R: 5′acattgttccaacatgccag3′, huKL-F: 5′tccatctgggatacgttcaccc3′, huKL-R: 5′tgtcgcggaagacgttgttg3′, huNPHS1-F: 5′gagagccccattcaaaggct3′, huNPHS1-R: 5′agaaggagctcacggtttcg3′, huHSPA5-F: 5′cgggcaaagatgtcaggaaag3′, huHSPA5-R: 5′ttctggacgggcttcatagtagac3′, huHSP90B1-F: 5′ggatggtctggcaacatgga3′, huHSP90B1-R: 5′ccgaagcgttgctgtttcaa3′, huuXBP1-F: 5′tccgcagcactcagactacg3′, huu/sXBP1-R: 5′agttgtccagaatgcccaaca3′, husXBP1-F: 5′ctgagtccgcagcaggtg3′, huDDIT3-F: 5′ggagcatcagtcccccactt3′, huDDIT3-R: 5′tgtgggattgagggtcacatc3′, mmGapdh-F: 5′tgttcctacccccaatgtgt3′, mmGapdh-R: 5′tgtgagggagatgctcagtg3′, mmNphs1-F: 5′cttttggcttcgctgtcacc3′, mmNphs1-R: 5′aaggcccagattgcatcgta3′, mmKl-F: 5′tctcaagaattcataatggaaacc3′, mmKl-R: 5′cagaaagtcaacgtagaagagtcct3′.Glomeruli were isolated from human and mouse kidneys as published previouslyKidneys were fixed with 4% paraformaldehyde and subsequently embedded with paraffin. Paraffin-embedded kidney sections (4μm) were processed for the following staining. For immunohistochemical staining of GRP78/BiP and CHOP, a HIER was performed using a Decloaking Chamber  set for 5 min at 110 °C in Citrate buffer pH 6  followed by a 15 min blocking step using Biocare Medical Background Sniper. The primary antibodies: mouse monoclonal anti-CHOP  and rabbit monoclonal anti-GRP78/BiP (clone [EPR4041(2)]; ab108615, 1:2000, Abcam) were diluted in Renior Red diluent  and incubated at 4 °C overnight in a humid chamber. For detection, the Mach-1 Universal HRP-Polymer Kit was used  according to protocol. For all the slides, nuclear counterstaining was performed using Mayer hematoxylin solution. Tissue sections were observed on a Zeiss Observer Z1. For immunofluorescent studies, after HIER treatment with Tris-EDTA pH9 (Citrate pH 6 for anti-GFP staining) for 20 min at 100 °C, sections were blocked using 10% goat serum + 0.1% Tween 20 solution for 1 h at room temperature. sections were incubated with anti-nephrin , anti-podocin  anti-vimentin , anti-WT1 , anti-Klotho , anti-αSMA – Cy3™ , anti-GFP  at 4 °C overnight, anti-synaptopodin  for 1 h at room temperature and fluorescein-labeled Lotus Tetragonolobus Lectin  for 1 h at 37 °C. Alexa Fluor conjugated secondary antibodies were used for visualization (Invitrogen). All the slides were co-stained with Hoechst 33342  for 10 min at room temperature. Sudan black B  solution was applied for 25 min at room temperature before mounting in Dako fluorescent medium .Immunofluorescent studies from Supplementary Fig. Immunofluorescent studies from Supplementary Fig. 64. In brief, formaldehyde/formalin fixed kidneys were embedded in 3% agarose in DI water and then sectioned to 200 µm thickness using a Vibratome. Slices were then incubated in clearing solution  at 50 °C overnight. Sections were washed in PBST  for 10 min before incubation in a sheep polyclonal antibody against nephrin  diluted at 1:100 and a rat monoclonal against Klotho , diluted at 1:200 in 1X PBS with 0.3% Triton X-100 (PBST) at room temperature overnight. After primary antibody incubation, samples were washed in PBST for 5 min at room temperature and were then incubated in a donkey anti-sheep secondary antibody conjugated to Alexa-488  diluted at 1:100 and a donkey anti-rat secondary antibody conjugated to Alexa-647  diluted at 1:100 in PBST at room temperature overnight. Samples were incubated in 80% wt/wt fructose (1 mL of dH20 added to 4 g of fructose with 0.25% 1-Thioglycerol) at 37 °C for 15 min and then mounted in a MatTek dish with a cover slip on top prior to imaging using a Leica SP8 3X gSTED system using either a 20 × 0.75 NA oil immersion objective or a 100 × 1.4 NA oil immersion objective.A slightly modified version of a previously published protocol was usedTEM was carried out by the electron microscopy unit core facility (EMil) at Karolinska Institutet, Huddinge University hospital per their standard protocols.TM ECL western blotting substrate  and the Licor Odyssey Fc imaging system (Licor).For Western blot analysis, glomeruli and rest of kidney fractions were lysed in RIPA lysis buffer (ThermoFisher Scientific) containing protease and phosphatase inhibitors (Roche) for 15 min on ice. The protein content of the samples was determined using Pierce™ bicinchoninic acid assay kit (ThermoFisher Scientific). Equal amounts of protein were separated on sodium dodecylsulfate–polyacrylamide gels, blotted onto polyvinylidene fluoride membranes and blocked in 5% nonfat dry milk in triethanolamine-buffered saline with 0.1% Tween-20. Western blots were probed with antibodies directed against klotho , podocin , calnexin , GRP78/BiP  and LC3B . Secondary antibodies were purchased from ThermoScientific. For detection method, we used PierceSerum and urine calcium, phosphate, and creatinine were measured using colorimetric kits . Intact FGF23 was measured using an intact FGF23 ELISA kit . The urinary albumin/creatinine ratio was measured by using the commercial kits Albuwell  and Quantichrome creatinine assay kit . The fractional excretion of phosphorus (FEPi) and calcium (FECa) were calculated as [/].63. Total RNA was extracted using TRIzolTM reagent (ThermoFisher Scientific). RNA quality was assessed by determining its RNA integrity number (RIN). mRNA-seq libraries were constructed using a TruSeq RNA SamplePrep kit (Illumina) according to the manufacturer’s instructions. Sequencing was conducted for 75 cycles on Illumina Nextseq 550 sequencer according to the manufacturer’s instructions.Glomeruli were isolated from mouse kidneys as published previously65 was used to index the mouse reference genome (mm10/GRCm38) and align the resulting fastq files. Mapped reads were then counted in annotated exons using featureCounts v1.5.166. The annotations and reference genome were obtained from Ensembl. The count table from featureCounts was imported into R/Bioconductor and differential gene expression was performed using the EdgeR67 package and its general linear models pipeline. Both conditions were analyzed by using four biological replicates. For the gene expression analysis, genes that had 1 count per million in 3 or more samples were used and normalized using TMM normalization. Gene expression analysis was also performed using voom/limma. The gene counts were transformed to log2-counts per million using voom68 with samples weights and subsequent differential gene expression analysis was performed in limma69. Genes with an FDR adjusted p value <0.05 were termed significantly regulated.Bcl files were converted and demultiplexed to fastq using the bcl2fastq v2.20.0.422 program. STAR 2.7.5b70. Annotation clusters identified by DAVID (clusters of related annotation terms) having an enrichment score of ≥1.3 were considered significant and a representative naming for the cluster was derived from the contained Gene Ontology terms and KEGG pathway analysis.Gene functional enrichments were determined using the DAVID Bioinformatics Resources (v. 6.8)49. Human kidney pieces were decapsulated and embedded in 3% agarose prewarmed at 40 °C. Slices were cut in ice-cold DPBS using a vibrating microtome Compresstome® VF310-0Z (Precisionary Instruments). Immediately after, the 300-μm-thick slices were pre-incubated containing Williams’ Medium E + GlutaMAX™-I  supplemented with 25 mM D-glucose and 1% Penn-Strep  for 1 h at 37 °C under 80% O2 and 5% CO2 atmosphere to allow the slices to restore their ATP levels71. Slices were then cultured for an additional 24 h with 1 μM tunicamycin72  and 0.05 μg/mL recombinant human Klotho  at 37 °C, 80% O2 and 5% CO2 on an orbital shaker at 80 RPM. Slices were harvested in 10% formalin solution  for histology analysis and in RNAlater for RNA isolation. All conditions were carried out as three technical replicates.A modified version of a previously published protocol was usedt-test or one-way ANOVA followed by Tukey’s multiple comparisons test when appropriate. Urinary albumin-creatinine ratio time course was analyzed using a mixed model ANOVA. Pearson correlation was used to analyze the association between the level of klotho transcript expression and ER/UPR transcripts expression in human. A p value inferior to 0.05 was considered statistically significant. All data are presented as mean ± SD.Statistical analysis was performed with GraphPad Prism software . For in vivo, in vitro, and ex vivo experiments, data were analyzed using Student’s Further information on research design is available in the Supplementary informationDescription of Additional Supplementary DataSupplementary Data 1Reporting summary


