Adaptive divergence leading to speciation is the major evolutionary process generating diversity in life forms. The most commonly observed form of speciation is allopatric speciation which requires that gene flow be prevented between populations by physical or temporal barriers, as they adapt to their respective environments. Eventually, these adaptive responses lead to accumulation of mutations in different lines. The increased genetic distance between the lines is known to lead to the populations becoming reproductively isolated. A widely accepted theory is that speciation simply occurs as a by-product of adaptive response of the populations. Several examples of allopatric speciation from ecology and laboratory exist. However, we know little about the nature (pre- or post-zygotic) of barriers that arise first in this process. Understanding the first barriers that arise between populations is key to understanding how the process of speciation initiates. In recent years, fungi have been used as model organisms to answer questions related to the evolution of reproductive isolation. Here, we show rapid evolution of pre-zygotic barriers between allopatric yeast populations. We further demonstrate that these pre-zygotic barriers arise due to altered mating kinetics of the evolved population. Moreover, our non-adaptive evolution experiments with yeast under limited selection pressure also show rapid emergence of reproductive isolation. Overall, our results show that evolution of pre-zygotic reproductive barriers can occur as a result of natural selection or drift.Saccharomyces cerevisiae. These defects arise because of the altered kinetics of mating of the participating groups. Thus, our work provides an understanding of how adaptive changes can lead to diversification among microbial populations.A population diversifies into two or more species—such a process is known as speciation. In sexually reproducing microorganisms, which barriers arise first—pre-mating or post-mating? In this work, we quantify the relative strengths of these barriers and demonstrate that pre-mating barriers arise first in allopatrically evolving populations of yeast,  Origin of Species explained natural selection as a mechanism for populations to adapt to prevailing environmental conditions, we do not yet understand the fundamental evolutionary forces leading to reproductive isolation. According to one idea, reproductive isolation, leading to speciation, is thought to evolve incidentally as a by-product of adaptation of populations to diverging selection pressures in allopatry  were evolved in glucose- and galactose-limited growth media for 600 generations, respectively. Three MATa and MATα lines were evolved in glucose, and three MATa and MATα lines were evolved in galactose—thus, making a total of 12 allopatric populations in the experiment . To study evolution in the absence of selection, 44 independent lines of haploids (22 lines of MATa and 22 lines of MATα) were also evolved in a mutation accumulation experiment for 70 transfers (~1500 generations). We demonstrate that metabolic specialization in the adaptive evolution experiment and the action of drift alone in the non-adaptive experiment leads to rapid evolution of pre-zygotic barriers, in the form of reduced mating efficiency as a result of altered mating kinetics, where the mating efficiency of different strains as a function of time varies quantitatively from each other. We demonstrate that, in specific environmental contexts, acquisition of a few SNPs only can establish significant barriers to mating. Overall, we demonstrate that evolution of reproductive barriers can start with pre-zygotic barriers among populations in allopatry.In this work, we ask the following question: which barriers arise first between populations in allopatry, evolving under selection and drift? To answer this question, we perform adaptive and non-adaptive evolutionary experiments with the yeast During allopatric evolution, an increase in fitness was observed due to a decrease in the lag phase duration and an increase in the growth rate  are as shown in Reproductive barriers between populations can be quantified via studying three processes in yeast: change in mating efficiency, mitotic growth of the hybrid, and meiotic efficiency of the hybrid. The pairwise mating efficiencies between all evolved haploid lines exhibit a marked decrease, as compared with those of the ancestor ; Fig. S2a was switched to α. A similar mating type switch was done for all haploid lines. We then quantified the mating efficiency of each evolved haploid with itself (carrying the opposite mating type). Each of the evolved haploids mates with their mating type switched counterpart with considerably higher efficiency, as compared with the average mating efficiency of the evolved haploid with all other evolved haploid lines . Seven hybrids, all from the glu1a and glu2a lines, could not be created. Thereafter, we characterized the growth kinetics of the 42 hybrid lines in glucose and galactose environments.From the 12 evolved haploid lines and the ancestor The hybrids were separated into five groups. Those resulting from mating between (i) two haploids evolved in glucose (glu-glu), (ii) haploids evolved in galactose , (iii) a glucose- and a galactose-evolved haploid , (iv) a glucose-evolved haploid with an ancestral haploid (glu-anc), and (v) a galactose-evolved haploid with an ancestral haploid . The performance of all five groups of hybrids was compared with that of the ancestral diploid.In glucose, the hybrids in the glu-glu, glu-anc, and the glu-gal group all exhibited a decrease in the growth rate, compared with the ancestral diploid . The fitOverall, our data suggest that, in allopatry, pre-zygotic barriers arise significantly faster as compared with post-zygotic barriers. In the framework of our evolution experiment, adaptation has two components: (i) decrease in lag phase duration and (ii) increase in growth rate. No correlation exists between these values of components of fitness and that of pre- or post-zygotic barriers to gene flow.To identify the genetic basis of adaptation and reproductive isolation between the evolved haploid lines, we sequenced the genomes of the 12 haploid lines, after adaptation for 600 generations , and compared with the ancestral sequence. The sequencing results show evidence of convergent evolution. Two glucose-evolved lines have mutations in MNS1, an ER membrane protein responsible for α-mannosidase activity (a stop codon and a frameshift mutation). MNS1 mutants exhibit a significantly longer life span in yeast  and in oa and their counterparts α (obtained by switching their mating type). As shown in While these similarities in genic targets exist, considerable difference in the mutational targets are also present between the different evolved lines (Table S3). These results lead us to hypothesize that altered mating kinetics in the evolved lines lead to establishment of first reproductive barriers. To test this possibility, we performed mating experiments where kinetics of mating between evolved haploids a and α each in a mutation accumulation (MA) experiment. The 44 lines (a1-a22 and α1-α22) were propagated for 70 transfers . Since a severe bottleneck is imposed at every transfer, the role of selection is largely absent and drift dictates evolutionary trajectory. MA experiments with microbial systems demonstrate that, under constant propagation under these conditions leads to a reduction in the fitness of MA lines , among the 44 lines evolved. There is no correlation between the adaptive response of the MA lines and the extent of change in the mating efficiency with the ancestral haploid. These results clearly demonstrate that reproductive isolation can, in addition to resulting from adaptation, also result as a by-product of drift .While the identity of mutations accumulated in the MA experiment is not yet known, genetic convergence in these lines is unlikely to have happened. MA experiments lead to evolution largely under the action of drift with minimal selection. As a result, the mutational targets’ identity is not related to adaptation in the environment of propagation. This leads to the observation that, in an MA experiment, fitness decreases with the number of propagations , 47.Speciation is the fundamental process that generates diversity of life forms on Earth. The most widely accepted explanation for speciation is that it occurs as a fallout of adaptation to diverging selection. Evolution of reproductive barriers among individuals in a population is a classic long-standing interest among biologists trying to understand the process of speciation. However, which barriers arise first as groups begin to diverge in allopatry?Our data suggest that upon starting with isogenic populations, adaptation in allopatry leads to rapid evolution of pre-zygotic barriers in yeast. In addition, drift alone can also lead to establishment of pre-zygotic barriers. Mating kinetics in yeast are intricately controlled . In factReproductive barriers could arise in several ways. Pre-zygotic barriers have been known to arise rapidly, in response to divergent selection , 54, altSaccharomyces cerevisiae strain used in this work is a derivative of ScPJB644 (MATa MEL1 ade1 ile trp1-HIII ura3-52)  and ScAM05 (MATa TRP1). The ancestral diploid was obtained by mating the two, and the resulting strain is referred to as ScAM06 (MATa TRP1/MAT α URA3). All strains were grown in YPD  at 30°C and shaking at 250 rpm in 25 × 100 mm test tubes unless specified otherwise. The freezer stocks of all strains are stored at −80°C in 25% (vol/vol) glycerol.URA3 was amplified from the plasmid p426GPD  using thThe GAL4c GAL80s-1 strains used in this work are described in , 68.The ancestral populations for the evolution experiment were started from the freezer stocks streaked on YPD plates. Single colonies of each ScAM04 and ScAM05 were inoculated in 5-mL YPD for 36 hours at 30°C and shaking at 250 rpm. These cultures were used as inoculum for the evolution experiment. Three replicate lines of each haploid strains were started in 0.2% (wt/vol) glucose or galactose in standard synthetic complete medium (SCM: 0.671% YNB with nitrogen base and 0.05% complete amino acid mixture). Thus, in total, we maintain 12 populations, three of each kind.Adaptive evolution experiments were performed by serial dilutions every 24 hours in SCM with the appropriate carbon source. Every 24 hours, growing cultures were diluted 1:100 in fresh SCM medium with appropriate carbon source yielding ~6–7 generations every 24 hours. Intermediate generations were cryopreserved in 25% (vol/vol) glycerol every 200 generations.ScAM04 was spread on YPD plates for single colonies. Prior to spreading, a small area was identified and marked on the plate. The colony inside (or closest to) the marked area, after 48 hours of growth at 30°C, was suspended in 2-mL PBS buffer. A fraction of this volume was spread on a fresh YPD plate for single colonies, and the process is repeated for 70 transfers. A total of 22 independent lines of ScAM04 were maintained in this experiment. Similarly, 22 independent lines for ScAM05 were propagated for the MA experiment.α haploids) from freezer stocks were revived in 5-mL YPD and incubated for 48 hours and then transferred 1:100 to a glycerol-lactate medium , 0.671% YNB with nitrogen base, and 0.05% complete amino acid mixture) and incubated for 48 hours at 30°C and shaking at 250 rpm. Two-mL SCM with appropriate carbon source  were inoculated for a final OD600 of 0.01. 