Recent studies on cancer cell metabolism have achieved notable breakthroughs that have led to a new scientific paradigm. How cancer cell metabolic reprogramming is orchestrated and the decisive role of this reprogramming in the oncogenic process and tumor adaptative evolution has been characterized at the molecular level. Despite this knowledge, it is essential to understand how cancer cells can metabolically respond as a living whole to ensure their survival and adaptation potential. In this work, we investigated whether different cancers and different subtypes display different metabolic phenotypes with a focus on breast cancer cell models representative of each clinical subtype. The potential results might have significant translational implications for diagnostic, prognostic and therapeutic applications.Metabolic reprogramming of cancer cells represents an orchestrated network of evolving molecular and functional adaptations during oncogenic progression. In particular, how metabolic reprogramming is orchestrated in breast cancer and its decisive role in the oncogenic process and tumor evolving adaptations are well consolidated at the molecular level. Nevertheless, potential correlations between functional metabolic features and breast cancer clinical classification still represent issues that have not been fully studied to date. Accordingly, we aimed to investigate whether breast cancer cell models representative of each clinical subtype might display different metabolic phenotypes that correlate with current clinical classifications. In the present work, functional metabolic profiling was performed for breast cancer cell models representative of each clinical subtype based on the combination of enzyme inhibitors for key metabolic pathways, and isotope-labeled tracing dynamic analysis. The results indicated the main metabolic phenotypes, so-called ‘metabophenotypes’, in terms of their dependency on glycolytic metabolism or their reliance on mitochondrial oxidative metabolism. The results showed that breast cancer cell subtypes display different metabophenotypes. Importantly, these metabophenotypes are clearly correlated with the current clinical classifications. In recent years, our concepts and knowledge of cancer cell metabolism have experienced a profound breakthrough that has led to a new scientific paradigm ,2,3. SinIn this context, a foundational scientific proposition is the concept of tumor metabolic reprogramming. This proposition was based on crucial experimental observations, such as the apparently paradoxical catalytic inefficiency of the glycolytic enzyme pyruvate kinase M2 (PKM2), which is expressed only in cancer and proliferative cells, relative to the more efficient PKM1, which is expressed in nontransformed somatic cells. PKM2 catalyzes the last glycolysis reaction; therefore, glycolytic flux is modulated by its lower catalytic velocity, which acts as a limiting factor. Consequently, many glycolytic intermediaries accumulate and can be used in branching anabolic routes, thus guaranteeing the high demand of biomass to sustain cell proliferation ,9,10,11.Metabolic reprogramming represents an orchestrated network of evolving adaptations during oncogenic progression. Cancer cells can adopt different metabolic landscapes in response to their own oncogenic evolution, changes in the cellular niche and environment, and resistance responses to chemo- and radiotherapy ,19. ImpoIn line with this research demand, we recently developed a novel metabolic nanosensor for robust measurements of mitochondrial pH. Interestingly, we found significant variations in intramitochondrial pH among different breast cancer cell lines that correlated with the current clinical subtype classification. Moreover, the variations in pH also correlated with differential responses to some metabolic drugs . In a seNevertheless, there are still relevant open questions from a functional perspective of breast cancer metabolic reprogramming. In this context, it is particularly interesting to investigate whether distinct metabolic profiles could correlate well with breast cancer clinical subtypes. The potential results from this type of investigation might have significant translational implications at different levels. Accordingly, the correlation between the current classification criteria for breast cancer subtypes and well-defined metabolic profiles could profoundly enhance the diagnostic and prognostic capacities. In addition, such work could pave the way for the development of novel therapeutic strategies based on “antimetabolic” subtype-specific drugs as well as novel technical applications with diagnostic and therapeutic purposes.Recently, foundational contributions initiated the investigation of key metabolic features in different breast cancer cell models in vitro and in vivo. In this context, the characterization of glutamine dependence tested by differential sensitivity responses to transaminase inhibitor aminooxyacetate (AOA) was of particular relevance. Interestingly, these results were in correlation with the oncogene c-Myc expression levels between breast cancer cell lines representative of clinical subtypes . In addiMoreover, there have been important contributions aiming to address whether breast cancer cell subtypes might feature different metabolic phenotypes. Most of these elegant works were performed by unlabeled metabolomic analysis to obtain quantitative data about total metabolite content. Some noticeable results were obtained but the data were not totally concordant displaying some discrepancies ,50,51,52Under these premises, as already proposed by the aforementioned authors, further steps to address metabolic phenotypic differences between breast cancer cell subtypes need to be performed by dynamic metabolic flux analysis. In this sense, isotope labeling experiments are required to identify dynamic flux differences in central metabolic routes, such as glucose preferential usage and TCA reliance, as well as to decipher key enzymatic activities promoting those preferential pathways.Notwithstanding the foundational and valuable information accounted by the aforementioned works, further steps are required in breast cancer cell metabolism research, particularly to accomplish the investigations about phenotypic and molecular metabolic differences among breast cancer cell subtypes.Accordingly, the present work was designed to gain further insights in that direction. First, breast cancer cell lines were profiled by data combined from different treatments with five metabolic inhibitors: AOA, EGCG, phenformin, the PI3K inhibitor BKM120, and the mitochondrial membrane uncoupler BAM15. This profiling approach, testing the five selected inhibitors rather than profiles based on a single drug-single metabolic question design, was accomplished as a combinatorial profile aiming to evaluate systemic functional responses of breast cancer cell lines representative of each clinical subtype. Thus, following some insights provided by previous works, where single experimental treatments with AOA, EGCG or Phenformin were performed, in the present study the degree of glycolytic flux dependence was tested by sensitivity responses to phenformin and BKM120 treatments. In addition, the reliance on tricarboxylic acid cycle (TCA) activity and its connection with the potential preferences for cytosolic versus mitochondrial energy supply was studied combining data by AOA, BAM15 and EGCG treatments. Secondly, potential dynamic metabolic flux differences between breast cancer cell lines were studied by stable-isotope dynamic labeling for key targets, such as pyruvate, based on preferential pyruvate dehydrogenase (PDH) or pyruvate carboxylase (PC) activities to quantitatively analyze the degree of TCA reliance, and other central pathways as glutamine/glutamate metabolism preferences based on anaplerotic input to TCA or their preferential use by reductive carboxylation.The results confirmed that there are differential dependences on glycolitiyc flux activity and TCA and anaplerotic activity reliance between hormone receptor-positive and triple-negative breast cancer subtypes. In addition, the cell lines representative of these clinical subtypes showed different dependencies on transaminase or glutamate dehydrogenase activity for recycling nonessential amino acids. Interestingly, the metabolic phenotypes were in correlation with the different breast cancer subtypes. Finally, as a mechanistic metabolic proof of principle, differences were found between hormone receptor-positive and triple-negative representative cell lines for preferential flux through pyruvate dehydrogenase (PDH) or pyruvate carboxylase (PC) by isotope labeling dynamic flux analysis. In addition, different