Bone marrow or hematopoietic stem cell transplantation (HSCT) is a curative treatment option for hematological malignancies, several non-malignant hematological disorders and severe combined immune deficiencies. However, relapse, graft-versus-host disease (GVHD) and opportunistic infections represent major complications following allogeneic HSCT which compromise the survival and quality-of-life of the recipient, limiting the wider application of this immunotherapy. Basic research has significantly advanced our understanding of the underlying mechanisms of these complications and has led to the development of promising pharmaceutical, immune and cellular therapies. Despite intensive research and development, GVHD, infection and relapse remain significant clinical problems and represent major unmet needs in HSCT.Frontiers In Immunology Research Topic presents the latest insights from preclinical studies of HSCT, highlighting opportunities and challenges, and sheds light on the future of HSCT. The collection is comprised predominantly of review articles which focus on GVHD pathophysiology, regulatory T cell-mediated immune tolerance, antigen presentation, microbiota/metabolite modulation of GVHD, intestinal immunopathology and infectious pulmonary complications, with an emphasis on the biological relevance to the clinic and translational potential.This Teshima and Hill provides a historical overview of major experimental concepts in bone marrow transplantation discovered using murine models and the translation of these findings into clinical practice, highlighting the pivotal insights afforded by murine models.The perspective article by Guo et al. review the biology of Tregs, their role in GVHD and GVL and the challenges related to Treg adoptive cell therapy to treat and/or prevent GVHD. The authors review Treg expansion strategies and discuss new sources of Tregs to address challenges encountered with this therapy. In the review by Ikegawa and Matsuoka, the authors detail the importance of Treg homeostasis post-HSCT and discuss strategies to manipulate this to modulate post-HSCT immunity to treat and/or prevent GVHD.Since the discovery of regulatory T cells (Tregs) by Sakaguchi in 1995, Tregs have been of great interest to researchers studying immune tolerance and regulation after HSCT. In this collection, Ara and Hashimoto review historical and recent advances in our understanding of the cellular and molecular mechanisms of GVHD-induced tissue damage in the gastrointestinal tract, providing important novel insights garnered from mouse models which inform translational potential. With technological advances in genomic/metagenomic sequencing and metabolomics, the ability to study intestinal microorganisms and their secreted molecules in HSCT has rapidly generated new knowledge in the field. In this collection, Fujiwara provides an in-depth review of the intestinal microbiota, metabolite and epithelial cell crosstalk critical in GVHD regulation, highlighting the complex relationships and potential microbiota-based therapeutic strategies.The cardinal feature of GVHD is immune-mediated tissue pathology. Gastrointestinal GVHD is the most challenging to treat and the greatest cause of GVHD-related mortality. In this collection, Zhou and Moore review murine models of infectious pulmonary complications after HSCT  focusing on host-pathogen interactions and the important insights garnered from these studies.Conditioning-induced toxicity, immunosuppressive therapies and alloimmunity permit opportunistic infections post-HSCT, a major cause of morbidity and mortality. Koyama and Hill review the field and discuss recent advances uncovered by the use of antigen-specific murine models of GVHD, providing novel insights for the translational potential of targeting this pathway. Critically, the authors review antigen presentation in xenograft transplantation models used to study GVHD and GVL and discuss the controversies associated with the interpretation of findings from these models.The therapeutic effect of HSCT, mediated by alloimmunity towards malignant cells , is compromised by detrimental alloimmunity towards recipient tissues (GVHD). Antigen presentation is central to these two processes, however the ability to delineate between these remains elusive and is considered the holy grail in HSCT. Song et al. review important mechanistic findings identified using murine models of chronic GVHD which reflect features characteristic of the disease in humans. The authors highlight low P-selectin glycoprotein ligand 1 expressing CD4 T cells as key players in cGVHD pathophysiology. Original research presented by Choi et al. revealed that deletion of the endoplasmic reticulum stress protein, XBP-1, reduced B cell activity and the ability to stimulate allogeneic CD4 T cells via a regulated IRE-1a-dependent decay pathway which led to a reduction in cGVHD. These preclinical findings demonstrate targeting XBP-1 a potentially useful strategy to ameliorate cGVHD.Chronic GVHD continues to be a major clinical problem in HSCT survivors. Shaikh et al. present a detailed description of the development of a murine model of orthotopic mesenteric lymph node transplantation to study immune cell responses and migration in the gastrointestinal tract. The authors determined its utility in a HSCT setting and suggest potential for broader application.Finally, In summary, murine models have proven to be instrumental for advancement of our understanding of the complications that occur following HSCT. As demonstrated in this collection, murine models of HSCT have led to the discovery of new scientific knowledge, permitted established concepts to be challenged and emerging concepts to be explored, enabled the development of new therapeutics and provided important insights that have guided clinical studies. With the development of new ideas, technologies and tools, murine models of HSCT will continue to empower academic research and translation into the clinic in the future.All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


As a cancer type potentially dominated by copy number variations, ovarian cancer shows hyperploid karyotypes and large-scale chromosome alterations, which might be promising biomarkers correlated with tumor metastasis and chemoresistance. Experimental studies have provided more information about the roles of aneuploids and polyploids in ovarian cancer. However, ploidy evaluation of ovarian cancer cell lines is still limited, even in some ploidy-related research. Herein, the ploidy landscape of 51 ovarian cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) were analyzed, and the ploidy statuses of 13 human ovarian cancer cell lines and 2 murine cell lines were evaluated using G-banding and flow cytometry. Most human ovarian cancer cell lines were aneuploid, with modal numbers of 52–86 and numerical complexity ranging from 5 to 12. A2780, COV434 and TOV21G were screened as diploid cell lines, with a modal number of 46, a low aneuploid score and a near-diploid ploidy value. Two murine cell lines, both OV2944-HM1 and ID-8, were near-tetraploid. Integrated information on karyotypes, aneuploid score and ploidy value supplied references for a nondiploid model construction and a parallel analysis of diploid versus aneuploid. Moreover, the gene expression profiles were compared between diploid and aneuploid cell lines. The functions of differentially expressed genes were mainly enriched in terms of protein function regulation, TGF-β signaling and cell adhesion molecules. Genes downregulated in the aneuploid group were mainly related to metabolism and protein function regulation, and genes upregulated in the aneuploid group were mainly involved in immune regulation. Differentially expressed genes were randomly distributed on all chromosomes, while chromosome 1 alteration might contribute to immune-related alterations in aneuploid cell lines. Chromosome 19 alteration might be potentially significant for aneuploid ovarian cancer cell lines and patients, which needs further verification in ploidy research. Ovarian cancer is the major cause of death among gynecological cancers. With an insidious onset and rapid progression, approximately 75% of ovarian cancer patients are already at an advanced stage when diagnosed, with less than a 30% five-year survival rate ,2. Nearlhttp://atlasgeneticsoncology.org (accessed on 1 October 2022)). Flow cytometry or image cytometry showed that nondiploid cancer cells existed in approximately 36%~75% of ovarian cancer patients, potentially affecting therapy choice and prognostic prediction [According to our previous summary of ploidy studies, many observational studies and correlation analyses of aneuploidy and chromosomal instability have been performed in patient samples . Karyotyediction ,13,14. Aediction ,16. Copyediction ,18,19. Vediction ,22,23. Tediction ,25. OverConsidering the prevalence and significance of karyotypic evolution in ovarian cancer, it is significant to take the ploidy status into consideration in experimental research, which is important for uncovering the underlying mechanisms of ploidy-mediated chemoresistance and exploring ploidy-targeted cancer therapy. The ploidy evaluation of cell lines is the first step in cancer ploidy research because the cell line models are the most basal tools in cancer research. Moreover, a comparison analysis between diploidy and aneuploidy cell lines is also inspiring and can help to explore potential new therapeutic targets based on aneuploid or polyploid cell lines in cancer. Recently, a comparative study based on diploid versus aneuploid cell lines overturned the concept that the knockdown of small GTPase Ran (Ras-related nuclear protein), a potential