150One hundred fifty µL of the cultures was then transferred to a clear flat-bottom 96-well plate (Costar) in triplicates and incubated at 30°C in an automated microplate reader (Tecan Infinite M200 Pro) until they reached a stationary phase. A gas-permeable Breathe-Easy (Sigma-Aldrich) sealing membrane was used to seal the 96-well plates. OD600 measurements were taken every 1 hour with 15 minutes of orbital shaking at 5 mm amplitude before the readings. Growth rates were calculated by plotting log (OD) from the exponential phase of growth against time. The slope of the straight-line fit was determined as the growth rate of the strain. The point of intersection of this straight line, with the straight line with the equation log , was taken as the duration of the lag phase.Evolved lines and ancestor  and incubated at 30°C for 24 hours and re-patched again on GNA plates for another 24 hours. Small lumps of cells were then patched onto sporulation medium plates containing 1% potassium acetate and 2% agar and incubated at 25°C for 5 days and 30°C for 3 days. Sporulated/un-sporulated cells were directly counted under ×400 magnification using a microscope to determine the sporulation efficiency (a minimum of 500 cells). The following formula was used to determine the sporulation efficiency.a-evolved haploid lines after 600 generations were transformed with a plasmid carrying the HO gene to obtain diploids of the evolved lines by selecting on URA dropout plates. The diploids were then sporulated as described previously. Tetrad dissection was performed via standard zymolyase treatment and manual dissection under the microscope. Haploids of both mating types were obtained from a single ascus and identified using colony PCR and primers described in  and resuspended in 100-μL autoclaved water. A volume of 1 mL of 70% ethanol was added along the sides of the vial while vortexing. The cells were then incubated at room temperature for an hour and then kept at 4°C overnight. The cells were then washed with 500 μL of RNase buffer  and resuspended in 100 μL of the same buffer. RNase A was added to a final concentration of 1 μg/μL. The cells were then incubated at 27°C for 4 hours and then washed with 1-mL PBS (centrifuged at maximum speed for 45 sec) and resuspended in 950 μL of the same buffer. Fifty μL of 1-mg/mL propidium iodide was added to a final concentration of 50 μg/mL. This was incubated at room temperature for 30 minutes. A volume of 500 μL of the cell suspension was gently vortexed and sonicated before analysis through flow cytometer .A 5-mL YPD culture was started from a single colony on a YPD plate. Saturated cultures were sub-cultured 1:100. When the cultures reached an OD of 1.00, a volume of 1.5 mL of the culture in the exponential phase was spun down  to perform all sequence data analyses. Illumina paired-end reads were uploaded into the server. The quality of the reads was assessed using FastQC (version 0.72). Raw reads were trimmed with Trimmomatic (version 0.38.1)   and mapp


KMT2A gene represent highly unfavorable prognostic factors and most commonly occur in patients less than 1 year of age. Rearrangements of the KMT2A gene drive epigenetic changes that lead to aberrant gene expression profiles that strongly favor leukemia development. Apart from this genetic lesion, the mutational landscape of KMT2A-rearranged ALL is remarkably silent, providing limited insights for the development of targeted therapy. Consequently, identifying potential therapeutic targets often relies on differential gene expression, yet the inhibition of these genes has rarely translated into successful therapeutic strategies. Therefore, we performed CRISPR-Cas9 knock-out screens to search for genetic dependencies in KMT2A-rearranged ALL. We utilized small-guide RNA libraries directed against the entire human epigenome and kinome in various KMT2A-rearranged ALL, as well as wild-type KMT2A ALL cell line models. This screening approach led to the discovery of the epigenetic regulators ARID4B and MBD3, as well as the receptor kinase BMPR2 as novel molecular vulnerabilities and attractive therapeutic targets in KMT2A-rearranged ALL.In acute lymphoblastic leukemia (ALL), chromosomal translocations involving the  KMT2A) gene in chromosome 11q23, in which the N-terminus of KMT2A fuses with the C-terminus of one of its translocation partner genes, such as AFF1 , MLLT1  or MLLT3  [KMT2A-rearranged infant ALL patients are at best 40% [Acute lymphoblastic leukemia (ALL) represents the most common type of cancer diagnosed in children and is currently curable in ~90% of patients . UnfortuKMT2A plays an essential role in regulating gene expression during early development and hematopoiesis [Functionally, wild-type opoiesis  regulatiopoiesis ,7,8. In opoiesis ,9,10, leopoiesis ,12.KMT2A-rearranged ALL (KMT2A-r ALL) may be improved by using epigenetic-based drugs targeting epigenetic vulnerabilities that are specifically essential to this type of leukemia. For instance, we recently showed that KMT2A-r ALL responds remarkably well to histone deacetylase (HDAC) inhibition [FLT3, a receptor tyrosine kinase, has been previously identified as a vulnerability in KMT2A-r ALL. A recent clinical trial with the FLT3 inhibitor lestaurtinib revealed that patients whose leukemia blasts exhibited sensitivity to FLT3 inhibition ex vivo experienced benefits from the addition of the FLT3 inhibitor to chemotherapy [KMT2A-r ALL is an epigenetically driven malignancy, combinations of epigenetic-based drugs and kinase inhibitors may well represent effective treatments for this elusive type of leukemia.In addition to the use of immunotherapeutic approaches such as blinatumomab , treatmehibition ,15. Intehibition ,17. Moreotherapy . Given totherapy , and thaKMT2A-r ALL cells by applying in vitro clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) knockout screens using synthetically designed single guide RNA (sgRNA) libraries [KMT2A-r ALL.Therefore, we set out to identify novel epigenetic regulators and kinases specifically essential to ibraries ,21 direcibraries ,23. CRISibraries . In the KMT2A-r ALL and those essential for ALL in general, we performed screens in KMT2A-r ALL cell lines  as well as wild-type KMT2A (KMT2A-w) B-cell precursor (BCP) ALL cell lines . The experimental outline  and kinome (504 genes). To distinguish between genes that are specifically essential to  outline A is base outline B.Approximately 67% or 76% of the sequenced reads at baseline (day 0) could be mapped to the epigenome or kinome sgRNA library, respectively. Read counts for non-targeting control sgRNAs remained stable between day 0 and day 21, indicating that the transduction of non-targeting sgRNAs did not affect cell viability or proliferation . Read coKMT2A-r ALL cell lines against those of KMT2A-w ALL cell lines , we have selected the receptor that exhibits the most differential response between KMT2A-r and KMT2A-w ALL cells for further validation, i.e., BMPR2.First, we determined the difference in z-scores between ll lines C,D. We i2A-w ALL C. In addLL cells D. Next, ll lines E,F. Thished data ,30,31,32d CREBBP E, as welBP . The location of these sgRNA sequences in ARID4B and MBD3, respectively, are indicated in + leukemic cells was progressively and significantly reduced over time in KMT2A-r ALL cells line SEM, but not in KMT2A-w ALL cell line 697  using the Synthego ICE Analysis tool. When genes that are essential for cell viability are targeted, knockout leads to cell death accompanied by the loss of the sgRNA sequence from the cell pool. In the case of ARID4B, all tested sgRNAs showed low KO scores ranging from 1% to 35% in the KMT2A-r ALL cell line SEM, with the commercially available sgRNA  producing the lowest score  revealed a significant loss of GFP signal in the line 697 E. Moreov97 cells F. Hence, BCP-ALL G–I.KMT2A-r ALL. These efforts and additional validation experiments revealed PSIP1, CREBBP, and kinase FLT3 representing known vulnerabilities in KMT2A-r, as well as ARID4B, MBD3, and BMPR2 as potential candidates to be considered as novel therapeutic targets in this aggressive type of leukemia. While the selected cell lines may not be a complete representation of KMT2A-r and KMT2A-w ALL, it is important to note that this approach successfully pinpointed well-established vulnerabilities specific to KMT2A-r ALL, such as CREBBP, PSIP1, and FLT3. These findings align with existing knowledge about the involvement of these genes in KMT2A-r ALL, supporting the reliability of our approach in detecting relevant targets.In this study we used CRISPR-Cas9 drop-out screens using sgRNA libraries against the human epigenome and kinome in various cell line models to identify novel molecular vulnerabilities in ARID4B, encoding AT-Rich Interaction Domain 4B (a member of the ARID family of chromatin remodeling proteins), is specifically lethal to KMT2A-r ALL cells. ARID4B plays a role in various cellular processes including embryonic development, cell proliferation, differentiation, and apoptosis [ARID4B plays a significant role in cancer development, metastasis, and cancer-related signaling pathways in different types of human cancers, including breast cancer, ovarian cancer, and hepatocellular carcinoma [ARID4B has the capability to interact with DNA sequences rich in AT base pairs, enabling it to recruit the SIN3A complex in specific regions of the genome. Once recruited, the SIN3A complex interacts with histone deacetylases (HDACs), resulting in the deacetylation of histones at the corresponding DNA locus, leading to chromatin condensation and transcriptional repression. Although we show here that ARID4B is essential to KMT2A-r ALL cells, the exact mechanistic role of ARID4B in this aggressive type of leukemia remains to be elucidated and further explored in the context of KMT2A-r ALL patient samples. From a therapeutic perspective, it would obviously be of interest to evaluate small molecule inhibitors of ARID4B in patient samples in vitro and in vivo using patient-derived xenograft (PDX) mouse models. To date, however, ARID4B inhibitors are not available.As shown, loss of poptosis ,49,50,51arcinoma ,47,52,53arcinoma ,55. WithARID4B, loss of MBD3 was not necessarily lethal to KMT2A-r ALL cells, but rather inhibited leukemic cell proliferation. MBD3 encodes a member of the methyl-CpG binding domain (MBD) protein family, which preferably binds to 5-hydroxymethylcytosine-marked genes, and has been implicated in various cellular processes, including cell differentiation, pluripotency, and cellular reprogramming [MBD3 expression or function has been observed in different human cancers, suggesting its involvement in tumorigenesis and cancer progression [MBD3 in KMT2A-r ALL as opposed to KMT2A-w BCP-ALL. Unfortunately, as is the case for ARID4B, no known MBD3 inhibitors are currently available.In contrast to gramming ,57,58. Mgression ,60,61,62BMPR2 to be lethal to KMT2A-r ALL cells. BMPR2 encodes a member of the bone morphogenetic protein (BMP) receptor family of transmembrane serine/threonine kinases. BMP signaling is activated by binding of TGF-beta superfamily ligands and is involved in various cellular processes, including embryonic development, tissue homeostasis, cell differentiation and hematopoietic stem cell (HSC) renewal [ACVR1 and BMPR1A to be potential molecular vulnerabilities in KMT2A-r ALL  mouse models. Moreover, conducting further exploratory experiments involving knockdown (instead of knockout) techniques such as siRNA- or shRNA-mediated RNA interference, might provide valuable insights. Additionally, it would be intriguing to investigate whether the recently identified therapeutic targets are under the influence of the KMT2A fusion complex. Furthermore, exploring the potential of MENIN inhibition, which targets the interaction between MENIN and the KMT2A fusion complex [KMT2A-r ALL.In summary, the present study provides novel molecular vulnerabilities of  complex , to effeKMT2A-rearranged B-ALL cell lines utilized in this study include SEM , KOPN-8  and ALL-PO [KMT2A::AFF1+). ALL-PO was a gift from the lab of Prof. dr. Cazzaniga . The KMT2A wildtype B-cell precursor (BCP) ALL cell lines utilized include 697  and NALM-6 ; ACC 128 DSMZ). All leukemia cell lines were cultured in RPMI-1640 medium containing GlutaMAXTM supplemented with 10% fetal calf serum, 100 IU/mL Penicillin and Streptomycin, and 0.125 µg/mL Amphotericin B , at 37 °C under a 5% CO2-containing atmosphere. HEK293T cells  were used for virus production and maintained in Dulbecco’s modified Eagle medium  with similar supplements and cultured under similar conditions. All cell lines were routinely tested for the absence of mycoplasma and DNA fingerprinted to assure cell line authenticity.The pediatric d ALL-PO   of <0.3 to minimize the number of cells with more than one genetic editing event. We aimed to obtain at least 500 sequencing reads per sgRNA by using 1000 cells per sgRNA at the starting point of the screen. Transduction was facilitated using 4 µg/mL polybrene (Millipore #TR-1003-G). To find the optimal virus volume for achieving an MOI of <0.3, each new cell type and virus lot was tested by a titration, which was assessed by measuring cell viability  on a Cytoflex S flow cytometer . 