preferences were also found in glutamine/glutamate paths between mitochondrial anaplerotic oxidative TCA recycling or cytosolic reductive carboxylation.Five breast cancer cell lines were selected, with each representative of a clinical subtype: MCF-7 (ER+PR+ve/HER2-negative); ZR751 (ER+PR+/HER2-negative); SKBR3 (ER/PR-negative/HER2+ve); MDA-MB-231 (ER/PR/HER2-negative or triple-negative); MDA-MB-468 (ER/PR/HER2-negative or triple-negative) ,53,54. M4 cells/cm2 for 72 h of culture or 1.46 × 104 cells/cm2 for 96 h. The SKBR3 cell line was cultured with McCoy’s 5A medium for optimal growth (Sigma). Culture media was reconstituted with 10% FBS, 1% GlutaMAX and 1% P/S. SKBR3 cells were routinely subcultured at a density of 1.8 × 104 cells/cm2 for 72 h of culture or 1.46 × 104 cells/cm2 for 96 h. The MDA-MB-231 and MDA-MB-468 cell lines were cultured with Dulbecco’s modified Eagle’s medium with GlutaMAX . DMEM was reconstituted with 10% FBS and 1% P/S. MDA-MD-231 cells were subcultured at a density of 1.8 × 104 cells/cm2 for 72 h of culture or 1.46 × 104 cells/cm2 for 96 h. MDA-MD-468 cells were subcultured at a density of 1.6 × 104 cells/cm2 for 72 h of culture or 1.2 × 104 cells/cm2 for 96 h. The ZR751 cell line was cultured with a slightly different protocol. This cell line has a longer doubling time; thus, ZR751 cells were regularly subcultured once a week, although the culture medium was changed twice between each cell seeding. ZR751 cells were cultured with RPMI 1640 (Sigma) for optimal growth. RPMI was reconstituted with 10% FBS, 1% GlutaMAX, 1% sodium pyruvate , and 1% P/S. The cells were habitually subcultured at a density of 2.4 × 104 cells/cm2 in T75 flasks.The cell lines were subcultured every 72–96 h. Cell culture medium was refreshed the day before subculture. Cells were routinely plated in T75 flasks. The MCF7 cell line was cultured with minimum essential media  for optimal growth. MEM media was reconstituted with 10% fetal bovine serum , 1% GlutaMAX (GIBCO), 1% nonessential amino acids  and 1% penicillin/streptomycin . Cells were routinely subcultured at a density of 1.8 × 10Each cell line was cultured strictly following the directions and protocol provided by the commercial brand suppliers to guarantee their optimal growth. The goal of this work was to study differential responses to metabolic inhibitors, and to perform dynamic flux analysis of metabolic activities and paths by isotope labeling approaches. Accordingly, the culture of all cell lines under the same media formulation have would affect their optimal proliferative and growth capacity, and thus possibly masking the results by an undesired challenging effect. Nonetheless, the original cell culture formulations provided by brand indications are supplied within optimal molar ranges for glucose, glutamine, pyruvate, amino acids, among other metabolites.The transaminase inhibitor aminooxyacetate (AOA), glutamate dehydrogenase inhibitor epigallocatechin gallate (EGCG), mitochondrial complex I inhibitor phenformin hydrochloride (Phem), and mitochondrial membrane uncoupler BAM15 were obtained from Sigma-Aldrich. The PI3K inhibitor BKM-120 was obtained from Caymanchemic. All the drugs were prepared following brand-protocol instructions. Concentrated stock solutions were routinely aliquoted and stored at −20 °C. Working solutions were freshly prepared for each set of experiments.4 cells/well, whereas the cell lines ZR751, SKBR3, MDA-MB-231 and MDA-MB-468 were seeded at 8 × 103 cells/well. All the cell lines were maintained in the wells with 100 μL of their corresponding culture medium. After plating, the metabolic drugs were added, and the plates were incubated for 96 h at 37 °C.Experiments were performed in real time by directly seeding the cells in flat bottom black 96-well plates for further fluorescence analysis. Each cell line was seeded in the wells with their corresponding culture medium. Specifically, MCF7 cells were seeded at 1 × 10® cell viability assay (Promega) under the conditions described in each experiment. CellTiter was assessed according to the manufacturer’s protocol, and the fluorescence amount obtained was proportional to the number of viable cells. Viability was expressed with respect to the percentage of untreated cells (100%). Blank wells were included on each plate to measure the fluorescence from serum-supplemented culture medium in the absence of cells. Then, the cell plates were incubated for 20 min at 37 °C, and the fluorescence was measured in a GloMax®-Multi+ Detection System (Promega).After 96 h of treatment, cell viability was monitored using a CellTiter-BlueAll experiments were performed five independent times in quadruplicate replicas for each experimental condition. Data analysis was performed by calculating the average value for all replicas of each experimental condition, and then media-blank values were subtracted from averages. Finally, data were expressed as percentages of viability relative to control untreated cells (100%).Cells were pulsed with heavy isotopes  dissolved in the corresponding routine media for MCF7 and MDA-MB-231 cell lines. Individual wells of a 6-well plate were washed with ice-cold PBS and then extracted in 0.5 mL extraction buffer . Extracts were centrifuged at maximum speed and stored at −80 °C.LC-MS was carried out using a Thermo Ultimate 3000 HPLC in line with a Q Exactive mass spectrometer. A 32 min gradient was developed over a 100 mm × 4.6 mm ZIC pHILIC column with a guard column (Merck-Millipore) from 10% buffer A (20 mM ammonium carbonate) and 90% buffer B (acetonitrile) to 95% buffer A and 5% buffer B. Samples were acquired in positive-negative switching mode, and a standard ESI source and spectrometer settings were applied . Metabolites were identified by standard metabolite matching to m/z and retention time. Integrated peak areas and label incorporation were quantified using AssayR .We have recently found that breast cancer cells representative of the clinical subtypes featuring positive expression to hormone receptors or triple-negative expression displayed significant differences in mitochondrial pH . MitochoChanges in breast cancer cell metabolism have already been documented, and foundational works have already shown differential responses to single metabolic inhibitor treatments, metabolite availability selective conditions, as well as by metabolomic studies primarily using unlabeled total metabolite content analysis. Accordingly, following some insights provided by previous works, in the present study the degree of glycolytic flux dependence was tested by sensitivity responses to phenformin and BKM120 treatments ,57. In aThe breast cancer cell lines showed the same pattern of response for the two experimental strategies selected for examining their glycolytic dependence . For theCollectively, these data initially indicate two differential phenotypes for glycolytic dependence between the tested breast cancer cell lines. Eventually, the SKBR3 cell line was found to show intermediate behavior between the other two phenotypes.Consistent with the findings described above, we further investigated whether these two phenotypes coincide with the cellular reliance on TCA and mitochondrial oxidative phosphorylation activities .Treatments with the transaminase inhibitor AOA short circuits the mitochondria-cytosol transitions of reducing power conducted by the aspartate-malate shuttle. Consequently, mitochondrial TCA activity is collapsed by AOA treatment. Again, the breast cancer cell lines showed two clearly different responses to AOA A. The MCSince TCA activity inherently requires a level of mitochondrial oxidative phosphorylation over a threshold, breast cancer cell lines that present sensitivity to TCA collapse should similarly be sensitive to mitochondrial membrane uncoupling. Accordingly, breast cancer cell lines were tested for the presence of the mitochondrial membrane uncoupler BAM15. In a recently published study, we tested BAM15 collateral toxicity in control experiments and did not observe side effects on breast cancer cells. The results of the BAM15 treatments corroborated the two phenotypic differences already found for the AOA treatments B. The MDThe results described above point to two potential metabophenotypes. Interestingly, the metabophenotypes correlated well with the proliferative capacity already reported for breast cancer cell lines. Moreover, the less proliferative MCF7 and ZR-751 cell subtypes displayed the same responses to the different metabolic inhibitors tested and the more proliferative MDA-MB-231 and MDA-MBD-468 cell subtypes displayed a common response between them that was clearly different from that of the other cell lines.Glutamine and glutamate present several metabolic destinations