therapeutic target in cancer, causes cancer cell death but does not affect normal cells . This reHowever, unlike CIN evaluations of patient samples, few basic studies have focused on the ploidy status and chromosomal alteration of ovarian cancer cell lines, and even fewer studies have assessed their basic ploidy status, even though some cell lines are commonly used, which makes current ploidy research in ovarian cancer confusing. Among various ovarian cancer cell lines, a previous CIN study simultaneously evaluated the response to CIN inducers with or without proteasome inhibitors, and tried to uncover the interplay between CIN and sensitivity to proteasome inhibition . HoweverHere, we evaluated the ploidy status and chromosomal alterations of 13 commonly used ovarian cancer cell lines for reference and inspiration for further research in ovarian cancer. To better understand the difference in expression profiles between the diploid and aneuploid groups and find potential targets for nondiploid cancer, high throughput RNA-sequencing and enrichment analysis are performed in this study. The effects of chromosome alterations on gene expression were analyzed via chromosome enrichment.www.cbioportal.org (accessed on 15 October 2022)). The pathway and process enrichment analyses of genes enriched in specific chromosomes in cell lines and patients were performed using a metascape analysis tool [A total of 51 ovarian cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) and 582 ovarian cancer patients were involved in this study . The anesis tool .2.A total of 15 cell lines were used for the experiments, including 13 human ovarian cancer cell lines  and 2 murine cell lines (OV2944-HM1 (HM-1) and ID-8). All cell lines were archived in our laboratory and the HM-1 cell line was a gift from Professor Haiou Liu . The cells were cultured in an RPMI-1640 or DMEM or MC5A medium containing 10% fetal bovine serum. All the cell lines were cultured at 37 °C in an incubator with 5% COA karyotype analysis was performed using the classic cytogenetic method ,36. The Ploidy analysis was performed according to previous analyses ,13. The ® Ultra™ Directional RNA Library Prep Kit for Illumina  according to the manufacturer’s instructions. After purification and enrichment, the final cDNA libraries were quantified by an Agilent2200. The tagged cDNA libraries were pooled in equal ratios and used for 150 bp paired-end sequencing in a single lane of the Illumina HiSeqXTen . The raw data were filtered to remove the adaptor sequences and low-quality reads. The clean reads were then mapped to the human genome (GRCh38_Ensembl104) using the Hisat2 [The total RNA was extracted from 6 ovarian cancer cell lines by Trizol reagent  separately. The RNA quality was checked by an Agilent 2200 . Only samples with an RIN (RNA integrity number) >7.0 were acceptable for cDNA library construction. The cDNA libraries were constructed using the NEBNexte Hisat2 . HTseq we Hisat2 .p-value < 0.05.A2780, COV434 and TOV21G were categorized as diploid cell lines and NIHOVCAR3, OVISE, OVCAR8 were categorized as aneuploid cell lines. DESeq2 (V1.6.3) was used to filter the differentially expressed genes (DEGs) among diploid cell lines versus aneuploid cell lines. The DEGs were determined by a log2FC of 1 and an adjusted FDR value of 0.05 . Gene onp < 0.05 was considered significant.The difference in ploidy value of 51 cell lines among different pathological types was analyzed with the Kruskal–Wallis Test. Fisher’s exact test was applied to identify the significant GO and KEGG pathways. The ploidy statuses of 51 ovarian cancer cell lines were obtained via cBioPortal online analysis, which is based on CNV data and ploidy estimation using the ABSOLUTE algorithm A. Most oFCM and G-banding were performed to assess the karyotypes and chromosome numerations of 13 commonly used human cell lines and 2 murine cell lines. According to the karyotype analysis using G-banding, only three ovarian cancer cell lines, A2780, TOV21G and COV434, were diploid cell lines with a modal number of 46 and karyotypical stability with relatively low numerical complexity A. The otHowever, some tetraploid cell lines presented low numerical complexity, such as SKOV3 and IGROV1. The ploidy analysis also revealed a significant difference between diploid and aneuploid cells using FCM, consistent with the karyotype analysis B. Most hConsidering the three parameters in A protein–protein interaction (PPI) enrichment analysis showed some interactions among the downregulated and upregulated genes in aneuploid cell lines A. A geneChromosome 1 (chr1) and chromosome 19 (chr 19) alterations are among the most frequent cytogenetic changes in epithelial ovarian cancer ,44,45,46To better understand the association between potential crucial chromosome alterations and ploidy status in ovarian cancer patients, we analyzed the genomic alteration between euploid and aneuploid ovarian cancer patients. A total of 582 ovarian cancer patients were included and ranked by aneuploid score from highest to lowest, which partially reflects the aneuploid level . The topIn this study we evaluated the karyotype and chromosomal numerical complexity of 13 human ovarian cancer cell lines and 2 murine cell lines using G-banding and flow cytometry. According to the integrated ploidy status based on three parameters, modal number, ploidy value and aneuploid score, three relatively stable diploid cell lines, A2780, COV434 and TOV21G, were screened in our study and can be used for nondiploid model construction for further aneuploidy and polyploidy research in ovarian cancer. However, most commonly used human and murine ovarian cancer cell lines are aneuploid or hyperploid, showing a relatively high ploidy value and high aneuploid score and large-scale numerical changes.Many ploidy studies have used the diploid colon cancer cell line HCT116 and the non-transformed diploid epithelial cell line RPE-1 to construct paired aneuploid and polyploid cell models ,32,47. HIn addition to supplying diploid alterations for model construction, the ploidy status of commonly used cell lines provides inspiration for future comparative analyses of diploid and aneuploid cell lines in ovarian cancer. Nondiploid cells are different from diploid cells in terms of cell fitness and characteristics, featuring worse genomic, metabolic, proteotoxic stress and immune stress, manifested as senescence and cell cycle arrest ,50. ThroSome commonly used chemotherapy drugs are not selectively lethal for cancer cells and usually cause the death of normal cells with rapid reproduction, such as carboplatin and paclitaxel. A previous study revealed that these two drugs showed no selectivity between diploid and aneuploid cells, although most cancers are highly aneuploidy ,52,53. TA comparative analysis of expression profiles showed that, compared with diploid cell lines, genes related to oxidative phosphorylation, lipid metabolism and regulation of protein function were downregulated in the aneuploid group. Consistent with the survival stress reported in nondiploid cells , metabolFor the integrated evaluation of ploidy status, it might be more comprehensive to combine cytogenetic methods and sequence or array data to obtain the overall and detailed chromosome variations. In our research, the cytological data of SKOV3 and IGROV1 indicated that they are near-tetraploid, but the ABSOLUTE estimation showed that the ploidy status was 1.9 and 2.0, respectively. The discordance of the ploidy evaluation results might be due to the difference between the two methods. The aneuploid score or the ploidy value using ABSOLUE estimation is based on copy number variations but cannot fully reflect cytogenetics . AlthougThe largest limitation of this study is that the functional regulation of differentially expressed genes was not verified; more work will be needed for clarification in ploidy research. Additionally, the number of ovarian cancer cell lines used in our study was limited. Despite these limitations, the ploidy information and comparison analysis in this study still provide inspiration for ploidy research in ovarian cancer.In conclusion, we analyzed the ploidy status of 51 ovarian cancer cell lines using the cBioPortal and evaluated the karyotype numerical complexity of 13 commonly used human ovarian cell lines and 2 murine cell lines using G-banding and flow cytometry. We explored the biological function of differentially expressed genes between a diploid and aneuploid group categorized by modal number, ploidy value and aneuploid score. Inflammation- and immune-related genes were overexpressed in the aneuploidy group while metabolism- and protein-related genes were downregulated in aneuploid cell lines. Chromosome enrichment suggested that chromosome 1 and chromosome 19 alteration might contribute to ploidy-related alterations in gene expression. In future research, on the basis of the heterogeneous ploidy status of cell lines, more efforts need to be made to uncover ploidy-mediated chemoresistance and explore ploidy-targeted cancer therapy in further ploidy studies in ovarian cancer.