24 h after transduction, the medium was replaced to remove polybrene and puromycin selection (1 µg/mL) was initiated one day later. Then, 48 h after puromycin selection, the cells were harvested, representing baseline (Day 0) samples. The remaining surviving cells carrying the sgRNA library were maintained for another 21 days and passaged every three to four days, to harvest cells on day 21. Genomic DNA was isolated using the Trizol reagent  according to the manufacturer’s instructions. The sgRNA sequences were recovered by a first round of PCR  by using 5 µg genomic DNA to ensure the sgRNA pool complexity. The PCR primer sequences were forward 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNGGCTTTATATATCTTGTGGAAAGGACG-3′ with NNNNNN as barcode and reverse 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACTGACGGGCACCGGAGCCAATTCC-3′ as described previously ,23,75. TKMT2A-rearranged and wild-type KMT2A ALL cell line models were assessed to identify genes specifically essential to either type of ALL.Analysis of the data was performed on the web-based analysis platform Galaxy version 0.5.8.4 , using default parameters. Sequencing reads were analyzed in a model-based analysis of genome-wide CRISPR-Cas9 knockout (MAGeCK) , a publiThe candidate molecular vulnerabilities emerging from our CRISPR-Cas9 sgRNA library screens were validated by examining cell survival upon gene knockout using a competition assay .https://ice.synthego.com (accessed on 22 March 2022)). The primers used are listed in The doxycyclin (DOX) inducible pCW-Cas9 vector (Addgene #50661) was utilized to express CAS9. For each target gene, one or two gRNAs were chosen from the CRISPR KO screen sgRNA libraries, complemented with one or two commercially available gRNAs against the genes of interest , IDT. EaThe levels of protein expression were determined by immunoblot analysis. For this, protein was extracted using RIPA buffer supplemented with protease inhibitors (ThermoScientific), and resolved on precast TGX™ gels and transferred to an 0.2 µm nitrocellulose membrane using a Transblot Turbo Transfer System (Bio-Rad). Blots were then probed with antibodies against MBD3 , ARID4B (#A302-233A-M Bethyl labs), B-actin (#ab6276 Abcam) or GAPDH . The membranes were then probed with infrared-labeled secondary antibodies IRDye 800 CW goat-anti-rabbit antibody  and IRDye 680 goat anti-mouse antibody . Proteins were visualized using an Odyssey Infrared Imaging System , and protein expression was quantified using the Odyssey software Image Studio Lite version 4.0.FACS analysis experiments for determination of BMPR2 expressing cells were performed on a CytoFlex Flow Cytometer (Beckman Coulter). Cells were fixed with 1% PFA before blocking with Human TruStain FcX™  and subsequently labeled with eBioscience™ Fixable Viability Dye eFluor™ 450  to select for viable cells. Subsequently the cells were stained with BMPR2 antibody  and PE goat anti mouse IgG as secondary antibody (#405307 BioLegend) according to the manufacturer’s recommendation. Raw CytoFLEX data were processed using CytExpert version 2.3 (Beckman Coulter).t-tests as indicated in the figure legends. All statistical analyses were conducted using GraphPad Prism8, version 8.3.4. p < 0.05 was considered statistically significant.Statistical significance of independent experimental replicates in the graphs were determined using two-sided Student’s


More than half of CRC patients develop metastatic disease (mCRC) with an average 5-year survival rate of 13%. Circular RNAs (circRNAs) have recently emerged as important tumorigenesis regulators; however, their role in mCRC progression remains poorly characterized. Further, little is known about their cell-type specificity to elucidate their functions in the tumor microenvironment (TME). To address this, we performed total RNA sequencing (RNA-seq) on 30 matched normal, primary and metastatic samples from 14 mCRC patients. Additionally, five CRC cell lines were sequenced to construct a circRNA catalog in CRC. We detected 47 869 circRNAs, with 51% previously unannotated in CRC and 14% novel candidates when compared to existing circRNA databases. We identified 362 circRNAs differentially expressed in primary and/or metastatic tissues, termed circular RNAs associated with metastasis (CRAMS). We performed cell-type deconvolution using published single-cell RNA-seq datasets and applied a non-negative least squares statistical model to estimate cell-type specific circRNA expression. This predicted 667 circRNAs as exclusively expressed in a single cell type. Collectively, this serves as a valuable resource, TMECircDB (accessible at   Tumor growth in the colon and rectum, classified as colorectal cancer (CRC), currently ranks third in terms of incidence and second in terms of mortality globally ,2. In thTo date, CRC research has primarily focused on the dysregulation of protein-coding genes to identify oncogenes and tumor suppressors as potential diagnostic and therapeutic targets . One emeet al. discovered that it may be able to function through alternative mechanisms depending on its specific location within the TME. They reported that ciRS-7 was completely absent in CRC cancer cells, but highly expressed in stromal cells. The putative ‘sponging’ target miR-7, on the other hand, was only found to be expressed in cancer cells, rendering the miRNA sponge mechanism implausible  sponges that counteract their target miRNA’s inhibitive effects . Howeverlausible . As thisMany recent studies have shown that many circRNAs are implicated in crucial hallmarks of CRC tumorigenesis, such as sustaining proliferative signaling, avoiding immune destruction, activating invasion and metastasis, and evading cell death and senescence ,14,19–58Furthermore, multiple obstacles have yet to be overcome in the study of cell-type specific expression of circRNAs. Most known single-cell RNA-seq (scRNA-seq) protocols require poly(A) selection, without which the sequencing data tend to be very noisy . As circhttps://www.maherlab.com/tmecircdb-overview.To address this, we performed RNA-seq of matched normal, primary and distant metastatic tissues from CRC patients and conducted a meta-analysis to discover differentially expressed (DE) circRNAs in metastatic tumors compared to primary tumors, termed circular RNAs associated with metastasis (CRAMS). Further, to overcome the limitations of determining cell-type specific expression of circRNAs in the TME, we performed cell-type deconvolution followed by a non-negative least squares (NNLS) model to estimate cell-type specific expression of circRNAs ,63. FromN = 14) were enrolled at Washington University School of Medicine in St Louis (WUSTL) and informed consent was obtained under an Institutional Review Board (IRB)-approved protocol. Adjacent normal colon , primary CRC , liver metastasis  and normal liver  tissues were resected from 14 stage IV mCRC patients and fresh frozen prior to RNA extraction. Input RNA quantity was 1 μg except for samples 15 (0.5 μg) and 27 (0.24 μg) due to low volume  supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. SW480 and SW620 cells were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin. LoVo cells were grown in DMEM/F12 (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Total RNA inputs of 1 μg were used to generate libraries using the New England Biolabs NEB Ultra II Directional RNA Library Prep for Illumina with rRNA Depletion kits. To construct the cell line reference database, 200 ng aliquots of each library were pooled and used as input for cDNA capture. The IDT xGen Exome Research Panel v2 including 415 115 exon probes was used for exome capture and paired-end sequencing was performed on Illumina HiSeq X.For each cell line sample, uniquely mapped reads and spliced reads were compared with exon probes from IDT xGen Exome Research Panel v2 using BEDtools v2.29.0 overlap to identify on- and off-target reads that were subsequently used to calculate cDNA-capture sequencing on-target rates .et al.  catalog as described in Silva-Fisher et al. . RedundaAs backspliced reads do not map to the linear transcriptome, chimeric alignments were extracted as a BED file from alignment results via STAR v2.7.3a . The chiA total of 740 513 nonredundant circRNAs were downloaded from circAtlas v2.0, which encompasses annotations (with necessary liftover from previous genome assemblies) from circbank, circBase, CIRCpedia v2, CircRiC, circRNADb, MiOncoCirc, deepBase v2.0, TCSD and CSCD . AdditioP < 0.05, |log fold change (FC)| > 0 and FDR adjusted P ≤ 0.15 was considered differentially expressed.To identify circRNAs associated with CRC tumor and metastatic progression, DE analysis was carried out comparing normal, primary and metastatic tissues via edgeR v3.30.3, using raw counts of linear genes and circRNAs . All linP < 0.05) in the same comparison group and in the same direction (up- or downregulated) and/or circRNAs found by CircTest to be differentially expressed (P < 0.05) in the same comparison group were considered validated in parallel.As means of orthogonal validation, the CIRI_DE_replicate function of CIRIquant v1.1.2  and the R package CircTest v0.1.1 were also run on the same cohort of detected circRNAs ,80. Circet al.  values Figure , step 6.To assess the performance of the NNLS models, we reconstructed ‘bulk’ expression from each model’s estimated cell-type specific expression of both linear genes and circRNAs, which was then compared with the ground truth to evaluate how accurately the model recaptures the real expression values Figure .L) of every gene in every sample was calculated through matrix multiplication of NNLS estimated coefficients (matrix B) and CIBERSORT estimated cell-type proportions (matrix p). The modeled ‘bulk’ expression (matrix L) was compared with the ground truth bulk expression (matrix l). A linear regression model was used (R function lm) to evaluate the correlation of expression between modeled and ground truth bulk data for each gene . Pearson correlation coefficients were calculated using R function cor.test for each sample  of every circRNA in every sample was calculated through matrix multiplication of NNLS estimated coefficients (matrix C) and CIBERSORT estimated cell-type proportions (matrix p). Modeled ‘bulk’ expression (matrix N) was then compared with the ground truth bulk expression (matrix n). A linear regression model was used (R function lm) to evaluate the correlation of expression between modeled and ground truth bulk data for each circRNA . Pearson correlation coefficients were calculated using R function cor.test for each sample ; 3360 were shared by normal and tumor samples . Overall, 1644 unique circRNAs were shared between all tissue types Figure .For downstream analyses of DE and cell-type specificity, RNA-seq reads mapping to linear genes were also quantified . A totalWe established a cell line reference catalog by sequencing five common CRC cell lines, including three derived from primary tumors  and two from metastatic tumors (SW620 and LoVo), termed WUSTL cell lines Figure . The tarA total of 32 469 unique circRNAs were detected in all cell lines, originating from 6666 parental genes. A total of 1325 circRNAs were found in common in all cell lines . Out of To identify circRNAs