and are key metabolites that sustain the production of nonessential amino acids (NEAA) that are crucial for cell proliferation. It has been recently demonstrated that glutamate is preferentially metabolized by transaminase or dehydrogenase activity depending upon the proliferative capacity of the cells A. SimilaAccordingly, to test whether breast cancer cells also showed a differential response to glutamate and NEAA requirements, the cells were treated with the GDH inhibitor epigallocatechin-3-gallate (EGCG). Breast cancer cell lines displayed the expected behavior depending upon their proliferative degree, i.e., the less proliferative MCF7 and ZR751 cells were sensitive to EGCG treatment, thus displaying almost a total loss of cell viability, whereas MDA-MB-231 and MDA-MB-468 cells showed a clearly milder decrease in cell viability of 42% and 45% respectively B.As already shown in Among these two metabophenotypes, the SKBR3 cell line interestingly seemed to confirm a different behavior in response to metabolic inhibition. On the one hand, it showed a milder dependence on glycolysis than the cell lines MCF7 and ZR751, as indicated by its intermediate level of sensitivity to phenformin and BMK120 treatments between the other two cellular metabophenotypes . On the In summary, breast cancer cell lines showed two clearly differentiated metabophenotypes . Of note13C-pyruvate isotope cell labeling experiments were performed to decipher whether the major dependence on glycolysis or TCA activity already observed was supported by a predominant metabolic route and/or enzymatic activity.Next, further experiments were performed to investigate the metabolic mechanisms, in terms of dynamic metabolic flux, that potentially generated the metabophenotypes found in this work. Consequently, 13C-pyruvate pulses were performed to investigate whether the breast cancer cell lines that already pointed to display different metabophenotypes might also show a preferential pyruvate destination via PDH or PC activities.Pyruvate labeling pulses were chosen to perform metabolic flux dynamics to directly analyze the preferential mitochondrial destination of pyruvate. TCA reliance and the preferential biosynthetic pathways are determined by pyruvate metabolic destinations that are regulated by PDH and PC enzymatic activities. Hence, to primarily focus on these two pathways, 13C-pyruvate labeled in its three carbon atoms can be metabolized in mitochondria by two main pathways that are regulated by pyruvate dehydrogenase (PDH) or pyruvate carboxylase (PC) activities. Pyruvate is decarboxylated by PDH to provide acetyl-CoA into TCA, whereas pyruvate is converted to oxalacetate (OAA) by PC, which in turn is mainly redirected to anabolic routes (As illustrated in c routes A. Acetylc routes A.Cancer cells with a higher energetic and biosynthetic demand (via TCA anaplerotic routes) predominantly exhibit high PDH activity ,61. The In contrast, hormone-positive MCF7 breast cancer cells, which were more dependent on glycolysis than on TCA activity, showed noticeably predominant PC activity A. The prImportantly, these results visibly support the presence of two different metabophenotypes: the PDH predominant phenotype displaying stronger reliance on TCA activity in MDA-MB-231 triple negative breast cancer cells and the phenotype showing larger PC activity that is associated with a stronger dependence on glycolysis in MCF7 hormone receptor-positive breast cancer cells.13C-glutamine isotope cell labeling experiments were conducted to further investigate metabolic mechanisms underlying the metabophenotypes found in this work  was of particular relevance. Interestingly, these results were in correlation with the oncogene c-Myc expression levels between breast cancer cell lines representative of clinical subtypes . In addiMoreover, there have been important contributions performed by unlabeled metabolomic analysis to obtain quantitative data about total metabolite contain breast cancer cell models. Some noticeable results were obtained but the data were not totally concordant displaying some discrepancies ,50,51,52Under these premises, as already discussed by the aforementioned authors, further steps to address metabolic phenotypic differences between breast cancer cell subtypes should be performed by dynamic metabolic flux analysis for concrete pathways, starting by central metabolic routes, such as glucose preferential usage and TCA reliance. Accordingly, the present work was designed to gain insights in that direction. First, to identify different functional responses (sensitivity or resistance primarily) to alterations provoked by the selected inhibitors that might be in concordance with breast cancer subtypes. Secondly, to investigate possible metabolic flux preferences by stable-isotope dynamic labeling for key targets, such as pyruvate, based on preferential PDH or PC activities to quantitatively analyze the degree of TCA reliance, and other central pathways as glutamine/glutamate metabolism.Thus, in the present work, the main goal was to investigate whether metabolic data obtained by this combination of functional and metabolic flux analysis approaches might complement the current clinical classification of breast cancer.Altogether, our data clearly displayed different metabophenotypes that interestingly correlated well with the clinical classifications. According to a systemic perspective, this study selected several inhibitors as key targets. Thus, the main goal was to generate a metabolic profile that could be used to search for different phenotypes and evaluate whether the potential metabophenotypes might be associated with the current breast cancer classifications. The profiling was designed to select principal metabolic pathways that support cellular function in terms of both energy and biosynthetic demands as targets. Accordingly, the metabolic dependence on glycolytic flux and/or mitochondrial oxidative metabolism via TCA activity was analyzed based on the cellular responses to small molecules that inhibit key enzymatic targets for sustaining the activity of the proposed pathways. In particular, BKM120 and phenformin were used to impact glycolysis while AOA and BAM15 were used to short circuit TCA activity and mitochondrial oxidative metabolism. These drugs have been extensively used for metabolic profiling. BKM120 is an inhibitor of PI3K activity, which in turn produces an inhibitory effect on the PI3K-Akt axis that ultimately impacts glycolysis by decreasing its flux ,57,62. I13C-pyruvate pulse experiments (Specifically, the cell lines MCF7 and ZR751, which are representative of the hormone receptor-positive breast cancer cell subtype, showed stronger glycolytic dependence . Therefoeriments . Labelederiments .On the other hand, the cell lines MDA-MB-231 and MDA-MB-468, which are representative of the triple-negative subtype, displayed a different metabophenotype featuring their main reliance on TCA activity and mitochondrial oxidative metabolism. This phenotype was clearly supported by the striking sensitivity to AOA and BAM15 treatments , whereas2 [Notwithstanding the reliability of these findings, several interesting questions remain open. In particular, the resistance of MCF7 and ZR751 cells to AOA treatments is intriguing. However, our data tentatively indicate that these cell lines might recycle cytosolic NAD+ to sustain glycolysis, even if the aspartate-malate shuttle was collapsed by AOA transaminase inhibition. Our data indicated high PC activity, which was clearly deduced from the presence of m+3 citrate and m+3 aspartate . Notably2 . Of note2 .The SKBR3 cell line, which is representative of the HER2+ breast cancer subtype, displayed an intriguing intermediate metabolic phenotype between the other two cell lines already discussed above. Overall, as illustrated by our results (summarized in Nevertheless, further studies should be performed to gain more detailed information to clarify whether SKBR3, as a representative cell line of the HER2-positive breast cancer subtype, represents another independent metabophenotype or is just a special type within one of the already well-described metabophenotypes.Confirming the scope of the proposed research, this work showed that breast cancer cell subtypes present different metabophenotypes. Importantly, these metabophenotypes are clearly correlated with the current clinical classification. These results pave the way for further studies to gain a detailed understanding of many particular metabolic routes and key targets that might be beneficial to sustain the already discovered metabophenotypes. Thus, in the future, important metabolic parameters could be crucial for potential diagnostic, prognostic and therapeutic developments.