The intratumor heterogeneity (ITH) of cancer cells plays an important role in breast cancer resistance and recurrence. To develop better therapeutic strategies, it is necessary to understand the molecular mechanisms underlying ITH and their functional significance. Patient-derived organoids (PDOs) have recently been utilized in cancer research. They can also be used to study ITH as cancer cell diversity is thought to be maintained within the organoid line. However, no reports investigated intratumor transcriptomic heterogeneity in organoids derived from patients with breast cancer. This study aimed to investigate transcriptomic ITH in breast cancer PDOs.We established PDO lines from ten patients with breast cancer and performed single-cell transcriptomic analysis. First, we clustered cancer cells for each PDO using the Seurat package. Then, we defined and compared the cluster-specific gene signature (ClustGS) corresponding to each cell cluster in each PDO.Cancer cells were clustered into 3–6 cell populations with distinct cellular states in each PDO line. We identified 38 clusters with ClustGS in 10 PDO lines and used Jaccard similarity index to compare the similarity of these signatures. We found that 29 signatures could be categorized into 7 shared meta-ClustGSs, such as those related to the cell cycle or epithelial–mesenchymal transition, and 9 signatures were unique to single PDO lines. These unique cell populations appeared to represent the characteristics of the original tumors derived from patients.We confirmed the existence of transcriptomic ITH in breast cancer PDOs. Some cellular states were commonly observed in multiple PDOs, whereas others were specific to single PDO lines. The combination of these shared and unique cellular states formed the ITH of each PDO.The online version contains supplementary material available at 10.1186/s13058-023-01617-4. There is mounting evidence that cancer cell heterogeneity within tumors plays an essential role in part of cancer resistance and recurrence , 2. In bIn recent years, three-dimensional organoid culture methods have been actively utilized in cancer research , 7 becauIn the present study, we use single-cell RNA-seq (scRNA-seq) to show that breast cancer PDOs are composed of several cell clusters with distinct cellular states. Furthermore, we define a cluster-specific gene signature (ClustGS) for each cell cluster in PDOs, and successfully capture one aspect of ITH. We found that seven ClustGSs were shared among multiple PDOs and some ClustGSs were specific to single PDOs only. The former represent cellular states related to the cell cycle, EMT, and estrogen response, whereas the latter represent cellular states often not clearly defined. These unique gene signatures specific to a certain PDO may reflect the biological characteristics of that PDO, indicating the assessment of cellular transcriptomic ITH may be helpful for characterizing PDOs.Surgical specimens were obtained by the core needle biopsy of surgically removed tumors. The specimens were dissociated into single cells using a MACS Tumor Dissociation Kit and a gentle MACS dissociator  in accordance with the manufacturer’s instruction. For pleural effusion specimens, drainage fluid was collected after trocar placement, centrifuged, and purified with Red Blood Cell Lysis Solution lysis buffer (Miltenyl Biotec). The organoids were established from the obtained cells as described previously . BrieflyOrganoids were dissociated into a single-cell suspension using harvesting solution and trypsin, and the cells were cryopreserved for the scRNA-seq experiment. Moreover, BC specimens were dissociated into single cells, as described above. The scRNA-seq libraries were prepared using the BD Rhapsody Single-Cell Analysis system  in accordance with the manufacturer's instruction. Briefly, after thawing, cells were labeled with BD Single-Cell Multiplexing Kit. The labeled cells were washed, pooled, and loaded onto the BD Rhapsody microwell cartridge; then cDNA was synthesized using the BD Rhapsody Whole Transcriptome Analysis Amplification Kit. The resulting gene expression and cellular label libraries were sequenced on Illumina NextSeq 550 platform  with paired-end reads . Sequencing data were processed using the BD Rhapsody Analysis pipeline on the Seven Bridges Genomics platform and converted to a gene expression count matrix.https://www.sevenbridges.com/bdgenomics/) to generate the count matrixes and Seurat [We utilized the web-based analysis pipeline for BD Rhapsody and BD Precise ASSAYS  were not considered for further analysis. Meta-clusters were manually defined by the Jaccard similarity index. All genes included in at least two ClustGS were defined as meta-cluster specific genes (meta-ClustGS).After clustering of each PDO, we identified differentially expressed features as cluster-specific gene expression signatures (ClustGS) by using the “FindAllMarkers” function with “only.pos = TRUE, min.pct = 0.25, logfc.threshold = 0”. We removed mitochondrial RNAs or ribosomal protein-encoding genes and selected the genes with an adjusted P-value.Gene enrichment analysis for each ClustGS was performed using the “enrichr” function of ClusterProfiler v3.18.1  with MsiGene overlap analysis between ClustGS and cluster-specific expressed genes of corresponding primary tumors or different passage organoids was performed using “GeneOverlap” package’s newGeneOverlap and testGeneOverlap functions. To obtain cluster-specific expressed genes in each dataset, we selected the genes with < 0.05 adjusted P-values calculated by Seurat’s FindAllMarkers with options of “only.pos = TRUE, min.pct = 0.25, logfc.threshold = 0”.The extracellular cytokines in the culture medium were comprehensively semi-quantified using the Proteome Profiler Array Human XL Cytokine Array Kit (R&D Systems) in accordance with the manufacturer’s instruction. Briefly, culture medium from four organoids  was collected 72 h after the change of fresh medium and frozen at − 20 °C. Between 59.5 and 846.2 µL of the culture medium, normalized by RNA quantity from the PDOs in the same culture, was loaded into the cytokine array. The Odyssey XF Imaging System  was used to detect fluorochromes; the absorbance was set to 685 nm and the exposure time was set to 10 min. The signal intensity corresponding to each factor in the array was quantified by ImageJ software, and the expression level was calculated relative to the positive control.Tumor sections were stained with hematoxylin–eosin (HE) and immunohistochemically examined for CHI3L1 and CST3 using a Nichirei Histofine system (Nichirei Biosciences Inc.). Tissue sections were incubated with primary anti-CHI3L1 antibody  and anti-CST3 antibody  at a 1:800 and 1:4000 dilution, respectively, and detected with HRP-labeled polymer-conjugated secondary antibody . Color development was achieved with 3,3′-diaminobenzidine tetrahydrochloride. Organoids were fixed and paraffin-embedded using Epredia HistoGel Specimen Processing Gel (Thermo Scientific) and Tissue-Tec VIP . Immunohistochemical staining on the organoid sections was performed by incubating with anti-CHI3L1 antibody  and anti-CST3 antibody  at a 1:400 and 1:500 dilution, respectively. Signals were detected using Dako EnVision + Dual Link System-HRP (Agilent).Ten PDO lines established during a certain period of time were subjected to the analysis in this study Fig. A. The clThe speed of organoid growth differed for each PDO. PDO155, 166, and 207P had the fastest growth rate, with a doubling time of approximately 7 days, which continued even after ten passages. PDO 165 and 180 grew more slowly than the three PDO lines listed above, with a doubling time of approximately 14 days. The other lines grew more slowly.Organoid morphology was also heterogeneous among PDOs Fig. B, C. SphCollectively, our results present the inter- and intratumor heterogeneity for organoid growth and morphology, suggesting that the PDOs reflect the biological properties of the original tumors.EPCAM and keratins, and we considered that the PDO lines used in this study contained nearly no stromal cells. Therefore, we hereby note that the subsequent analysis assesses the ITH of cancer cells.We performed single-cell transcriptome analysis of 10 PDOs by multiplexing scRNA-seq using the BD Rhapsody Single-Cell Analysis System. After assignment and quality filtering, we obtained the high-quality expression profiles of 9548 cells: an average of 6635 counts and 2203 genes per cell  were not considered for further analysis. Each PDO had at least one cluster with ClustGS, and in total, 38 clusters with a ClustGS were identified ; PDO165#C2 contained TGFB2 and DKK1; PDO165#C3 contained cell cycle-related genes ; and PDO165#C4 contained basal keratins   and two different passages of PDO lines (180 and 210). Subsequently, we performed the same analysis as above, and confirmed the existence of ITH in each sample  in meta-ClustGS 3, mesenchymal marker ACTA2 in meta-ClustGS 4, and the metastasis-associated gene ANXA1 in meta-ClustGS 6. There were also two meta-ClustGSs associated with estrogen response: luminal-lineage specific TFs FOXA1 and GATA3 in meta-ClustGS 5, CCND1 in meta-ClustGS 7. These results suggested that each PDO contained several cell populations that were regulated by the defined gene expression programs that were common across PDOs.To compare the degree of ITH between PDOs, we calculated the Jaccard similarity index across 38 ClustGS from each cluster. Based on the similarity of ClustGS, seven meta-clusters were identified with the hierarchical clustering guide, representing a set of cell clusters characterized by the same expression programs across PDOs  and meta-ClustGS 5 (estrogen response 1) containing luminal-lineage TFs (FOXA1 and GATA3) were observed in four PDO lines  derived from patients with ER+ /HER2– or ER–/HER2+ tumors, suggesting that these PDOs had distinct ITH of co-existing of basal-like and luminal cells  was present in 7 of 10 PDO lines , pleural effusion sample (PDO207P), and early recurrence (PDO210). These suggested that meta-ClustGS 7 may reflect a different cellular state to normal ER-positive breast cancer cells with a good prognosis, even though it is annotated with the same estrogen response by MsigDB analysis. For example, meta-ClustGS 7 contained  outcome , 20, sugThese results indicate that meta-ClustGSs can describe each PDO with a qualitative and quantitative assessment of ITH.In the process of identifying meta-clusters, we also identified nine PDO-specific clusters . We then compared the cellular states within and between PDOs by examining the similarity of the ClustGSs. Seven meta-ClustGSs, reflecting cellular states, were common among multiple PDOs, and nine ClustGSs were found to be unique to individual organoids. The common meta-ClustGSs included cell states related to the cell cycle, EMT, and estrogen response. In contrast, the cellular states unique to the individual PDOs appeared to display unique characteristics for that specific PDOs. These results indicated that we may be able to estimate the unique characteristics of a particular PDO by examining the ITH within the PDO; a comparison with other PDOs may not be necessary.Using single-cell RNA-seq technology, we confirmed the existence of ITH in breast cancer PDOs. Several studies have reported the ITH of cancer cells and its characteristics in cancer tissues and cell lines , 18, 28.We also observed patient-specific ITH patterns in several PDOs. Previous studies tended to focus on the recurrent patterns of ITH and ignore unique ITH , 29. HowMYC amplification (data not shown) and may have enhanced ribosome biogenesis [From this perspective, we focused on PDO155 derived from a patient with aggressive inflammatory breast cancer (IBC). IBC is a cancer subtype with a poor prognosis characterized by extensive lymphatic embolism and cutaneous inflammation, and its etiology and molecular mechanisms are poorly understood , 30. PDOogenesis . The abnogenesis , 32. AltWe described the landscape of transcriptomic ITH within breast cancer PDOs. We observed cell populations with cellular states common to various PDOs and cell states specific to individual PDOs. The latter could reflect the unique biology of the tumor of origin.Additional file 1. Figure S1: A and B. QC metrics showing unique feature counts and percentage of mitochondrial RNA per cell. C. UMAP visualization of scRNA-seq profiles from 10 PDOs. Each dot represents a single-cell colored by its corresponding PDOs. D and E. UMAP overlay representing expression levels of EPCAM and KRT8.  Figure S2: Hallmark terms and adjusted p-values detected by MSigDB hallmark gene enrichment analysis for each ClustGS in PDO165.  Figure S1: A and C. UMAP visualization of 1,822 and 6,362 cells annotated as EPCAM-positive cells within scRNA-seq profiles of primary breast cancer tissues of patients 165 (A) and 210 (C). B and D. Heatmaps showing the number (top) and significance (bottom) of overlapping genes between each ClustGS identified in indicated PDOs and each cluster-specific expressed gene list identified in its original tumor tissues.  Figure S4: A and B. UMAP visualization of scRNA-seq profiles of PDO180 P6 (A) and PDO210 P8 (B). C and D. Heatmaps showing the number (top) and significance (bottom) of overlapping genes between each ClustGS identified in indicated PDOs and each cluster-specific expressed gene list identified in PDOs.  Figure S5: Hallmark terms and adjusted p-values detected by MSigDB hallmark gene enrichment analysis for 7 meta-ClustGSs.  Figure S6: Heatmap showing which meta-ClustGS are present or absent in each PDO.  Figure S7: HE staining and immunohistochemistry (CHI3L1 and CST3) of PDO155 and its original tumor tissue.Additional file 2.Table S1: 38 ClustGSs across 10 PDOs.Table S2: Cluster-specific expressed gene lists in P165 and P210.Table S3: Cluster-specific expressed gene lists in PDO180 P6 and PDO210 P8.Table S4: Meta-ClustGSs.Table S5 PDO-specific ClustGSs.