that have yet to be characterized in CRC patient samples, we compared our results with the CRC cohort from the pan-cancer circRNA consortium MiOncoCirc Figure  74). In. In74). To investigate novel circRNAs unannotated to date, we compared all unique backsplice junctions detected in the WUSTL patient cohort and WUSTL cell lines with the integrative circRNA resource circAtlas encompassing multiple existing circRNA databases, dubbed ‘other databases’ Figure  69–77)..69–77). It is worth noting that many circRNA databases only characterize unique circRNAs by their backsplice junction coordinates, without taking into consideration possible different isoforms arising from the same backsplice junction. Hence, the true percentage of novel, unannotated circRNAs in our datasets may be >14%.To characterize circRNAs altered in the mCRC progression, we performed DE analysis using edgeR comparing normal, primary and metastatic tissues from the WUSTL patient cohort Figure . All expTo provide additional evidence for the DE status of these candidates, we performed two parallel DE tests using CIRIquant and CircTest on the same cohort of circRNAs detected from the WUSTL patient cohort ,80. AmonSeveral previously characterized circRNAs that are associated with mCRC or other types of metastatic cancer were shown to be differentially expressed with supporting evidence from at least one other DE test Figures . This inIn general, more circRNAs showed lowered expression in primary or metastatic tumors compared with normal tissues Figure . For exaTo better understand circRNAs dysregulated in CRC in the context of TME, we evaluated their cell-type specific expression in bulk patient RNA-seq. To do so, we leveraged existing patient scRNA-seq data from two independent cohorts, termed the Lee 2020 scRNA-seq cohort  and the Li 2017 scRNA-seq cohort , to build signature matrices Figure  81,82)..82). UsiThe resulting cell-type proportion estimates from CIBERSORT were then used to train an NNLS statistical model to estimate the cell-type specific expression value of circRNAs based on their bulk expression  values Figure , step 6.P < 0.05) was predicted by the Lee 2020 model (93%) than by the Li 2017 model (90%). The percentage of circRNAs (P < 0.05) from the Lee 2020 model (55%) was also greater than that from the Li 2017 model (46%). There was an overall increase in the linear regression R2 values in the Lee 2020 model as compared to the Li 2017 model in linear genes with significant P-values, with R2 ranging from 0.126 to 0.997 with a mean of 0.592 in the Lee 2020 model and R2 ranging from 0.126 to 0.997 with a mean of 0.586 in the Li 2017 model  in the Lee 2020 model and 1725 (6%) in the Li 2017 model, and a consensus of 1217 (4%) between models  unique circRNAs only showed expression in one cell type, while the Li 2017 model predicted 7458 (26%). The consensus between both models showed 4123 (14%) circRNAs with exclusive expression in a single cell type. To select those with significant cell-type specificity, we required the following criteria: s Figure . To furts Figure .P-values, was reported to be specific to CRC cells . Th. Thde noOur study has addressed many limitations of existing CRC research. To date, there has not been a comprehensive, spatial expression profile for circRNAs in mCRC. For example, the pan-cancer database of expressed circRNAs across cancers, MiOncoCirc, consists of 868 patient samples, of which 217 (25%) are from prostate cancer, whereas only 13 (1.5%) samples are from CRC. Further, since these CRC samples were not curated from adjacent sites of normal, primary and metastatic tissues from the same patients, it proved challenging to systematically identify dysregulated circRNAs throughout mCRC progression . Our uniWe performed transcriptome profiling of a matched cohort of mCRC patients and characterized CRAMS. We detected 28 670 unique circRNAs, 51% of which were isoforms unannotated in MiOncoCirc Figure . ThroughA vast majority of DE circRNAs were downregulated in primary or metastatic tumors, which could be attributed to the faster cell proliferation diluting their global abundance as the cancer progresses, or increased export to exosomes due to their high stability ,74. CompIdentifying the spatial origin and cell-type specific expression of circRNAs is paramount to understanding their biological roles as potential TME regulators. To address the lack of cell-type deconvolution methods involving circRNAs, we devised a unique computational approach to identify cell-type specific circRNAs associated with the TME of CRC. By combining deconvolution analysis using two published scRNA-seq datasets and building an NNLS statistical model, we discovered that 667 circRNAs were exclusively expressed in one cell type only. Our results were congruent with earlier findings by highlighting known cell-type specific circRNAs such as ciRS-7 being exclusive to stromal cells Figure  18). In. In18). R2 range was similar to that of the linear genes but with lower maximum R2 values  and GitHub (https://github.com/ChrisMaherLab/TMECircDB). The RNA-seq data generated in this study (WUSTL patient cohort and WUSTL cell lines) have been deposited in the NCBI Gene Expression Omnibus database under the accession code GSE221240. The Lee 2020 scRNA-seq data referenced during the study are available in a public repository from the NCBI Gene Expression Omnibus under the accession code GSE144735. The Li 2017 scRNA-seq data referenced during the study are available in a public repository from the NCBI Gene Expression Omnibus under the accession code GSE81861. All the other data supporting the findings of this study are available within the article and its The cell-type specific expression data of identified circRNAs are available at the TMECircDB resource: zcad021_Supplemental_FilesClick here for additional data file.


The combinations of esculetin with epirubicin and vemurafenib showed antagonistic interactions, the combinations of esculetin with cisplatin, docetaxel and paclitaxel showed additive interactions. For the combinations of esculetin with mitoxantrone, the isobolographic analysis displayed synergy. (4) In the treatment of malignant melanoma, esculetin should not be combined with epirubicin or vemurafenib, due to the reduction of their anticancer effects, while the synergistic interactions (esculetin + mitoxantrone) deserve a preclinical recommendation as a beneficial combination during anticancer therapy.(1) Malignant melanomas are dangerous skin cancers, and the treatment of melanomas with various cytostatic drugs often causes side effects and after their prolonged use resistance to these drugs appears. The aim of this study was to evaluate the anticancer effects of esculetin (a simple coumarin) and to assess pharmacodynamic interactions between esculetin and six commonly used cytostatic drugs  using an isobolographic analysis. (2) The experiments were carried out on four human malignant melanoma cell lines . The effects of esculetin on viability, cell proliferation and cytotoxicity were verified in the range of concentrations of 2–200 μM. (3) Esculetin inhibited, in a dose-dependent manner, malignant melanoma cell viability and proliferation. The IC Malignant melanomas are cancers that arise from melanocyte cells. Among skin cancers, they cause the most deaths of patients and, due to the distribution of melanocytes to various tissues, they can occur almost anywhere on the body, which also proves the high metastasis ratio of this cancer. For the fair-skinned Caucasian population, the origin of cancer is associated with exposure to sunlight and its ultraviolet (UV) radiation . EstimatAn interesting compound of natural origin, commonly used as a herbal medicine in Asian countries is esculetin (aesculetin) . EsculetCitrus limon, Euphorbia lathyrism, Ceratostigma willmottianum, Aesculus hippocastanum [Fraxinus rhynchophylla—extracts from these plants are used by herbalists as traditional astringents and medicines for liver and gallbladder ailments [Esculetin is present in many plants, such as castanum , and Fraailments . Esculetailments ,10,11. Eailments ,9, and iailments ,13.Both in vivo and in vitro studies confirm that the mechanism of induction of apoptosis of cancer cells by esculetin is associated with an increased percentage of DNA fragmentation and arrest of the cell cycle in the G1 phase. This is related to the upregulation of p21WAF1 and p27KIP1 expression, leading to a decrease in cyclin D1/cyclin-dependent kinase (CDK) 4 and cyclin E/CDK2 complexes ,15.In the MTT test, it was observed that all the tested drugs  inhibited the viability of four malignant melanoma cell lines a–d. Addi50) values for esculetin, cisplatin, epirubicin, docetaxel, paclitaxel, mitoxantrone and vemurafenib in various melanoma cell lines are presented in 50 values for cisplatin, mitoxantrone, docetaxel and paclitaxel were determined experimentally in our previous studies [Significant inhibition of malignant melanoma cell viability was observed already from the concentrations of 2–10 µM esculetin, and at higher concentrations of esculetin up to 200 µM, viability was observed reaching 20–25% for the FM55P and FM55M2 cell lines and about 30–40% for the A375 and SK-MEL28 cell lines. Esculetin had a slight effect on the viability of normal keratinocyte and melanocyte cell lines, a significant inhibition of viability was observed above concentrations of 60–100 µM. The experimentally derived median inhibitory concentration  and keratinocytes (HaCaT). The treatment of all the investigated cells with increasing concentrations of esculetin reduced DNA synthesis that was evaluated by measuring BrdU (5-bromo-2′-deoxyuridine) incorporation into cellular DNA in proliferating cells .Esculetin from the concentration of 10–20 µM inhibited significantly malignant melanoma cell proliferation. In a concentration of 100 µM and higher, esculetin inhibited almost 100% of melanoma cell proliferation. In the case of normal melanocyte and keratinocyte lines, inhibition of proliferation of cell lines was also observed, but much less compared to malignant melanoma cell lines. In the case of the HEMa-LP melanocyte line, esculetin (at a concentration of 150–200 µM) significantly reduced cell proliferation to approximately 50–40%. In the case of the HaCaT keratinocyte line, a significant inhibition of proliferation by esculetin was observed from a concentration of 40 µM, however, a reduction of proliferation below 50% was observed only for esculetin at a concentration of 200 µM .Cytotoxicity of esculetin to normal human melanocytes (HEMa-LP), normal human keratinocytes (HaCaT) and four malignant melanoma cell lines  was detected by means of LDH assay. The release of lactate dehydrogenase into the medium, as a consequence of cell membrane destruction and cell death after exposure to esculetin, was estimated in the LDH assay . In this50 values ± SEM). In such a situation, the drug concentrations were transformed to logarithms and their anti-proliferative effects (measured by the MTT test) were transformed to probits. The linearly related concentration–response effects for the studied drugs  were plotted into the Cartesian system of coordinates (50) values for tested compounds are presented in From the linear log-probit concentration–response inhibitory effects for esculetin and six commonly used chemotherapeutics on four malignant melanoma cell lines, it was possible to determine the median inhibitory concentrations . This is also due to the close location of points A (IC50add) and M (IC50mix) on the isobologram. The combination of esculetin and epirubicin (at the fixed ratio of 1:1) showed a statistically significant antagonistic interaction in the FM55P, A375, FM55M2 cell lines . Graphically, Point M was placed drastically above the line of additivity and Point A.Isobolographic analysis of interactions for parallel concentration–response lines revealed that the combination of esculetin with cisplatin (at the fixed ratio of 1:1) exerted additive interactions in