CS-M08sh exhibited the highest K concentration (14.50 g kg−1), an increase of 29% and 49% over TM-1 and AM UA48, respectively. Other CS lines had higher Mg, P, and S than the controls. A similar trend was found at the MS location. This research demonstrated that chromosome substitution resulted in higher seed macronutrients in some CS lines, and these CS lines with a higher content of macronutrients can be used as a genetic tool towards the identification of desired seed nutrition traits. Also, the CS lines with higher desired macronutrients can be used as parents to breed for improved nutritional quality in Upland cotton, Gossypium hirsutum L., through improvement by the interspecific introgression of desired seed nutrient traits such as Ca, K, P, S, and N. The positive and significant (p ≤ 0.0001) correlation of P with Ca, P with Mg, S with P, and S with N will aid in understanding the relationships between nutrients to improve the fertilizer management program and maintain higher cottonseed nutrient content.Nutrients, including macronutrients such as Ca, P, K, and Mg, are essential for crop production and seed quality, and for human and animal nutrition and health. Macronutrient deficiencies in soil lead to poor crop nutritional qualities and a low level of macronutrients in cottonseed meal-based products, leading to malnutrition. Therefore, the discovery of novel germplasm with a high level of macronutrients or significant variability in the macronutrient content of crop seeds is critical. To our knowledge, there is no information available on the effects of chromosome or chromosome arm substitution on cottonseed macronutrient content. The objective of this study was to evaluate the effects of chromosome or chromosome arm substitution on the variability and content of the cottonseed macronutrients Ca, K, Mg, N, P, and S in chromosome substitution lines (CS). Nine chromosome substitution lines were grown in two-field experiments at two locations in 2013 in South Carolina, USA, and in 2014 in Mississippi, USA. The controls used were TM-1, the recurrent parent of the CS line, and the cultivar AM UA48. The results showed major variability in macronutrients among CS lines and between CS lines and controls. For example, in South Carolina, the mean values showed that five CS lines  had higher Ca level in seed than controls. Ca levels in these CS lines varied from 1.88 to 2.63 g kg Macronutrients are involved in different physiological and metabolic processes. For example, K, Zn, and P are involved in CO enzymes ,2; P is  enzymes ,4; Mg, S enzymes  due to t enzymes ,6. Nutri enzymes ,7,8. Mac enzymes . In addi enzymes . The sur enzymes .Medicago truncatula [Nutrient accumulation in seeds is controlled by several processes, including nutrient uptake, translocation, redistribution, and accumulation ,12. Inheuncatula ; limiteduncatula . While Quncatula ,21, and uncatula ,23, it iuncatula .Molecular and genetic research on cottonseed macronutrients is needed. Chromosome substitution can be used to infuse new genetic variation into a crop species, some of which could significantly affect seed macronutrient contents. Since the genetics of cottonseed traits associated with chromosome substitution germplasm lines is scarce , the objp < 0.0001) influenced by location, line, and their interactions (p < 0.0001) influenced by line, and location × line interactions. The effects of line, location, and their interactions on some macronutrients such as Mg, P, C, N, and S indicated the importance of the environmental factors of each location and the genetic makeup of each line on the accumulation of these nutrients in the cottonseed.ANOVA analysis showed that Mg, P, C, N, and S were significantly , a significant increase over TM-1 (−1. Line CS-T04 had the highest concentration of Mg (2.85 g kg−1). Lines CS-M04 and CS-T04 had higher P concentrations than the controls and the rest of the CS lines, with CS-M04 being the highest (5.38 g kg−1). Both CS-M08sh and the control AMUA48 exhibited the highest concentration of C, but four CS lines  were higher than TM-1. Seven CS lines were higher in S than the controls, with CS-T04 being the highest. Four  out of nine lines had higher N than the controls, with CS-T02 having the highest concentration (47.79 g kg−1). Lines CS-B02, CS-B04, CS-B08sh, CS-M02, CS-T08sh, CS-T04, and CS-T02 had higher S than the controls, with CS-T04 being the highest (5.39 g kg−1).In South Carolina, the mean values showed that five CS lines  had higher Ca levels in seed than both controls . Ca levever TM-1 . Lines CIn MS, the concentration of Ca was higher in CS-M08sh, CS-T02, and CS-T04, with CS-M08sh being the highest . PotassiTwo CS lines (CS-T02 and CS-T04) showed higher Ca in both locations compared to the controls. CS-T04 had higher Mg concentrations in both locations. Three CS lines  showed superiority over TM-1, but only CS-T02 and CS-T04 showed superiority over both controls. Cultivar AM UA48 was the best performer for C concentration in both locations. CS-T02 exhibited a higher N concentration than the controls. Four CS lines  exhibited a higher concentration of S than the controls.p ≤ 0.0001) correlation in both locations; for example, P positively correlated with Ca, P positively correlated with Mg, S positively correlated with P, and S positively correlated with N . A similar observation was noticed for lines CS-B02, CS-B04, CS-B08sh, CS-M02, CS-M04, CS-M08sh, CS-T02, and CS-T04 for Mg concentration; lines CS-M04 and CS-T04 for P concentration; and lines CS-B08sh, CS-T02, CS-T04, and CS-T08sh for N concentration. Other CS lines showed negative responses for some macronutrients; for example, CS-M02 and CS-M04 showed reduced N compared to the controls.Since cotton chromosome substitution lines are near-isogenic to the recurrent parent TM-1 with one chromosome or chromosome arm introgressed from sly used ,29,30,31ronments . In thisronments ,33. In aIn MS, similar responses for macronutrients in CS lines were shown when compared to the controls, exhibiting positive, negative, or non-responsive effects of chromosome or chromosome arm substitution as in the SC location. The higher concentration of some nutrients in some CS lines reinforced our explanation that the accumulation of seed nutrients in CS lines were due to introgressed chromosomes as these CS lines are near-isogenic and the only differences between these CS lines and the parent or control are the chromosomes or chromosome arms under study. For example, Ca concentration was high in lines CS-M08sh, CS-T02, and CS-T04 compared with the controls. A similar observation was recorded for K in CS-M02 compared to TM-1; CS-T04 and CS-T08sh for Mg compared with the controls; lines CS-M04, CS-M08sh, CS-T02, and CS-T04 for P compared with the controls; lines CS-B08sh, CS-M08sh, and CS-T02 for N compared with the controls; and lines CS-B02, CS-B04, CS-B08sh, CS-M04, CS-M08sh, CS-T02, CS-T08sh for S content. The negative response of nutrients in some CS lines was shown in, for example, CS-B02, CS-B04, and CS-M04 for C. It must be noted here that some seed nutrients showed different location responses in some CS lines, reflecting the significant effects of environmental conditions at each location. Previous research on other seed traits also showed different responses, depending on the environmental factors of each location, including soil type, temperature, drought, and other environmental conditions . For exaAmong the macronutrients evaluated in the CS lines, some showed a positive response at one location, while others showed a negative response at the same location, and others showed a positive response in both locations. Previous research conducted on other seed traits found similar observations. For example, ref.  found hoA similar observation was noted at the Mississippi location, with different responses of the CS lines due to location effects. These observations demonstrated that chromosome substitution resulted in a higher concentration of macronutrients in some CS lines, with no change or reduced concentrations in others. The positive or negative response of these macronutrients was due to both location and CS line genotype, as explained by others above ,34. The p ≤ 0.0001) positive correlation between P and Ca, P and Mg, S and P, and S and N in both locations may