Genes associated with growth factors were previously analyzed in a radiation- and estrogen-induced experimental breast cancer model. Such in vitro experimental breast cancer model was developed by exposure of the immortalized human breast epithelial cell line, MCF-10F, to low doses of high linear energy transfer (LET) α particle radiation (150 keV/μm) and subsequent growth in the presence or absence of 17β-estradiol. The MCF-10F cell line was analyzed in different stages of transformation after being irradiated with either a single 60 cGy dose or 60/60 cGy doses of alpha particles. In the present report, the profiling of differentially expressed genes associated with growth factors was analyzed in their relationship with clinical parameters. Thus, the results indicated that Fibroblast growth factor2 gene expression levels were higher in cells transformed by radiation or in the presence of ionizing radiation; whereas the fibroblast growth factor-binding protein 1gene expression was higher in the tumor cell line derived from this model. Such expressions were coincident with higher values in normal than malignant tissues and with estrogen receptor (ER) negative samples for both gene types. The results also showed that transforming growth factor alpha gene expression was higher in the tumor cell line than the tumorigenic A5 and the transformed A3 cell line, whereas the transforming growth factor beta receptor 3 gene expression was higher in A3 and A5 than in Tumor2 cell lines and the untreated controls and the E cell lines. Such gene expression was accompanied by results indicating negative and positive receptors for transforming growth factor alpha and the transforming growth factor beta receptor 3, respectively. Such expressions were low in malignant tissues when compared with benign ones. Furthermore, Fibroblast growth factor2, the fibroblast growth factor-binding protein 1, transforming growth factor alpha, the transforming growth factor beta receptor 3, and the insulin growth factor receptor gene expressions were found to be present in all BRCA patients that are BRCA-Basal, BRCA-LumA, and BRCA-LumB, except in BRCA-Her2 patients. The results also indicated that the insulin growth factor receptor gene expression was higher in the tumor cell line Tumor2 than in Alpha3 cells transformed by ionizing radiation only; then, the insulin growth factor receptor was higher in the A5 than E cell line. The insulin growth factor receptor gene expression was higher in breast cancer than in normal tissues in breast cancer patients. Furthermore, Fibroblast growth factor2, the fibroblast growth factor-binding protein 1, transforming growth factor alpha, the transforming growth factor beta receptor 3, and the insulin growth factor receptor gene expression levels were in stages 3 and 4 of breast cancer patients. It can be concluded that, by using gene technology and molecular information, it is possible to improve therapy and reduce the side effects of therapeutic radiation use. Knowing the different genes involved in breast cancer will make possible the improvement of clinical chemotherapy. Breast cancer is a complex disease in which multiple genetic alterations are present. However, its origin is not known, and it is generally associated with the accumulation of multiple anomalies in individual cells related to specific sequences of genetic changes that are important in tumor progression ,2. CanceThe risk of breast cancer is associated with endogenous factors such as hormones and exogenous factors such as radiation exposure (atomic bomb) or chemical factors from the environment. It is known that the transformation of a normal cell to a malignant one is induced by mutations in the genes that codify proteins, important in the regulation of cell processes that contribute to carcinogenesis through complex biochemical pathways . There ePhysical carcinogens such as radiation have been used as initiators and promoters in cell models of human and animal cell lines ,7,8. In The identification of genes involved in cancer stages in estrogen and radiation-induced breast carcinogenesis is of critical importance for a better understanding of the mechanisms involved. An experimental breast cancer model has been established where a normal human breast epithelial cell line such as MCF-10F underwent a stepwise transformation into malignant cells by the effect of low doses of alpha-particles in the presence of estrogens .FGFBP1) induces the FGF receptor (FGFR). Fibroblast growth factor2 (FGF2)-mediated angiogenesis is hypothesized to play a part in the development of squamous cell, colon, and breast cancers [The fibroblast growth factor-binding protein 1  and Transforming Growth Factor beta (TGFB) are secreted by a wide range of altered cells and malignancies [TGF) receptor [TGFBR3) is a membrane proteoglycan that often functions as a coreceptor with other TGF-beta receptor superfamily members.Transforming Growth Factor Alpha  is a tyrosine kinase receptor that has been linked to the genesis and progression of breast cancer tumors, and its IGF1R expression is associated with clinical–pathological factors in breast carcinomas [FGF2, FGFBP1, TGFA, TGFBR3, and IGF1R in a radiation- and estrogen-induced experimental breast cancer model.The insulin growth factor receptor  α particle radiation and subsequent growth in the presence or absence of 17β-estradiol. The MCF-10F cell line was analyzed in different stages of transformation after being irradiated with either a single 60 cGy dose or 60/60 cGy doses of alpha particles that then showed gradual phenotypic changes, including altered morphology, an increase in cell proliferation relative to the control, anchorage-independent growth and invasive capability, before becoming tumorigenic in nude mice. The cell lines used in this model were: (i) the parental cell line MCF-10F (C), (ii) an Estrogen cell line (E), (iii) a malignant and nontumorigenic cell line (60/60 cGy) named Alpha3 (A3), (iv) a malignant and tumorigenic cell line (60/60 cGy plus estrogen) named Alpha5 (A5), and (v) the Tumor2 cell line (T2) derived from a xenograft of the A5 cell line injected into nude mice; these cell lines were cultured in the presence or absence of E for periods of up to 10 months post-irradiation [FGF2, (B) FGFBP1, (C) TGFA, (D) TGFBR3, and (E) IGF1R in the following cell lines: MCF-10F/Estrogen (C/E), Control/Alpha3 (C/A3), Estrogen/Alpha5 (E/A5), Alpha3/Alpha5 (A3/A5), Alpha5/Tumor2 (A5/T2), and Alpha3/Tumor2 (A3/T2). Growth factor gene expression was determined by the profiling of differentially expressed genes through an Affymetrix array (U133A) , such asThe results indicated that high LET radiation such as that emitted by radon progeny in the presence of estrogen induced a cascade of events indicative of cell transformation and tumorigenicity in human breast epithelial cells. The studies are associated with the genes involved in the control of changes induced by growth factors that take place by the effect of radiation and hormones in the normal nontumorigenic MCF-10F and the tumorigenic cell lines.FGF2 and FGFBP1 gene expression, the results indicated that both had opposite results. Thus, while FGF2 was higher in A3 (60cGy/60cGy) than in the T2 cell lines, FGFBP1 was higher in T2 than in the A3 cell lines. Such results indicated that radiation (60cGy/60cGy doses) increased FGF2, whereas, in T2, 60cGy/60cGy doses plus estrogen injected in the immunologically depressed nude mouse. FGFBP1 was higher than the A3 cell line, indicating that the gene is at a higher level in the tumor and is influenced by estrogens, being a good marker for breast carcinogenesis. FGF2 was also higher in the A3 cell line than in the control MCF-10F (C) cell line. It is important to mention that, when single doses of alpha radiation (60cGy) were used to develop this model, there were no signs of transformation; then, the double (60cGy/60cGy) doses induced signs of transformation such as invasive capabilities and anchorage independence, but it was not tumorigenic in the SCID or nude mice [FGFBP1 was higher in the C than A3 cell line and in E than the A5 cell line.When a comparison was made between genes affected in the model as TGFA and TGFBR3 gene expression, studies indicated opposite results. Thus, the TGF alpha gene expression was higher in T2, the tumor cell line, than the nontumorigenic A3 cell line, as well as the tumorigenic cell line A5, indicating radiation in the first case, and the environment of the tumor formed in the second case added other factors that it could also be growth factors present in the animal. However, TGFBR3 gene expression was higher in the A5 than E cell lines and in the A3 than C cell lines, indicating the presence of radiation in the role of such a gene. It was also