all the studied melanoma cell lines a,c,e,g. ll lines b,d,f andll lines h. This i50mix value (point M on the isobologram) with IC50add value  with Student’s t-test with Welch’s correction. On the isobolograms, the proximity of point M to point A or A′/A″ indicates an additive interaction.The combinations of esculetin and docetaxel a,c,e,g at-test with Welch’s correction. On the contrary, the antagonistic interaction was observed between esculetin and vemurafenib (at a fixed ratio of 1:1) in all the tested melanoma cell lines  in the A375, FM55M2 and SK-MEL28 melanoma cell lines c,e,g. Thll lines b,d,f,h. 50mix and IC50add) values for all the studied combinations of esculetin with six chemotherapeutics  were calculated. The interaction indices for antagonistic interactions between esculetin and epirubicin ranged from 1.75 to 2.16, while those between esculetin and vemurafenib ranged from 2.11 to 2.13  which represents a two-drug combination . With su50 for esculetin was 100 μM [50 values for esculetin against malignant melanoma cells ranging from 18.20 μM to 120.64 μM depending on the cell lines. A much higher concentration of IC50 = 400 μM (after 48 h incubation) was obtained by scientists testing esculetin on colon cancer LoVo cell line [50 concentrations of esculetin in the range of 1.96–12.88 µM were observed against laryngeal carcinoma cells  [The results of this study confirmed that esculetin inhibits the proliferation of malignant melanoma cell lines. Many studies confirm the anticancer properties of esculetin. For instance, esculetin at concentrations of 9.3–600 μM inhibited the proliferation of several prostate cancer cell lines  . The mols 100 μM , which iell line . The mecell line . Anotherell line . Low IC5and M4e) . Esculetand M4e) .50 concentrations of esculetin against melanoma (18.20–120.64 μM) obtained herein are comparable to the IC50 concentrations of 95–142.5 μM obtained against endometrial cancer cells (HEC-1B and Ishikawa cell lines). Esculetin promoted cell apoptosis by upregulating expression levels of cleaved caspase 3 and cleaved PARP [Esculetin at concentrations up to 80 µM reduced the proliferation of Lewis lung carcinoma (LLC) cells and reduced the expression of c-myc, cyclin D1 and NF-κB . After cved PARP .50 = 37.8 µM) by inducing their apoptosis, as evidenced by the production of reactive oxygen species (ROS) and the accumulation of cells in the sub-G1 phase [Esculetin (at a concentration of 50 mM) was effective against HepG2 liver cancer cells, by binding to three potential targets (phosphoglycerate kinase 2 (PGK2), glycerol-3-phosphate dehydrogenase (GPD2) and glucose-6-phosphate isomerase (GPI)). Esculetin significantly inhibited the rate of glycolysis, which affects the production of lactate and glucose utilization in HepG2 liver cancer cells. Upregulation of caspase-3 and Bax, and downregulation of Bcl-2 were also observed, indicating that esculetin induced cell apoptosis. Animal tests  showed that esculetin inhibited tumor growth compared to the control group. Measurement of the target protein in tumor tissue showed an increase in PGK2, GPD2 and GPI in response to esculetin treatment . EsculetG1 phase . EsculetG1 phase .50 = 43 µM) of the key enzyme of melanogenesis, which is the fungal tyrosinase. In addition, esculetin at a relatively low concentration of 5 µM (compared to concentrations tested in our experiment in the range of 2–200 µM) significantly inhibited melanin production in B16 murine melanoma cells without affecting their growth. In addition, it was proved in experiments on patches of split epidermis, which were treated with esculetin solutions, that the number of melanocytes decreased in them (compared to the control), which additionally confirmed the inhibitory effect on tyrosinase activity and the process of melanogenesis [50 = 42.86 μg/mL ≈ 240 μM), which exceeds the IC50 concentrations we obtained for four melanoma cell lines. However, scientists proved that esculetin induced nuclear condensation and fragmentation, induced apoptosis by reducing the level of Sp1 protein and stopped the cell cycle. Esculetin increased protein levels of p21, p27, Bax and active caspase 3 and decreased protein levels of cyclin D1, procaspase 3 and PARP [It is interesting to note that esculetin shows strong inhibition  is an effective cytostatic drug used in the treatment of melanoma , however, resistance is observed after 6–10 months of therapy, and the therapy itself is associated with side effects, including joint pain, headache, fatigue and fever. There are also alarming observations that some patients develop other skin cancers after monotherapy: squamous cell carcinoma or keratoacanthoma ,40. For In the present study, the interactions between esculetin  and several cytostatic drugs with different mechanisms of action were evaluated. Since the cell lines we tested have the BRAF mutation, vemurafenib was included in the tested combinations. This compound has the ability to inhibit mutated BRAF serine-threonine kinase and selectively binds to the ATP binding site of BRAF V600E kinase and inhibits its activity. Vemurafenib potently inhibits ERK phosphorylation and proliferation of BRAF mutant cells only . The stuThus, our experiments and isobolographic analysis showed that the combinations of esculetin and cisplatin are characterized by additive interactions. An additive interaction was also observed for the combination of cisplatin and osthole for two melanoma cell lines, FM55P and FM55M2 . Our exp50mix values considerably differed from the theoretically calculated IC50add (accepted as additive) values. Theoretically, it is possible to statistically assess and differentiate the significance between the analyzed IC50 values , but this differentiation is not precise. For instance, a synergistic interaction assessed at p < 0.0001 is more powerful than that at p < 0.05. Analogously, an antagonism at p < 0.05 is less powerful than that observed at p < 0.0001. Of note, the interaction indices were calculated in this study to determine the power (strength) of interactions [50 values did not differ significantly  and metastatic (FM55M2 and SK-MEL28) malignant melanoma cell lines, which were purchased from the European Collection of Authenticated Cell Cultures (FM55P and FM55M2), and the American Type Culture Collection (A375 and SK-MEL28), respectively. Experimental conditions on growth of the in vitro culture were described previously .Esculetin (ESC), mitoxantrone, docetaxel, paclitaxel and vemurafenib  were dissolved in DMSO. Cisplatin (CDDP) and epirubicin (both from Sigma-Aldrich) were dissolved in phosphate buffered saline (PBS–CDDP) or in sterile hot water (epirubicin), respectively. To avoid any toxic effects of DMSO on cell proliferation, the concentrations of DMSO were maintained up to 0.1%.4 cells/mL depending on the cell line), normal human keratinocytes (HaCaT) (density 1 × 104 cells/mL) and normal human melanocytes (HEMa-LP) (density 5 × 103 cells/mL), the respective cells were placed on 96-well plates , as described in detail earlier [To determine the cell viability in all the studied malignant melanoma cell lines   after esculetin application, the ELISA BrdU Kit  was used. In this test, the optimized amounts of cells were placed on a 96-well plate (NEST) (100 µL/well). After 24 h, the cells were exposed to increased concentrations of esculetin for 48 h .To confirm cell cytotoxicity in all the studied malignant melanoma cell lines , after esculetin application, Cytotoxicity Detection KitPLUS LDH  was used. In this test, the optimized amounts of all the studied malignant melanoma cells, normal human keratinocytes (HaCaT) and normal human melanocytes (HEMa-LP) were placed on 96-well plates (NEST). After 24 h, cells were washed in PBS, and increasing concentrations of esculetin were added to the cells. The cytotoxicity of esculetin was assessed by measuring cytoplasmic lactate dehydrogenase (LDH) activity released from damaged cells after 72 h of exposure to the former drug ,17.50 values for every tested drug. Parallelism of two concentration–response lines  was assessed with the test for parallelism, as described previously [50 values for the tested compounds when used alone. However, if the concentration–response effect lines are not parallel, the additivity on the isobologram is illustrated as an area bounded by two (lower and upper) lines of additivity [50 values for the drugs administered alone, median additive inhibitory concentrations for the mixtures of esculetin + cisplatin, esculetin + epirubicin, esculetin + docetaxel, esculetin + paclitaxel, esculetin + mitoxantrone and esculetin +vemurafenib at the fixed ratio of 1:1 (IC50add) were calculated [50mix and IC50add values), synergy can be observed if the interaction index is lower than the arbitrary accepted value of 0.7 [To precisely assess pharmacodynamic interactions between esculetin and six chemotherapeutics in the malignant melanoma cell lines  we used the isobolographic analysis in the MTT assay. Based on log-probit analysis according to Litchfield and Wilcoxon , we deteeviously ,57. If tditivity ,59. Fromlculated ,59,60,61lculated . Owing te of 0.7 ,50,51. Ae of 0.7 ,50,51. Ae of 0.7 ,50,51. Fe of 0.7 .p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, respectively). The inhibitory concentration (IC50 and the IC50mix) values for esculetin and six commonly used chemotherapeutics  were computed with the log-probit method [t-test with Welch’s correction was used to compare the experimentally derived IC50mix values  with their respective, theoretically calculated and presumed to be additive IC50add values, as recommended elsewhere [Results were analyzed with a one-way ANOVA test followed by Tukey’s post hoc test. Values were presented as means ± standard errors (SEM). Statistical significance was marked graphically using asterisks  deserves a preclinical recommendation as a very beneficial combination for anticancer therapies.Esculetin inhibited melanoma cell viability and proliferation in a dose-dependent manner. The IC


Breast cancer is the most prevalent cancer among females with different molecular subtypes. Corosolic acid is a pentacyclic triterpenoid with anti-cancer properties. The MTT assay was used to assess the cytotoxic activity of corosolic acid on MDA-MB-231 and MCF7 cell lines. To determine the apoptotic cells, the flow cytometry technique was utilized. The expression levels of apoptosis-related genes and proteins were quantified using quantitative real time-PCR (qRT-PCR) and Western blotting methods. The activity of caspase enzymes was measured by spectrophotometry. Corosolic acid significantly inhibited the proliferation of both cell lines compared with controls. This agent markedly induced apoptosis in MDA-MB-231 cells but did not affect MCF7 cells compared with controls. Treating the MADA-MB-231 and MCF7 cell lines with corosolic acid showed an inducing effect on apoptosis-associated caspases, including Caspase-8, 9, and -3, in MADA-MB-231 cells with no effect on apoptotic markers in MCF7 cells. Further experiments uncovered corosolic acid-induced apoptosis in MADA-MB-231 cells by decreasing the expression of the phosphorylated form of JAK2 and STAT3 proteins.The present data suggested that corosolic acid is an apoptosis-inducing phytochemical in triple-negative breast cancer MADA-MB-231 cells. Also, corosolic acid triggered apoptosis in these cells by stimulating both pathways of apoptosis and inhibiting the JAK/STAT signaling. Furthermore, corosolic acid was found to inhibit MCF7 cell proliferation by a non-apoptotic mechanism. Breast cancer is the most prevalent type of cancer among females and ranks as the second cause of cancer-dependent death in women worldwide . Breast Lagerstroemiaspeciosa with antiinflammatory, antiobesity, antidiabetic, antihyperlipidemic, anti-cancer, and antiviral activities . Both MCF7 and MDA-MB-231 cell lines were seeded at the density of 2×104 cells/well into 96-well cell culture plates. Subsequently, the seeded cells were treated