suggest the stability of these traits across locations, and these traits may help growers for fertilizer management and advance our knowledge of nutrient uptake and translocation. The wide distribution, especially in Ca, K, N, and S, and significant variability of nutrients among CS lines and between CS lines and the controls signified the potential use of CS lines with high levels of these nutrients for future breeding programs to increase the nutritional quality of cottonseed. The right skewed, left skewed, or peak trends, shown by the nutrient distribution between CS lines, showed complex interactions of these nutrients in these lines.The positive correlation of P with Ca and Mg; N with Ca and K; S with Ca, Mg, P, and N in SC; the positive correlation of P with Ca and Mg; N with Mg; the negative correlation of N with P; and the negative correlation of S with P and positive correlation with N in MS indicated that the correlation between some nutrients can be influenced by complex genetic and environmental factors . The conG. tomentosum and G. mustelinum is possible. This is the first report on the effects of chromosome or chromosome segment substitution on macronutrients in cottonseed. We were able to discover specific chromosomal associations with cottonseed macronutrient contents gene(s) and developed some novel germplasm with the gene(s) of improved macronutrient contents from interspecific crosses using the wild species.This research demonstrated that the improvement of cottonseed nutritional quality using chromosomes or chromosome segments from the wild species G. barbadense L. (CS-B), G. tomentosum Nutt.Ex Seem (CS-T), and G. mustelinum Watt (CS-M), respectively, into Upland cotton , the recurrent parent of the CS lines, and AM UA48, Reg. No. CV−129, . The nine CS lines were: CS-B02, CS-B04, CS-B08sh, CS-M02, CS-M04, CS-M08sh, CS-T02, CS-T04, CS-T08sh; TM-1 and AM UA48 were used as controls. The developed lines are described in detail elsewhere [262829Nine cotton chromosome substitution lines were used and represented by the substitution of chromosome two, four, and the short arm of chromosome eight (8sh) from three tetraploid species of ewhere 26282930,403 in a microwave digestion system. The concentrations of nutrients were determined by inductively coupled plasma spectrometry (ICP) (Thermo Jarrell-Ash Model 61E ICP and Thermo Jarrell-Ash Autosampler 300) [The macronutrients Ca, K, Mg, P, C, N, and S were analyzed in the ground, dried whole seed samples. Collected seeds from each CS line and control were ground with a Laboratory Mill 3600 , and analyzed by digesting 0.5 g of dried ground seed in HNOler 300) . The nutler 300) ,42. Seedv/v HCl. Then, 5 mL of 5 M HCl and 5 mL of ammonium molybdate–ammonium metavanadate was used. The concentration of P was determined by a Beckman Coulter DU 800 spectrophotometer as previously described by others [The concentration of P in cottonseed was determined by the yellow phosphor vanado–molybdate complex method , and as y others ,42. The G. barbadense, G. tomentosum, and G. mustelinum, because an individual CS line is considered to be near-isogenic in a background of TM-1. Correlation analyses were conducted in SAS [Cotton lines were grown in a randomized complete block design with four replications within each location. Proc Mixed   was condd in SAS  using PrThe current research demonstrated the possibility of increasing major macronutrient  levels in cotton using wild species germplasm introgression. In this research, we used a comparative method  to detec


Compounds reduced cell colony formation of both cell lines and inhibited the growth and viability of IGR39 cell spheroids more efficiently compared to triple-negative breast cancer spheroids.Heterocyclic compounds are one of the main groups of organic compounds possessing wide range of applications in various areas of science and their derivatives are present in many bioactive structures. They display a wide variety of biological activities. Recently, more and more attention has been focused to such heterocyclic compounds as azoles. In this work, we have synthesized a series of new imidazole derivatives incorporating a benzenesulfonamide moiety in their structure, which then were evaluated for their cytotoxicity against human triple-negative breast cancer MDA-MB-231 and human malignant melanoma IGR39 cell lines by MTT assay. Benzenesulfonamide-bearing imidazole derivatives containing 4-chloro and 3,4-dichlorosubstituents in benzene ring, and 2-ethylthio and 3-ethyl groups in imidazole ring have been determined as the most active compounds. Half-maximal effective concentration (EC After huge breakthrough in pharmaceutical chemistry in early twentieth century, treating more and more previously incurable or difficult-to-treat diseases has become common practice in doctor’s offices nowadays. Research and synthesis of various functionalized heterocycle compounds have contributed significantly to this progress. Heterocycles are one of the largest classical divisions in organic chemistry and they play important role in modern drug design due to their wide range of biological properties . A lot oTherefore, based on recent studies, benzenesulfonamide-bearing imidazole derivatives were synthesized in this work. Two cancer cell lines, namely TNBC cell line MDA-MB-231 and human melanoma cell line IGR39 were selected to test cytotoxic activity and compound effect on cell colony-forming ability. Both cancer types are considered very aggressive and have a low survival prognosis. Resistance to drugs occurs quite often in the case of melanoma treatment with targeted therapeutics, due to BRAF mutations . More th2) was prepared in the reaction of amine 1 with 1-chloropropanone in small amount of water at reflux  was treated with various α-haloketones in water/1,4-dioxane solution to obtain compounds 4–9, which were later used as precursors for synthesis of corresponding imidazoles 10–15 in similar reaction conditions as were in product 3 synthesis (10–15 (or the rest in this work) can be purified without using any organic solvents. For purification, these imidazole derivatives can be dissolved into 5% sodium hydroxide aqueous solution instead. Then, after filtration, the acidification of the obtained solution with glacial acetic acid will provide purer forms of the desired products without any significant loss to the purified amount of it. We also wanted to modify some of the pharmacokinetic properties of 1H-imidazole derivatives 10–15 to see, how it would affect the cancer cells. We decided to perform S-alkylation of 1H-imidazole 11 to reach higher solubility in organic solvents and lipophilicity. As a result, in the first attempt, imidazole 11 was treated with ethyl iodide in DMF overnight to obtain compound 16. In a 1H NMR spectrum for 16, the triplet assigned to the protons in the CH3 group at 1.30 ppm and quartet assigned to the protons in the CH2 group at 3.15 ppm have proved the presence of ethyl moiety in the molecule. Elemental analysis data of 16 confirmed that imidazole 16 did not form hydroiodide salt. Moreover, to increase reaction rate, we used triethylamine as a base catalyst, which shortened the reaction time to 3 h.The 3-((2-oxopropyl)amino)benzenesulfonamide (t reflux . The strtomerism . Howeversolution . Moreoveynthesis  25]. We. We2) wa4–9 were dissolved in glacial acetic acid and treated with urea to afford oxoimidazoles 17–22  was used in reactions with α-haloketones in water/1,4-dioxane mixture to obtain precursors 25–30 for further synthesis of imidazoles 31–36 , that reduced cell viability below 50%, have been identified between non-hydroxylated compounds, especially with imidazolone fragment. 