important to see that IGF1R gene expression was higher in the tumor cell line T2 than the nontumorigenic A3 cell line and this one compared to the control cell line.When a comparison was made between FGF2, FGFBP1, TGFA, TGFBR3, and IGF1R expression levels .Data on the n levels A–E were FGF2, FGFBP1, TGFA, and TGFBR3 gene expression levels were significantly (p < 0.001) lower in malignant breast cancer than in normal tissues; however, the IGF1R expression levels were not significantly different between both types of tissues  correlated with FGF2, FGFBP1, TGFA, and TGFBR3 in all BRCA patients, except for the IGF1R expression levels in Her2. The results showed a significantly (p < 0.05) positively correlation with all these genes in BRCA-Basal and BRCA-LumA and BRCA LumB, except for TGFBR3, which was negative for BRCA-Her2 and BRCA-LumB and positive for BRCA LumB.The results in FGF2 and FGFBP1 gene expression levels in breast invasive carcinoma subtypes with purity adjustment was statistically significant in its positive correlation in all BRCA, except for BRCA-Her2. Such results showed that EGFR was significantly (p < 0.05) correlated with FGF2 and FGFBP1 gene expression levels in all BRCA patients and significantly (p < 0.05) positively correlated with all these genes in BRCA patients. Thus, such expression was higher in BRCA-Basal, BRCA-HER, BRCA-LumA, and BRCA–LumB patients. Among all BRCA patients, TGFBR3 was positive in all BRCA patients and other types such as BRCA-Basal and BRCA-LumA. The correlation between EGFR expression and FGF2, FGFBP1, TGFA, TGFBR3, and IGF1R expression was explored using the TIMER2.0 Gene Outcome Module adjusted by the clinical stage factors across various cancer types. The association between the gene expression and the stage of the disease is shown in FGF2, FGFBP1, TGFA, TGFBR3, and IGF1R gene expression levels were significantly (p < 0.001) higher in all BRCA in stages 3 and 4 than in other clinical stages of patients. Such levels were non-significantly different in any BRCA-Basal patients. In addition, elevated FGF2 and TGFA gene expression levels were significant (p < 0.001) in stage 4 in BRCA-LumA in comparison with other clinical stages. There was a significant difference in stage 4 of patients in BRCA-LumB (p < 0.01) and in BRCA-Her2 (p < 0.05). The FGFBP1 gene expression levels were also significantly (p < 0.05) higher in stage 4 in BRCA-Her2 and BRCA-LumB patients, and it was significant (p < 0.01) in stage 4 in BRCA-LumA patients. The TGFBR3 gene expression levels were significantly (p < 0.001) higher in stage 4 in BRCA-LumA patients, and there was also a significant (p < 0.01) difference in stage 4 in BRCA-Her2 and BRCA-LumB patients. The IGF1R gene expression levels were also significantly (p < 0.001) higher in stage 4 in BRCA-LumA patients and there was a significant (p < 0.05) difference in stage 4 in BRCA-Her2 and BRCA-LumB patients. When the clinical stage was compared with BRCA-Basal, they were all negative; The IGF1R gene expression was significantly higher in Stages 3 and 4 than in the other clinical stages of patients. The IGF1R gene expression levels were also significantly (p < 0.001) higher in stage 4 in BRCA-LumA patients, and there was a significant (p < 0.05) difference in stage 4 in BRCA-Her2 and BRCA-LumB patients; however, this expression was not significant in BRCA-Basal patients, corroborating the difficulties of designing drugs for such patients, and there was a correlation between EGFR and IGF1R gene expression. Among all BRCA patients, IGF1R was negative in all BRCA patients but positive in BRCA-Basal, BRCA-LumA, and BRCA-LumB.The results in The present report shows growth factor genes linked to human breast cancer through an experimental breast cancer model that derived from a normal breast cell line transformed into malignant ones. These studies indicated that high LET radiation such as that emitted by radon progeny, in the presence of estrogen, induced a cascade of events indicative of cell transformation and tumorigenicity in human breast epithelial cells. Overexpression of several oncoproteins and tumor suppressor genes was reported where growth factors were thought to be involved in such processes ,21. GeneTo find novel prognostic/diagnostic markers for different forms of cancer, in vitro model systems have been widely employed to obtain insights into the molecular events of cancer, such as initiation, promotion, and prevention. All of these elements have biological consequences that either result in cellular death or cell survival, such as various mutations  that havThe soil and porous rocks that contain uranium give off the radioactive gas known as radon. All people are exposed to radon to some extent, although it is more prevalent in regions with high uranium concentrations, because most ground sources contain trace amounts of the element. Radon builds up in poorly ventilated or enclosed spaces when it migrates from soil and rock into the surrounding air. These regions serve as the main settings in which people are exposed to radiation from radon and suffer negative health effects. Since it is so common, residential radon is crucial. Even though miners may be exposed to much higher levels of ionizing radiation, even a small amount of extra risk from radon exposures in a residential setting has the potential to cause far more cancer cases in the general population than the exposure of a relatively small number of miners .Ionizing radiation consists of ions, as well as positively and negatively charged particles, such as alpha particles and electrons . The radReports have indicated that early defects in proliferation increase cancer risk, as it occurs in hyperplasia, since it perturbs the pathways usually regulated by local growth factors and systemic hormones, such as estrogens . The estOur experimental breast cancer model showed that transformed MCF-10F cell lines had a very complex pattern of gene and protein expressions after being exposed to double doses of alpha particles and then treated with estrogen as compared to a single dose of radiation and control; during the transformation process, several changes were induced, such as anchorage independency and invasive capabilities by single or double doses of radiation, either in the presence or absence of estrogen .The association between breast cancer development and prolonged exposure to estrogen suggests that this hormone may have an etiologic role in the causation of this disease ,15,49. EEGFR (HER1), HER2, HER3, and HER4, are part of the human epidermal growth factor receptor (EGFR/ErbB) family and are crucial in controlling cell proliferation, differentiation, and migration [EGFR and FGF2, FGFBP1, TGFA, TGFBR3, and IGF1R in breast-invasive carcinoma subtypes. Thus, EGFR was correlated with FGF2, FGFBP1, TGFA, and TGFBR3 in all BRCA patients, except for the IGF1R expression levels in BRCA-Her2. The results also showed a positive correlation with all these genes in BRCA-Basal and BRCA-LumA and BRCA LumB, except for TGFBR3, which was positive in BRCA-Her2 and negative for BRCA-Her2 and BRCA-LumB.Four receptor tyrosine kinases, known as igration ,56. ThesFGF2 plays important roles in tissue development and repair. Using heparan sulfates (HS)/heparin as a cofactor, FGF2 binds to FGFR and induces downstream signaling pathways, such as the ERK pathway, that regulate cellular behavior. FGF-BP1 is a secreted chaperone that mobilizes paracrine-acting FGFs, stored in the extracellular matrix, and presents them to their cognate receptors. FGFBP1 enhances FGF signaling, including angiogenesis during cancer progression, and is upregulated in various cancers.The amplification of different members of the FGF family of genes was shown to be a better independent prognostic indicator of breast cancer in humans ,58, wherTGFA gene encodes a growth factor that is a ligand for the epidermal growth factor receptor, which activates a signaling pathway for cell proliferation, differentiation, and development. This protein may act as either a transmembrane-bound ligand or a soluble ligand. The TGFBR3 is a membrane proteoglycan that often functions as a co-receptor with other TGF-beta receptor superfamily members and may inhibit TGFB signaling. A decreased expression of this receptor has been observed in various cancers. It is known that TGF stimulates anchorage-independent cell growth in soft agar in the presence of transforming growth factor β [TGF binds to EGFR and causes tyrosine phosphorylation of the receptor [The encoded receptor named factor β ,59,60,61IGF-1) and by a related hormone called IGF-2. It is important to mention that all gene expressions analyzed in these studies showed that the growth factors were positive in BRCA-Basal patients, an important fact