with different concentrations of corosolic acid  for 24, 48, and 72 hr.Both MCF7 and MDA-MB-231 cell lines were purchased from National Center for Cell Science . The cells were then cultured and incubated in a humidified incubator at 37 Cell viability After incubation of the cell lines with increasing concentrations of corosolic acid, the viability of the cells was quantified using Vybrant 3--2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay kit  according to the manufacturer’s instructions. Briefly, the cell lines were incubated with 5 mg/ml MTT solution for 4 hr to evaluate the influence of corosolic acid on their viability. Subsequently, the MTT solution was removed and 200 µl of dimethyl sulfoxide (DMSO) solution was added to each well. After 15 min, the quantity of formazan product was detected by measuring the absorbance at 570 and 630 nm using a plate reader spectrophotometer .Real-time PCR-ΔΔCt method and the GAPDH transcript was used as the housekeeping gene. The primer sequences used in the present study are shown in The primary primer sequences were extracted from the NCBI database and the specific primers were designed by using Primer Express software v1.5 (Applied Biosystems). The total RNA content of both cell lines was extracted using an RNeasy mini kit . To synthesize the complementary DNA (cDNA) from 1 µg/ml of single-stranded RNA, the oligo-d (T) 15 primer  and M-MLV reverse transcriptase were used. Real-time PCR analyses were carried out on cDNA samples by using SYBR-green master mix  on ABI PRISM 7900HT  under thermocycling conditions. Furthermore, melt curve analysis was carried out for each gene to prove the specificity of the primers and the lack of non-specific products. The expression levels of all genes were analyzed using the 2Flow cytometry°C) for 5 min, washed once with 5 ml of a phosphate-buffered saline (PBS), and resuspended in the binding buffer. Afterward, 5 µl of PI and 5 µl of FITC-annexin V were added to the cell suspension and incubated in the dark for 15 min at room temperature. Analysis of FITC-annexin V binding was carried out on a FAC scan flow cytometer (BD Biosciences) with an excitation wavelength of 488 nm and an emission wavelength of 350 nm. PI-negative/Annexin V-positive cells were presented as early apoptotic cells and the cells with positive PI and Annexin V were reported as late apoptotic cells. The effect of corosolic acid on apoptosis was studied using a FITC/annexin-V-propidium iodide (PI) kit  according to the manufacturer’s protocol. Briefly, MCF7 and MDA-MB-231 cells were treated with corosolic acid at concentrations equal to IC50 and IC25 for 48 hr. Then, the treated cells were centrifuged (1000 g) at room temperature  to prepare the whole-cell lysates. The lysates were then centrifuged and the supernatants were utilized for Western blot analysis.To measure the expression of proteins by Western blotting technique, both MCF7 and MDA-MB-231 cell lines were seeded in 6-well culture plates at the density of 2×10°C. All membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody in the dark for 1 hr at room temperature. All antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Finally, the immunoblots were developed by using an enhanced chemiluminescent kit  followed by exposure to autoradiography film.A bicinchoninic acid (BCA) assay kit  was used to estimate the total protein content of the samples. 40 µg of the total protein was loaded onto 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane  through electroblotting. The membranes were blocked with 5% non-fat milk for 1 hr at room temperature. Subsequently, the membranes were exposed to Caspase-8, Caspase-9, Caspase-3, Bax, Bcl-2, STAT3, JAK2, Phosphorylated STAT3, Phosphorylated JAK2, and GAPDH primary antibodies overnight at 4 Measurement of the activity of caspase enzymes°C. Subsequently, 200 μg of the total protein content of the cell lysate was mixed with the assay buffer  to a final volume of 100 μl. Then, 20 μl of each substrate , ,  was added to the mixture. The reaction mixtures were incubated for 30 min at 37 °C and the absorbance of the enzyme products was measured at 405 nm using an Epoch microplate spectrophotometer.To measure the activity of Caspase-8, -3, and -9, both MCF7 and MDA-MB-231 cell lines were lysed in the lysis buffer  with vortexing for 30 min at 4 Statistical analysispost hoc test. These statistical analyses were done using SPSS software version 19 . The statistical difference between the data was considered significant at P<0.05.All experiments were repeated at least three times and the obtained data were expressed as mean±standard deviation (SD). The results were analyzed using the one-way analysis of variance (ANOVA) test followed by Duncan’s multiple range tests as the Effect of corosolic acid on the viability of MDA-MB-231 and MCF7 cell linesThe results of the MTT assay revealed that treating both MDA-MB-231 and MCF7 cell lines with increasing concentrations of corosolic acid for 48 and 72 hr could mitigate the proliferation of these breast cancer cell lines in a dose-dependent manner compared with the untreated control cells. Corosolic acid treatment for 24 hr had no significant effects on either cancer cell line  and 2A. Effects ofcorosolic acid on apoptosis induction in MDA-MB-231 and MCF7 cell linesTo evaluate the apoptosis-inducing activity of corosolic acid on MDA-MB-231 and MCF7 cell lines, the cells were treated with IC25 and IC50 concentrations of this natural compound. In the MDA-MB-231 cell line, a marked increase in the late and early stages of apoptosis was observed after treatment of the cell line with 10 μM (IC25) and 20 μM (IC50) of corosolic acid for 48 hr . CorosolEffects of corosolic acid on caspase enzyme activity in MDA-MB-231 and MCF7 cell linesAfter corosolic acid treatment, the activity of three key caspase enzymes, Caspase-8, -3, and -9 was measured in MDA-MB-231 and MCF7 cell lines to evaluate the activation and function of these enzymes in the presence of this compound. As depicted in Effects of corosolic acid on expression of apoptosis-related factorsin MDA-MB-231 cell lineTo assess the possible effect of corosolic acid on key factors involved in the apoptosis of MDA-MB-231 cells, the expression levels of some apoptosis-related genes and proteins were measured. The results of quantitative real time-PCR analyses provided evidence to show that corosolic acid, at concentrations equal to IC25 and IC50 for 48 hr treatment, considerably elevated the transcript levels of CASP-8, -9, and -3 in the MDA-MB-231 cell line in comparison with untreated control cells. However, it had no significant effect on the expression levels of JAK2 and STAT3 genes compared with the controls . The resEffects of corosolic acid on expression of apoptosis-related factorsin MCF7 cell lineTo unravel the potential effect of corosolic acid on apoptosis markers in MCF7 cells, the gene and protein expression of these markers were quantified using real time-PCR and western blotting. The results of gene expression analysis uncovered that corosolic acid treatment of MCF7 cells with IC25 (14 µM) and IC50 (28 µM) concentrations of this agent for 48 hr did not alter the transcript expression of CASP-8, -9, and -3 as well as BAX and BCL-2 as compared with the untreated control cell line . The resAs described before, TNBC is the most aggressive type of breast cancer and develops resistance to treatment methods due to the absence of ER, PR, and HER-2 receptors . AlthougIn the first step, MDA-MB-231 and MCF7 cell lines were treated with increasing concentrations of corosolic acid for 24, 48, and 72 hr. Corosolic acid treatment of both MDA-MB-231 and MCF7 cell lines led to a significant decrease in the viability of the cells in a dose- and time-dependent manner. Of note, this triterpenoid hindered the MDA-MB-231 cell proliferation rate at lower concentrations  than those in the MCF7 cell line . This suggests that the TNBC cell line, MDA-MB-231, is more sensitive to corosolic acid than the PR- and ER-positive MCF7 cell line. Additionally, it can be implied that the anti-breast cancer effect of corosolic acid is not necessarily receptor-dependent. In the second step, we hypothesized that the antiproliferative activity of corosolic acid may arise from its effect on the induction of apoptosis in both cell lines. Hence, the cell lines were treated with IC50 and IC25 concentrations of corosolic acid. The results clarified that corosolic acid considerably augmented both early and late apoptotic cell populations of the MDA-MB-231 cell line, however, it showed no remarkable pro-apoptotic activity against MCF7 cells. To affirm the role of the key caspase enzymes in this effect, the cell lines were exposed to IC50 and IC25 concentrations of corosolic acid, and then the activity of Caspase-8, -9, and -3 was measured. The obtained findings confirmed the elevated activity of all three caspases in the MDA-MB-231 cell line due to corosolic acid treatment. However, the compound did not affect the activity of the enzymes in the MCF7 cell line. The process of apoptosis functions through two main pathways that include mitochondria-dependent (intrinsic) and death receptor-mediated (extrinsic) pathways. The former pathway initiates apoptosis via activating Caspase-9 and the latter induces apoptosis by stimulating Caspase-8. These activated caspases trigger the activation of Caspase-3, as the executioner in apoptosis . AccordiIn the third step, the effect of corosolic acid treatment on the expression levels of some apoptosis-related genes and proteins was quantified in MDA-MB-231 and MCF7 cell lines. The results of real-time PCR and western blotting methods evidenced that corosolic acid had no marked effect on the expression levels of Caspase-8, -9, -3, Bax, and Bcl-2 at either mRNA or protein levels. However, treating the MDA-MB-231 cell line with IC50 and IC25 concentrations of corosolic acid significantly amplified the levels of Caspase-8, -9, and -3 in comparison with untreated control cells. et al. identified that corosolic acid decreased the proliferation and increased apoptosis of MDA-MB-231 cells in a dose-dependent manner. They revealed that the apoptosis-inducing effect of corosolic acid on MDA-MB-231 cells was mediated by a mechanism that involves reactive oxygen species (ROS)  activity. However, they did not study the apoptosis-inducing activity of corosolic acid in this cell line . Son et es (ROS) . However pathway . They me pathway . Sung an pathway . However pathway -29. It set al. demonstrated that corosolic acid suppressed the proliferation of glioblastoma cells and tumor-associated macrophages by blocking STAT3 and NF-κB in these cells (An increasing body of evidence in the literature shows that targeting the JAK/STAT signaling pathway in TNBC cell lines is a novel and efficient approach to stimulating apoptotic cell death in these cancer cells -32. Consse cells . It has se cells . CorosolNatural products that target both pathways of apoptosis are the most competent anti-cancer drugs because they prevent drug resistance that arises from the cross-talks between the signaling pathways in the cancer cells . Therefoin vitro investigation of the effects of corosolic acid on breast cancer cell lines. Thus, future works should focus on the development of in vivo models of breast cancer using both cancer cell lines. Then, the present factors can be evaluated in those animal models to establish the present results.Although the present study has valuable data and offers beneficial contributions to current literature, we note some limitations of our research and suggest some extra evaluations for future studies. As indicated in the title of the manuscript, the present study is an KH, ATJ, and SZ designed the experiments and methodology. SAJ, OZK, RSZ, QAQ, and YFM performed the experiments and collected the data. SHA, CMW, YABJ, and MSM were involved in data analysis and manuscript writing. KH edited the manuscript. The authors have no conflicts of interest to declare.