4-Hydroxybenzenesulfonamide derivatives with thioimidazole ring (compounds 31–36) were practically inactive against both tested cell lines. It is worthy to notice, that the most active compounds in separate groups contained either 4-chloro  or 3,4-dichloro  substituents. The least active compounds were those containing cyano group in phenyl ring (the only active compound was 8). Two compounds (23 and 16) contain 2-ethylthio and 3-ethyl substituents in imidazole ring, which could contribute to their high anticancer activity, and the importance of this fragment in this position could be of great interest in the future research.Tested imidazole derivatives showed different activity against human triple-negative breast cancer and human melanoma cell lines at 100 µM concentration . Almost 8, 16, 19, 23, 25, and 28, which reduced cell viability of at least one cell line below 10%, and they were then tested more thoroughly both against human cancer cell lines and normal cells lines (fibroblasts (HF) and endothelial cells (EC)). The effective concentrations that reduce cell viability by 50%  have been established  [Compound s series B. Moreov >50 µM) . In gene >50 µM) , and for >50 µM) . However >50 µM)  and ther >50 µM) . Meanwhi >50 µM) .Doxorubicin, which is considered as a chemotherapeutic agent against triple-negative breast cancer, reduces MDA-MB-231 cell viability below 50% at 5 μM concentration , and str8, 16, 19, 23, 25, and 28 were tested for their effect on IGR39 and MDA-MB-231 cell colony formation at 50% of their established EC50 concentrations.Both types of cancer are characterized as recurrent, thus we decided to test the compound effect on single cell ability to survive and proliferate using clonogenic assay. The most active agents 23, which was the most cytotoxic in MTT assay, showed comparable activity with compounds 8 and 16 against both cell lines. In the presence of compound 23, MDA-MB-231 cell colony area dropped down to 27.1 ± 6.6%, compared to the control (p < 0.05)  . All thrAs previously mentioned, the strategy of combinational therapy is being extensively studied and investigated whether this approach could reduce the resistance of cancer to available therapy. It has been established MERTK inhibitor UNC2025 reduces colony formation of BRAF mutant and BRAF wild-type cell lines at 300–500 nM concentration . Combina50 value could reduce MDA-MB-231 cell colony formation up to 40% [Many studies have been done on MDA-MB-231 cell colony formation in the presence of drugs used for triple-negative breast cancer treatment, novel compounds, or their combinations. Franco et al.  establisp to 40% . Combinap to 40% . In summ8, 16, 19, 23, 25, and 28 on melanoma IGR39 and triple-negative breast cancer MDA-MB-231 cell spheroid growth . Melting points were determined on a MEL-TEMP  melting point apparatus and are uncorrected. IR spectra  were recorded on a Perkin–Elmer Spectrum BX FT-IR spectrometer  using KBr pellets. The 1H and 13C-NMR spectra were recorded in DMSO-d6 on a Bruker Avance III  spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) calibrated from TMS (0 ppm) as an internal standard for 1H NMR, and DMSO-d6 (39.43 ppm) for 13C NMR. Elemental analyses  were performed on an Elemental Analyzer CE-440 . Compounds 2-23 and 25-42 synthesis methods, identification data and NMR spectra can be found in Reagents were purchased from Sigma-Aldrich . The reaction course and purity of the synthesized compounds were monitored by TLC using aluminum plates pre-coated with silica gel 60 F2. They were used until the passage of 20.The human melanoma IGR39 and the human triple-negative breast cancer MDA-MB-231 cell lines, as well as the human endothelial cells (EC) CRL-1730 were obtained from the American Type Culture Collection . Human foreskin fibroblasts (HF) CRL-4001 were originally obtained from ATCC and kindly provided by Prof. Helder Santos . IGR39, MDA-MB-231, EC, and HF were cultured in Dulbecco’s Modified Eagle’s GlutaMAX medium ). Medium was supplemented with 10,000 U/mL penicillin, 10 mg/mL streptomycin (Gibco), and 10% fetal bovine serum (Gibco). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO3 cells/well). After 24 h, the cells were treated with 100 μM of various concentrations of tested compounds. Only medium without cells was used as a positive control, and the medium with 0.5% DMSO (Sigma-Aldrich Co.) served as a negative control. After 72 h, the medium from cells was discarded and the fresh medium containing 0.5 mg/mL of MTT solution (Sigma-Aldrich Co.) was added. Cell were incubated for the next 4 h. Then the liquid was aspirated, and the colored formazan product was dissolved in 100 μL DMSO (Sigma-Aldrich Co.). The absorbance was measured at 570 and 630 nm using a multi-detection microplate reader. Compound effect on cell viability was calculated using a formula:Cell viability was tested using 3--2,5-diphenyltetrazolium bromide  assay, as described elsewhere . BrieflyA—mean of absorbance of tested compound,A0—mean of absorbance of blank ,NCA—mean of absorbance of negative control .50 of the most active compounds, the same MTT procedure has been applied, only the compound serial dilutions from 50 µM to 1.56 µM have been made in a medium and added to the cells in triplicate repeats. EC50 value that represent the concentration of a compound causing 50% reduction of cancer cell metabolic activity has been calculated using Hill equation.For establishing EC2 cells/well) and grown in DMEM GlutaMAX supplemented with 10% FBS  and 1% antibiotics  at 37 °C in a humidified atmosphere containing 5% CO2. After 24 h, the fresh medium containing tested compound 8, 16, 19, 23, 25, and 28 at a concentration representing 50% of calculated EC50 values were added to cells, followed by incubation at 37 °C in a humidified atmosphere containing 5% CO2 for the next 10 days (for IGR39 cell line) and 12 days (for MDA-MB-231 cell line), replenishing the media with the compounds once a week. Cells treated with medium containing 0.5% DMSO served as a negative control. After incubation, the colonies were stained with 0.1% Crystal Violet (Sigma-Aldrich Co.) solution. To start with, the media from cells was removed and the cells were washed once with sterile PBS. Then, the cells were fixed in a 4% formaldehyde (Thermo Scientific) solution and washed with PBS two times to remove the fixative, and stained with Crystal Violet solution for 20 min. After the stain had been removed, the remaining stain residues were washed three times sterile water. Lastly, the plates were dried overnight, and imaged with SYNGENE G:BOX gel doc system , using GeneSys software version 1.5.5.0, followed by quantification Gene tools software version 4.3.8.Colony formation assay was applied to evaluate the inhibitory effect of the most active compounds on cell ability to survive, and proliferate by forming colonies, as described elsewhere . Briefly2. Then, cells were trypsinized, centrifuged, and seeded into ultra-low attachment 96-well plate in a volume of 100 µL . The plate was placed on a magnetic drive  and incubated for 2 days at 37 °C in a humidified atmosphere containing 5% CO2. Then the fresh medium containing tested compound concentration representing 100% of calculated EC50 values was added to the wells. The spheroids were captured every two days using the Olympus IX73 inverted microscope (OLYMPUS CORPORATION). The quantitative analysis of compound anticancer activity in spheroids was performed using ImageJ  and Microsoft Office Excel 2016 software.Magnetic 3D Bioprinting method was used to form spheroids, as described elsewhere . Brieflyp < 0.05.All biological experiments were repeated at least three times, calculating the mean and standard deviation. The data was processes using Microsoft Office Excel 2016 software . Statistical analysis was performed by using Student’s t-test. The level of significance was set as In this study, the chemical transformations of benzenesulfonamides were carried out and a series of 1-substituted imidazoles with aliphatic, aromatic fragments were synthesized and evaluated for their anticancer activities in 2D and 3D models in vitro.In the MTT assay, compounds were more effective against human triple-negative breast cancer MDA-MB-231 cell line, compared to malignant melanoma IGR39 cell line. Benzenesulfonamide-bearing imidazole derivatives containing 4-chloro and 3,4-dichlorosubstituents in benzene ring, and 2-ethylthio and 3-ethyl groups in imidazole ring have been determined as the most active compounds. These compounds reduced cell colony formation of both cell lines. Tested derivatives inhibited the growth and viability of IGR39 cell spheroids more efficiently compared to triple-negative breast cancer spheroids.The obtained results suggest that benzenesulfonamide-bearing imidazole could be a suitable core for the anticancer compounds against such aggressive and invasive tumour types as malignant melanoma and triple-negative breast cancer.