considering the absence of treatment in those patients negative for estrogen receptors.IGF-1R is a protein found on the surface of human cells that belongs to the large class of tyrosine kinase receptors. It is a transmembrane receptor that is activated by a hormone called insulin-like growth factor 1  medium supplemented with antibiotics ), and 10 μg/mL and 5% equine serum , 0.5 μg/mL hydrocortisone , and 0.02 μg/mL epidermal growth factor  were added ,65,66,67The breast cancer model (Alpha-model) consists of: (i) MCF-10F, (ii) Estrogen, (iii) Alpha3, (iv) Alpha5, and (v) Tumor2 cell lines that were used to analyze the gene expression by the Affymetrix U133A oligonucleotide microarray , which contains 14,500 genes. The arrays were quantitatively analyzed for gene expression using Affymetrix GeneChip Operating Software (GCOS) with a dual global scaling option in the Genes@Work software platform of the discovery algorithm SPLASH  with a false discovery rate of 0.05 .TIMER2.0, an information web resource, evaluates the clinical impact of different immune cells in various cancer types through three components: immune association, cancer exploration, and immune estimation, each with several modules to investigate tumor immunological, clinical, and genomic features. The Gene_DE module of the exploration component provided statistical analysis carried out by the Wilcoxon test used in p < 0.05 was considered significant.UCSC Xena, a web source of information, provided the statistical significance computed by the one-way ANOVA test. The UCSC Xena web source allows users to explore functional genomic data sets for correlations between genomic and/or phenotypic variables and focuses on the integrative visualization of multi-omics data sets across different genomic contexts, including genes, genomic elements, or any genomic region, for both coding and noncoding parts of the genome . p < 0.0FGF2, FGFBP1, TGFA, TGFBR3, and IGF1R could give a clue on how to define combined drug treatments. It is important to mention that there is no drug therapy for negative estrogen receptor patients; therefore, searching for markers for this type of cancer is essential for breast cancer patients. It can be summarized that, in the breast cancer model, the cell line transformed only by radiation independently of estrogen was characterized by greater growth factor gene expression than in the other cell lines. Understanding the effect of radiotherapy on different types of cells will help us improve the clinical outcome of radiotherapy. Thus, gene signatures have been demonstrated to be specific to tumor types; hence, cell dependency must be considered in future treatment planning. Molecular and clinical features affect the results of radiotherapy. Thus, using gene technology and molecular information, it is possible to improve therapy and reduce the side effects of therapeutic radiation use. Knowing the different genes involved in tumorigenesis will make possible the improvement of clinical chemotherapy. Thus, gene signatures of the growth factors involved in different tumor types should be considered in future treatment planning, developing an original approach never considered before for any type of cancer.An experimental human breast cancer model derived from the MCF-10F cell line treated with different doses of high-LET (α-particle) radiation and estrogen exposure was used in these studies. It can be concluded that finding new markers such as


Glandora rosmarinifolia appears to involve a pro-oxidant mechanism in hepatocellular carcinoma (HCC) and in triple-negative breast cancer (TNBC) cell lines. Its most abundant compound is a hydroxy-methyl-naphthoquinone isomer. Important pharmacological activities, such as antitumor, antibacterial, antifungal, antiviral and antiparasitic activities, are attributed to naphthoquinones, probably due to their pro-oxidant or electrophilic potential; for some naphthoquinones, a mechanism of action of topoisomerase inhibition has been reported, in which they appear to act both as catalytic inhibitors and as topoisomerase II poisons. Our aim was to evaluate the cytotoxic activity of the essential oil on an acute myeloid leukemia cell line HL-60 and on its multidrug-resistant (MDR) variant HL-60R and verify its ability to interfere with topoisomerase II activity. MTS assay showed that G. rosmarinifolia EO induced a decrease in tumor cell viability equivalent in the two cell lines; this antitumor effect could depend on the pro-oxidant activity of EO in both cell lines. Furthermore, G. rosmarinifolia EO reduced the activity of Topo II in the nuclear extracts of HL-60 and HL-60R cells, as inferred from the inability to convert the kinetoplast DNA into the decatenated form and then not inducing linear kDNA. Confirming this result, flow cytometric analysis proved that EO induced a G0-G1 phase arrest, with cell reduction in the S-phase. In addition, the combination of EO with etoposide showed a good potentiation effect in terms of cytotoxicity in both cell lines. Our results highlight the antitumor activity of EO in the HL-60 cell line and its MDR variant with a peculiar mechanism as a Topo II modulator. Unlike etoposide, EO does not cause stabilization of a covalent Topo II-DNA intermediate but acts as a catalytic inhibitor. These data make G. rosmarinifolia EO a potential anticancer drug candidate due to its cytotoxic action, which is not affected by multidrug resistance.It was previously shown that the antitumor and cytotoxic activity of the essential oil (EO) extracted from the aerial parts of  The plant kingdom represents one of the most important sources of active principles in which numerous biological activities have been recognized. In particular, EOs play important functions both in plant life, such as protection against plants or vermin, repellent or recall action for pollinating insects and as raw materials for the synthesis of new drugs . The bioGlandora rosmarinifolia (Ten.) D.C. Thomas (Boraginaceae) has previously shown antitumor and cytotoxic activities involving a pro-oxidant mechanism in HCC and TNBC cell lines , EO  or etoposide (60 µg/mL). The order of addition was assay buffer, DNA, EO or etoposide, and then, nuclear extracts. The reaction mixture was incubated at 37 °C for 30 min, quenched with 1% (v/v) SDS/25 mM Na2EDTA and then treated with 0.25 mg/mL proteinase K  at 55 °C for 60 min. The samples were separated by electrophoresis on a 1% TAE ethidium bromide agarose gel, and the linear pBluescript II SK (+) DNA was identified by comparison with linear pBluescript II SK (+) DNA produced by the action of the restriction enzyme Pst I  acting on a single site on pBluescript II SK (+).DNA cleavage assays using nuclear extracts (200 ng) from untreated cells were performed in 20 µL of reaction mixture containing 150 ng of supercoiled pBluescript II SK (+) plasmid DNA, 0.5 mM ATP in assay buffer [10 mM Tris–HCl, 50 mM KCl, 50 mM NaCl, 0.1 mM EDTA, 5 mM MgCl5) were treated for 48 h with G. rosmarinifolia EO or etoposide (used at the respective IC50 for the two lines). After treatment, cells were collected and washed twice with ice-cold PBS and then resuspended at 1 × 106 /mL in a hypotonic fluorochrome solution containing propidium iodide (PI) 50 μg/mL and RNase (10 mg/mL) in 0.1% sodium citrate plus 0.03% (v/v) Nonidet P-40. After 45 min at room temperature (in the dark) of incubation in this solution, the samples were filtered through a nylon cloth, 40 μm mesh, and samples were analyzed using a FACSCanto instrument . The data were analyzed with BD FACSDiva software v.6.1.2. (Becton Dickinson). Cell distribution was determined by evaluating the percentage of events accumulated in the different phases of the cycle.To determine cell-cycle distribution, HL-60 and HL-60R cells  followed by Tukey’s test using Statistics ver. 12 .G. rosmarinifolia EO caused cytotoxicity in terms of cell growth inhibition and cell-cycle variation both in the HL-60 and HL-60R cell lines. EO was not subject to cell resistance through P-gp and sensitized the MDR cell line to the cytotoxic effects of P-gp substrates, doxorubicin and etoposide. The antitumor activity of EO on the HL-60 cell line and its MDR variant depends on a pro-oxidant action.G. rosmarinifolia EO acts as a modulator of Topo II through a different mechanism to etoposide; EO acts as a catalytic inhibitor rather than a Topo II poison. Unlike etoposide, which induces arrest in the G2-M phase of the cell cycle, EO induces a cell-cycle arrest in the G0-G1 phase of the cell cycle. The reduction in the S-phase cells by EO supports the hypothesis that it works by preventing Topo II from functioning properly at an early stage.G. rosmarinifolia EO could be used as an adjuvant in chemotherapy regimens.In light of these considerations,