The optimal radiobiological model, which assesses the biological effects of novel radiotherapy techniques that concurrently modify multiple physical factors, has not yet been defined. This study aimed to investigate the impact of intensity‐modulated radiotherapy (IMRT) and volumetric‐modulated arc therapy (VMAT) on cellular response in head and neck cancer and melanoma models.Clonogenic analysis, DNA double‐strand break analysis, apoptosis, and cell cycle analysis were performed on cancer stem cell models, cancer models, and normal tissue cell models to assess radiation sensitivity.The segmented radiation approach used in IMRT applications enhanced radiosensitivity and cytotoxicity in the cancer models, while changes in dose rate had varying effects on cytotoxicity depending on the tumor cell type. VMAT increased cellular resistance, favoring treatment outcomes.The biological processes were influenced differently by dose rate, IMRT, and VMAT depending on the tumor cell type. The selection of the most appropriate technique is crucial in representing new radiotherapy approaches. The obtained data can serve as a model to address clinical questions in daily practice. The integration of non‐standard outcomes with standard applications should be considered in clinical settings. Among the primary treatment modalities for cancer patients, radiotherapy plays a vital role.The main objective of this study was to develop a radiobiology model that accurately reproduces the biological effects of advanced radiation procedures. Previous literature has identified the lack of a suitable model for these state‐of‐the‐art techniques. Therefore, our investigation focused on assessing the cellular response to complex dose distributions generated using the IMRT approach, considering various physical factors. Our ultimate goal was to establish a model that closely reflects the daily applications of these techniques.In this study, we conducted a comprehensive examination of the impact of parameters commonly used in everyday clinical practice, utilizing novel technological devices, on in vitro cell survival. To simulate real‐life scenarios, we employed three distinct cell models: two tumor cell lines known for their robust radioresistance properties, tumor stem cell lines recognized as the primary contributors to radiotherapy resistance, and normal cell lines susceptible to the effects of radiotherapy. After establishing appropriate dose ranges, we meticulously investigated the effects of dose, dose rate, and non‐uniform dose distribution on cellular survival using various assays, including clonogenic assay, DNA double‐strand break assay, apoptosis assay, cell cycle assay, and autophagy assay. Additionally, we explored the in vitro effects of volumetric‐modulated arc therapy (VMAT) application using a phantom that closely mimics daily clinical applications. Through this comprehensive study, our aim was to address the existing gap in the literature by developing an appropriate radiobiology model for advanced radiation techniques. The findings from our research contribute to a better understanding of the cellular response to complex dose distributions and have significant implications for improving treatment planning and enhancing patient outcomes in clinical practice.22.13), it provided a complete scattering medium with varying density levels for irradiating the flasks  was used to verify the calculated monitor unit (MU) values, ensuring that the dose values fell within ±3% of the planned values. Additionally, Gafchromic EBT3 films were placed at the bottom of each cell flask during irradiation to confirm the targeted doses using a secondary dosimetry method. The dose values obtained from the Gafchromic films also confirmed that the doses delivered to the flasks were within ±5%. All analyses were performed in triplicate.A cylindrical water phantom was specifically designed for the VMAT irradiation technique to accommodate T25 cell culture flasks, allowing for the simultaneous irradiation of two flasks. The phantom was designed to maintain physical conditions similar to the VMAT technique, ensuring reproducible irradiation setups. To mimic different density levels of the human body and create a scenario similar to daily applications, materials with four different densities were placed inside the phantom. When the phantom was filled with water  and human head and neck CSC line  were utilized as CSC models. Additionally, a melanoma cell line (NM2C5/RRID:CVCL_B064) and head and neck cancer cell lines (A253−/RRID:CVCL_1060 and FADU/RRID:CVCL_1218) were established as cancer cell models. Fibroblast cell line (WI‐38/RRID:CVCL_0579) and epithelial cell line (WSS‐1/RRID:CVCL_2767) were employed as control models representing normal tissue cells. All cell lines were cultured in a standard cell culture incubator at 37°C, under a 5% CO2.2.2All cancer cell models and control cells were exposed to irradiation at doses of 2, 4, 6, 8, and 10 Gy for clonogenic assays. The untreated group consisted of 100 cells, while the 2 Gy treatment group had 200 cells, the 4 Gy treatment group had 600 cells, the 6 Gy treatment group had 1000 cells, the 8 Gy treatment group had 1200 cells, and the 10 Gy treatment group had 1400 cells. After 3 weeks of irradiation, colonies containing at least 50 cells (>1 mm) were evaluated in the clonogenic assay. All analyses were conducted in triplicate. Statistical analyses were performed using the SPSS program with the following procedures. For the quadratic term, “D2” was defined as the product of the dose and dose (D2 = dose × dose). For survival analysis, the variable “S” was calculated as the natural logarithm of the ratio of colonies to cells (ln(colonies/cells)) minus the natural logarithm of the plating efficiency (ln(PE)). The weight of observed colonies was calculated using the formula “W” = colony × cells/(cells−colony). Linear regression analysis was conducted using the SPSS software . The dependent variable “S” was used, while the independent variables “dose,” “D2,” and “W” (under WLS in SPSS) were included. The analysis included confidence intervals and R‐squared values (Statistics in SPSS). Since the regression line passes through the origin, the option to include a constant in the equation (Options in SPSS) was not selected.2.2.34 cells/100 μL. The cells were then exposed to various doses of ionizing radiation and incubated for 24, 48, and 72 h. Cell proliferation was assessed using the WST‐1 assay method, which involved adding the WST‐1 Cell Proliferation Reagent to each well, followed by measuring the absorbance at 450‐nm and 620‐nm wavelengths using a microplate reader. The effects of irradiation on cell proliferation were quantified by analyzing the dose–response curve using Sigmoidal dose–response curve analysis in the Calcusyn 2.0 software.Cells were seeded in 96‐well plates at an initial density of 1 × 102.2.45 cells/mL and exposed to radiation doses ranging from 0 to 10 Gy. Annexin V (Annexin V‐EGFP Apoptosis Detection Kit‐Biovision) and Mitoprobe JC‐1 (MitoScreen JC‐1 kit‐BD Biosciences) methods were employed to study apoptosis in three replicates, following the manufacturer's instructions. The untreated group served as the control group. Flow cytometry analysis was conducted using the BD Acuri 6 system to evaluate the results.Cell lines were seeded in six microplates at a concentration of 5 × 102.2.55 cells/mL in six microplates. The analysis was performed at the 48th hour, a time point at which significant DNA double‐strand breaks could be detected. Flow cytometry was employed for evaluation.The impact of varying radiation doses on DNA double‐strand breaks in cancer models was examined using γ‐H2AX analysis (Alexa Fluor 488 Mouse anti‐H2AX kit). Cell lines exposed to radiation doses ranging from 0 to 10 Gy were seeded at a concentration of 5 × 102.2.6Cell lines were seeded at a concentration of 5 × 105 cells/mL in six microplates and exposed to radiation doses ranging from 0 to 10 Gy. The cell cycle distribution in cancer cells was investigated using cell cycle analysis (BD Cycletest Plus kit) at 24, 48, and 72 h after radiation exposure. Propidium iodide (PI) bound to DNA was quantified using flow cytometry with 488‐nm excitation and 586/42 bandpass emission filters. The FACSuite software was used to determine the number of cells in the sub G0/G1, G0/G1, S, G2/M, and >4n stages, and the percentages were calculated accordingly.2.2.7p < 0.05.Descriptive statistics were employed for data analysis. Nonlinear regression analysis based on the linear‐quadratic model was used to investigate the relationship between radiation doses and the survival fraction of cells. The same analysis was conducted to assess the impact on cell survival response using the phantom. A one‐way analysis of variance (ANOVA) was performed to compare the effects of different dose rates after 4 Gy irradiation. Apart from the aforementioned statistical analyses, all other statistical analyses were carried out using GraphPad Prism 9.5. The significance level was set at 33.13.1.1R2 = 0.978, p = 0.011, Figure In the head and neck cancer model, the coating efficiency was 17.20% in the A253 cell line and 40.33% in the FADU cell line. The melanoma cancer model (NM2C5 cell line) exhibited a coating efficiency of 36.33%. A significant correlation was observed between the dose and survival  head and neck CSC line was 99.67%. In contrast, the coating efficiency in the human  melanoma CSC line was 66.33%. A significant correlation was observed between the dose and survival . The head and neck cancer model demonstrated higher radiosensitivity compared to the melanoma model. The WSS‐1 epithelial cell line and WI‐38 fibroblast cell line were chosen to represent early and late responding normal tissues in radiobiological modeling due to their distinct behavioral traits, accurately reflecting the characteristics of normal tissues. Notably, CSC models displayed different radiosensitivity properties in comparison with tumor cell models . Interestingly, apoptosis was not observed in the NM2C5 cell line when exposed to radiation doses of 2–10 Gy from 24th to 72nd hours. In the human (parent) head and neck CSC line, apoptosis was triggered at 2–10 Gy after 48th hours, and in the human  melanoma CSC line, apoptosis occurred at 6–10 Gy after 72nd hours, both compared to the control group. Additionally, in the WI‐38 cell line, apoptosis was induced at 2–10 Gy after 24th hours, while in the WSS‐1 cell line, apoptosis was induced at 8 and 10 Gy from 24th to 72nd hours compared to the control group  head and neck CSC line, and at 6–8 Gy at 72nd hours in the human  melanoma CSC line compared to the control group. Consistent with the annexin V results, compared to the control group, apoptosis was induced at 2–10 Gy at 24th hours in the WI‐38 fibroblast cell line and induced at 6–10 Gy at 24th and 72nd hours in the WSS‐1 cell line  head and neck CSC line at doses of 2–10 Gy and in the human  melanoma CSC line at doses of 4–10 Gy. Interestingly, DNA double‐strand breaks were not detected in the WI‐38 and WSS‐1 cell lines  melanoma CSC line and the human (parent) head and neck CSC line exhibited cell cycle arrest at the G2/M stage at doses of 2–10 Gy after 24 and 72 h. No significant difference was found in the cell cycle of the WI‐38 cell line compared to the control group at doses of 2–10 Gy after 24 and 72 h. The WSS‐1 cell line showed differences in the G0/G1, S, and G2/M phases of the cell cycle compared to the control group at doses of 2–10 Gy after 24 and 72 h.3.22 flasks. A comparison of cell survival responses between non‐uniform and uniform areas was