Gastric adenocarcinoma with peritoneal carcinomatosis (PC) is therapy resistant and leads to poor survival. To study PC in depth, there is an urgent need to develop representative PC-derived cell lines and metastatic models to study molecular mechanisms of PC and for preclinical screening of new therapies.PC cell lines were developed from patient-derived PC cells. The tumorigenicity and metastatic potential were investigated by subcutaneously (PDXs) and orthotopically. Karyotyping, whole-exome sequencing, RNA-sequencing, and functional studies were performed to molecularly define the cell lines and compare genomic and phenotypic features of PDX and donor PC cells..We established three PC cell lines  and characterized them in vitro. The doubling times were 22, 39, and 37 h for GA0518, GA0804, and GA0825, respectively. Expression of cancer stem cell markers  and activation of oncogenes varied among the cell lines. All three PC cell lines formed PDXs. Interestingly, all three PC cell lines formed tumors in the patient derived orthotopic (PDO) model and GA0518 cell line consistently produced PC in mice. Moreover, PDXs recapitulated transcriptomic and phenotypic features of the donor PC cells. Finally, these cell lines were suitable for preclinical testing of chemotherapy and target agents in vitro and in vivoWe successfully established three patient-derived PC cell lines and an improved PDO model with high incidence of PC associated with malignant ascites. Thus, these cell lines and metastatic PDO model represent excellent resources for exploring metastatic mechanisms of PC in depth and for target drug screening and validation by interrogating GAC for translational studies.The online version contains supplementary material available at 10.1186/s13046-021-02003-8. Gastric adenocarcinoma (GAC) is the fifth most common cancer and the third leading cause of cancer-related mortality worldwide . GAC is The resources to study PC are starkly limited. Patient-derived tumor cell lines, patient-derived xenografts(PDXs), and orthotopic models will be indispensable in this respect. There are 38 gastric cancer cell lines in the cancer cell line encyclopedia (CCLE); the majority of these are from Asian patients . HoweverSeveral human GAC cell lines have been used to create orthotopic mouse models of PC –20. HoweHere, we report on three new GAC cell lines established from PC cells from the Western patients. All three patient-derived PC cell lines are highly tumorigenic in the subcutaneous PDX model. Furthermore, whole exome sequencing (WES) and RNA sequencing (RNA-seq) revealed that the PDXs recapitulate the transcriptomic profile and conserve some of the mutational and copy-number variations (CNVs) similar to donor PC cells. Moreover, these cell lines are suitable for studying cytotoxics and target inhibitors. Most importantly, one cell line (GA0518) reliably formed PC in mice within 30 days in the PDO model. Thus, these new cell lines and the PDX and PDO models provide an opportunity to investigate mechanisms of PC and validate therapeutic targets.Three PC cells were collected from volunteering patients with GAC and malignant ascites; and patient clinical characteristics including therapy, tumor types, site of metastases, and histopathology are listed in Supplemental Table 5 cells in 30 μl in 50% Matrigel–phosphate-buffered saline) were injected into the distal stomach wall. We applied an improved orthotopic model to avoid the leakage into the peritoneal cavity . More details are provided in the results. The injection site and PC were monitored by in vivo BLI imaging weekly with intraperitoneal injection of 150 μg/g of D-Luciferin (PerkinElmer).To assess orthotopic tumor growth and PC in mice, we first transduced the cell lines with a lentivirus expressing Luciferase and mCherry for in vivo bioluminescence (BLI) imaging of tumor growth in SCID mice. Luciferase-expressing cells .Data were analyzed using the Student t-test and Fisher exact test (for colony formation and cell migration assays). Other assay results are presented as mean ± standard error of the mean and represent the results of at least three experiments. The significance of differences between groups was judged using a two-tailed Student t-test. Results were considered statistically significant if the More detailed materials and methods can be found in the online The three PC cell lines had different morphologies under the microscope Fig. A. To visDoubling time was estimated during the exponential phase of growth. GA0518 cells had the shortest doubling time, 22 h. GA0804 and GA0825 cells had similar doubling times, around 39 h and 37 h, respectively  with more mutations  staining of several notable CSC makers  in three cell lines Fig. B and itsAs the three cell lines expressed CSC markers in varying proportions, to define the similarity between the cell lines and their corresponding donor PC cells, we determined the activation oncogenic pathways in three cell lines and the corresponding donor PC cell samples by CyTOF. As shown in Fig. We then performed functional in vitro assays in the three cell lines. The colony formation capability was highest in GA0518 (256.67 colonies/1000 cells), followed by GA0804 (105.33 colonies/1000 cells), and then GA0825 (56.33 colonies/1000 cells)  of SCID mice injected with 1 × 104 cells  was injected into the anterior wall, each injection lasting 30 s. After the injection, an alcohol swab was used to sterilize the posterior wall. The stomach was repositioned into the abdominal cavity, and the wound was closed  of mice with GA0518 cells developed bloody ascites within 4 weeks  of 13.39 nM. GA0518 cells were sensitive to RAD001 with IC50 of 28.91 nM but were less sensitive to 5-fluorouracil, with IC50 values of 45.03 nM, and were resistant to erlotinib, at an IC50 of 261.9 nM , and JQ1 . As shown in Fig. We are reporting three new GAC cell lines derived from three patients with PC. All three cell lines were highly tumorigenic and aggressive in PDX and PDO models compared with commercially available GAC cell lines (Snu-1 and MKN45), and GA0518 cells were the most consistent and aggressive. WES and RNA-seq analyses revealed that the transcriptome and phenotypic features of PDXs closely recapitulated those of the donor cells from patients. Importantly, in the improved PDO model, GA0518 cells formed PC including malignant ascites in mice within 4 weeks. These findings suggest that the cell lines and PDO PC model can be reliably used for studying PC metastatic mechanisms and for target validation to advance therapeutics for GAC.KMT2C, CDKN2A, ABL1, and SC3H12A) than GA0825 donor PC cells (with only KMT2C mutation). It has been reported that most gastric cancers in advanced stage have abnormal chromosomes [Chromosomal instability, including abnormal numbers and structural rearrangements, is a prominent feature of cancers. Through cytogenetic analysis, we found that GA0518 has a near-diploid karyotype and cytogenetic complexity. This cell line is highly heterogeneous and comprises at least four cell subtypes, including 40% of cells with a karyotype of 45,X, 20% with 46,XX,− 12,+der , and 10%omosomes , 24 and Similarly, the three cell lines established from PC patients with GAC also had high heterogeneity, with multiple subclones, and one of them had high hyper-diploidy with 91–106 chromosomes . AnotherCD44 is a well-recognized CSC marker in GAC –31 and iWe also improved the surgical technique for the PDO model to avoid PC due to cell leakage in the peritoneal cavity. It has been well documented that the PDX and PDO models from the same source may