conducted using the clonogenic assay method.The effective dose of 4 Gy was determined for the cancer cell models, and the cancer cells were exposed to this dosage in partially and fully open 25 cm3.2.1t = −0.378, p = 0.742). Similarly, in the FADU cell line, the coating effectiveness and survival rates were 2.89% and 57.78% following fractionated 4 Gy treatment, and 2.11% and 42.22% after non‐fractionated 4 Gy exposure. Again, the difference was not significant . In the NM2C5 cell line, the coating effectiveness and survival rates after fractionated 4 Gy exposure were 0.87% and 7.74%, respectively, while they were 3.4% and 30.36% after non‐fractionated 4 Gy exposure. The difference was significant . This indicated a 22.62% decrease in survival following fractional irradiation in the melanoma cell line NM2C5. In summary, it was observed that the fractional irradiation technique improved cytotoxicity in the melanoma cell model, but it did not produce a significant difference in the head and neck tumor models . In the human  melanoma CSC line, no colonies were detected after fractionated 4 Gy treatment, resulting in a 0% survival rate. After non‐fractionated 4 Gy treatment, the coating efficiency and survival rates were 35.00% and 38.18%, respectively. The difference was highly significant . There was a 38.18% reduction in survival due to fractional irradiation in the melanoma stem cell line. Overall, it was observed that fractional irradiation increased cytotoxicity in the melanoma CSC model, while the survival effect of gradual irradiation was not observed in the CSC models, similar to the tumor cell models  head and neck CSC line, the coating efficiency and survival rates were 70.42% and 83.17% after fractionated 4 Gy, and 68.08% and 80.41% after non‐fractionated 4 Gy, respectively. However, the difference was not significant . In the WSS‐1 cell line, the coating efficacy and survival rates were 2.67% and 11.11%, respectively, after fractionated 4 Gy exposure, and 3.67% and 15.28% after non‐fractionated 4 Gy exposure. However, the difference was not significant . Overall analysis of the data showed that fractional irradiation in the WI‐38 cell line resulted in a 27.35% reduction in survival. It was observed that the more radioresistant normal tissue cells (WI‐38) exhibited a higher level of cytotoxicity, similar to what was seen in the melanoma tumor and CSC model , 300 MU/min (standard irradiation), or 600 MU/min (high‐dose rate) in 25 cm3.3.1t = 0.281, p = 0.805). Similarly, in the FADU cell line, the survival rates at 4 Gy were 45.2% with a dose rate of 50 MU/min, 42.23% with a dose rate of 300 MU/min, and 34.32% with a dose rate of 600 MU/min. Again, the difference was not significant . In the melanoma cancer model, the survival rates after treatment at 4 Gy were 13.15% with a dose rate of 50 MU/min, 22.02% with a dose rate of 300 MU/min, and 51.02% with a dose rate of 600 MU/min. The difference was significant . It was observed that cytotoxicity decreased as the dose rate increased. When considering all the data from the tumor cell models, it was found that the cytotoxicity of head and neck tumor models increased with increasing dose rate, while the cytotoxicity of melanoma cell models decreased with increasing dose rate . Overall, the findings indicate that compared to standard dose‐rate treatments, cell survival decreased at high‐dose rates in head and neck stem cells, as expected. It was observed that low‐dose rates had a more negative effect on survival in melanoma stem cells compared to standard dose‐rate irradiation  head and neck CSC line was 28.28% at a dose rate of 50 MU/min, 42.91% at a dose rate of 300 MU/min, and 33.24% at a dose rate of 600 MU/min. However, the difference was not significant. In the human  melanoma CSC line, the survival rate after treatment at 4 Gy was 29.24% with a dose rate of 50 MU/min, 65.30% with a dose rate of 300 MU/min, and 43.33% with a dose rate of 600 MU/min. The difference was significant . In the FADU cell line, there was a 38.67% increase in survival after 2 Gy irradiation. However, this difference was no longer present at 4 Gy, and no significant difference in viability was detected using the VMAT technique (p = 0.06). In the melanoma cancer model, there was an 8.21% decrease in survival after 2 Gy irradiation compared to the control group. However, there was a 32.17% improvement in survival following high‐dose 6 Gy irradiation compared to the control group. In the NM2C5 cell line, significant differences in vitality were observed compared to the control group at 2 Gy, 6 Gy, and 8 Gy (p < 0.0001). Radiosensitivity in epithelial cell lines increased with the use of VMAT at 2 Gy, while radiosensitivity in the melanoma cell model increased at low doses and decreased considerably at doses above 4 Gy . In the melanoma CSC line, there was an 81.87% decrease in survival at 2 Gy compared to normal cells. Radiosensitivity was observed to increase at higher dosages (p < 0.001), and it was also observed to increase at 2 Gy with the use of VMAT technique. Furthermore, the VMAT technique in the phantom was observed to enhance radiosensitivity and viability in the head and neck stem cell model, similar to the effects observed in tumor cell models. In melanoma stem cell lines, the VMAT technique exerted different effects at low and high doses  head and neck CSC line, with a 40.76% decrease in survival observed after 2 Gy irradiation. This decrease in survival was found to be significant compared to the control group . On the contrary, the WI‐38 cell line exhibited a 62.02% increase in survival compared to the control group after 2 Gy irradiation, and this difference was found to be significant (p < 0.0001). When the results of normal tissue cell models were analyzed collectively, it was observed that the VMAT technique reduced radiosensitivity in normal tissue cells, unlike in tumor and CSCs , normal tissues (lung epithelium and fibroblast cells), and CSC models (head and neck and melanoma CSCs). Upon reviewing the collective results, it was observed that the radiosensitivity differed among the various tumor cells, normal tissue cells, and CSCs. Notably, the radiosensitivity of CSC models was found to be lower compared to tumor cell models. CSCs have been shown to exhibit increased resistance to radiation compared to non‐CSC cells, and their presence is believed to contribute to treatment failure and tumor recurrence.4.2The multi‐leaf collimator (MLC) system is employed in daily radiotherapy applications to deliver radiation to the target at different intensities and at different times, enabling the modification of dose intensity in both tumors and normal tissues and achieving desired dose distributions. This technique, known as IMRT, involves delivering the radiation beam as a cumulative sum of segmented irradiation to the tumor and surrounding normal tissues throughout the treatment course. Interplay between intercellular signaling systems and this segmented irradiation could influence the cellular response, potentially leading to either an increase or decrease in radiation sensitivity.The application of IMRT has provided valuable insights into its physical characteristics, such as its ability to deliver high doses to the target while minimizing radiation exposure to nearby critical organs. This capability has enabled the successful treatment of patients with complex‐shaped tumors, surpassing the limitations of traditional radiation therapy. Nevertheless, it should be noted that most IMRT techniques involve the delivery of treatment fractions over extended time periods and at lower dose rates. The radiobiological implications of reduced dose rates and fractionated irradiation techniques are not yet fully elucidated, and further research is needed to comprehensively understand their impact on treatment outcomes.In our study, we investigated the effects of two‐dimensional fractionated irradiation techniques, representing both uniform and non‐uniform irradiation patterns, on the survival curve's 4 Gy shoulder region as the test dose. Following the completion of cell clonogenic survival studies, the findings revealed that the fractionated irradiation approach resulted in increased radio‐cytotoxicity and radiosensitivity in both melanoma and melanoma stem cell models. To the best of our knowledge, this is the first study to demonstrate that segmental irradiation enhances the radio‐cytotoxicity of melanoma cells. While long‐term therapy involving segmental irradiation has been reported to decrease radiosensitivity in various tumor types, it is noteworthy that our study observed an increase in radiosensitivity.4.3The biological response to radiation is influenced by factors such as radiation energy, dose, and dose rate.In this study, the efficacy of dose rate was evaluated in CSC models, marking the first instance in the literature. Interestingly, it was observed that lower dose rates had a more detrimental impact on survival compared to standard dose‐rate irradiations. It is advisable to consider these variances for therapeutic advantages in clinical practice and verify them through appropriate clinical protocols.4.4In this paper, we employed a cylindrical water phantom that is compatible with a reproducible irradiation system to investigate the impact of complex dose distributions obtained through IMRT techniques on cellular response. This system created physical conditions that are in line with the VMAT technique. However, determining the optimal experimental design that closely resembles daily clinical practice is not straightforward. Various researchers have proposed solid phantom models in the literature for this purpose.In volumetric irradiation techniques such as the VMAT technique, high doses are physically delivered to the tumor tissue, resulting in sharp dose gradients in normal tissues. While a high dose is maintained within the tumor, reduced dose regions are applied to normal tissues. One of the crucial radiobiological considerations associated with these new physical parameters is that irradiation can cause side effects in normal tissues beyond the expected levels. Dose constraints for normal tissues have been established based on clinical observations obtained from three‐dimensional radiotherapy applications.In conclusion, the findings of this study need to be validated through in vivo testing before incorporating them into the current treatment regimens of clinical practice. It is essential to consider the physical and radiobiological aspects of applying stereotactic radiotherapy, especially for melanoma cells, which involves modifying the dose rate and segmental irradiation at high rates. By doing so, we can better understand and manage the potential side effects in normal tissues and optimize treatment outcomes for patients.Serra Kamer: Funding acquisition (lead); investigation ; methodology ; resources ; supervision ; validation ; writing – review and editing (supporting). Sunde Yilmaz Susluer: Methodology ; validation ; writing – original draft (lead); writing – review and editing (lead). Tugce Balci Okcanoglu: Methodology ; validation . Cagla Kayabasi: Methodology ; validation . Besra Ozmen Yelken: Methodology ; validation . Sinan Hoca: Investigation ; methodology ; visualization . Emin Tavlayan: Investigation ; methodology ; visualization . Nezahat Olacak: Conceptualization ; investigation . Yavuz Anacak: Conceptualization ; supervision . Murat Olukman: Conceptualization ; supervision . Cumhur Gunduz: Investigation ; methodology ; supervision ; validation .The authors have no conflicts of interest to declare.No ethical approval statement required.