respond differently to the same treatments . FurtherIn summary, three new GAC cell lines were established from GAC patients with PC from the United States. All three cell lines formed PDXs with high efficiency, and the molecular features of the PDXs mimicked the donor PC cells from patients, making these cell lines highly suitable for validation of targets to be advanced in human trials. Furthermore, GA0518 cells stand out in their performance as a PC metastatic model in our improved PDO model and may provide an advantage for studying metastatic mechanisms, and for target drug screening and validation by interrogating GAC for translational studies.Additional file 1. Supplemental Materias & Methods.Additional file 2: Supplemental Fig. 1. Genetic alteration of three donor PC cells and cell lines. A. Whole-exome sequencing was performed in three donor PC cells IP-013 (GA0518), IP-107-02 (GA0804) and IP-116 (GA0825) directly from patients according to the Materials and Methods. Mutations in three donor PC cells were analyzed by a bioinformatician (S.Z.). B&C. Karyotyping analyses of GA0518 (B) and Snu-1 (C) were performed by the Cytogenetics and Cell Authentication Core at MD Anderson as described in Materials and Methods. Abnormal chromosomal changes were observed in GA0518 (B) and Snu-1 (C). Red arrows indicate some of the chromosomal changes in each cell lines. Supplemental Fig. 2. Expression of CSC markers in the corresponding donor PC cell samples. Expression of several notable CSC markers  in the corresponding donor PC cell samples  was determined by immunofluorescence staining as described in Materials and Methods. Supplemental Fig. 3. Expression of CD44, CD133, and EpCAM was evaluated by flow cytometry in the three new cell lines. Supplemental Fig. 4. Activation of stemness and oncogenic markers in three corresponding donor PC cells by CyTOF. CyTOF was performed in the donor PC cell samples IP-013, IP-107-2, and IP-116 according to the Materials and Methods. Activation of CSC markers (CD44 and ALDH1) and oncogenic pathways such as pS6, p-AKT, and mTOR was analyzed and identified by bioinformatician (R.W). Supplemental Fig. 5. Representative BLI images for GA0804 PDO model A. Representative BLI images from Day 1 to Day 36 in a GA0804 PDO mouse without PC metastasis; B. Representative BLI images in GA0804 PDO mouse with PC metastasis from Day 1 to Day 36. Supplemental Fig. 6. Representative BLI images for GA0825 and MKN45 PDO models. A. Representative BLI images from Day 1 to Day 27 in two PDO mouse models of GA0825 with no PC. B. Representative BLI images from Day 9 to Day 26 in two PDO mouse models of commercial GAC cell line MKN45 with no PC. Supplemental Fig. 7. IC50 of three new cell lines on chemo and target inhibitors. The cell line GA0518 (A), GA0804 (B), and GA0825 (C) were seeded onto 96-well plates with 3000 cells/well. After overnight incubation, cells were treated with the indicated doses of two chemo-5-FU and Docetaxel; and two target inhibitors- Erlotinib and RAD001 for 6 days and then CellTiter96 Aqueous one solution was added to each well (20ul to 100ul culture media) followed by 2 h incubation at 37 °C and absorbance reading at OD 490. GraphPad Prism7 was used for IC50 analysis by following the instruction from GraphPad.com (https://www.graphpad.com/support/faq/how-to-determine-an-icsub50sub/).Additional file 3: Supplemental Table 1. Patients’ characteristics.


Urothelial carcinoma (UC) accounts for > 90% of canine tumors occurring in the urinary bladder. Toceranib phosphate (TOC) is a multi-target receptor tyrosine kinase (RTK) inhibitor that exhibits activity against members of the split kinase family of RTKs. The purpose of this study was to evaluate primary UC tumors and UC cell lines for the expression and activation of VEGFR2, PDGFRα, PDGFRβ, and KIT to assess whether dysregulation of these RTKs may contribute to the observed biological activity of TOC.Transcript for VEGFR2, PDGFRα, PDGFRβ, and KIT was detected in all UC tissue samples and UC cell lines. The Proteome Profiler™ Human Phospho-RTK Array Kit (R & D Systems) provided a platform to assess phosphorylation of 42 different RTKs in primary UC tumors and UC cell lines. Evidence of PDGFRα and PDGFRβ phosphorylation was present in only 11% or 33% of UC tumors, respectively, and 25% of UC cell lines. Treatment of UC cell lines with TOC had no significant impact on cell proliferation, including UC cell lines with evidence of PDGFRβ phosphorylation.Phosphorylation of several key RTKs targeted by TOC is present in a small subset of primary UC tumors and UC cell lines, suggesting that these RTKs do not exist in a state of continuous activation. These data suggest that activation of RTKs targeted by TOC is present in a small subset of UC tumors and UC cell lines and that treatment with TOC at physiologically relevant concentrations has no direct anti-proliferative effect on UC cells.The online version contains supplementary material available at 10.1186/s12917-021-03027-0. Canine urothelial carcinoma (UC) represents 1.5% to 2% of all canine neoplasia and accounts for ≥ 90% of urinary bladder tumors in dogs. The majority  is reporGiven the proclivity of UC tumors to develop in the trigone region of the urinary bladder, adequate surgical excision remains challenging and the majority of UC tumors are considered inoperable . AlternaIt is well-established that alterations in processes involved in tumor growth, progression and metastasis are mediated by ligand-receptor interactions involving receptor tyrosine kinases (RTKS) and downstream signaling molecules. Given the role of RTKs in the control of numerous cellular processes  and the importance of aberrant RTK signaling in cancer, RTKs have emerged as important targets for the development of anticancer therapies. Toceranib phosphate (TOC)  , Maltese (N = 1), Shetland Sheepdog (N = 1), West Highland Terrier (N = 1), with the remainder being mixed breed dogs (N = 4). The tumor locations most frequently involved the bladder trigone (N = 4), the apex (N = 4), and the dorsal bladder wall (N = 1) with evidence of prostatic (N = 1) and urethral (N = 4) involvement on ultrasonographic imaging. None of the patients had evidence of locoregional metastasis at the time of diagnosis and none had evidence of distant metastasis. All of the patients were presented with clinical signs which included stranguria, dysuria, hematuria, and/or pollakiuria. All of the patients received therapy with a non-steroidal anti-inflammatory  with 6/9 patients receiving some form of cytotoxic chemotherapy. Additionally, 3/9 patients received therapy with toceranib phosphate  tumor samples were collected from 9 canine patients that were presented to the Ohio State Veterinary Medical Center (OSU-VMC). The mean age was 10.3 years . The patient breeds included the Scottish Terrier  and established canine UC cell lines (N = 5). Transcript for PDGFRa, PDGFRb, VEGFR2, and KIT was detectable in all of the primary UC tumor samples and UC cell lines  accounts for over 90% of canine malignancies occurring in the urinary bladder and despite aggressive therapies including partial cystectomy, radiotherapy, and systemic chemotherapy, long term outcomes for dogs remains poor. While clinical signs often improve following systemic medical therapy, objective response rates are noted in 18–36% , 25 of tWhile transcript for PDGFRα, PDGFRβ, VEGFR2 and KIT was detected in all tumor samples and UC cell lines, the pattern of expression was variable. Furthermore, while mRNA was present in UC tumor tissues and UC cell lines, the presence of RTK phosphorylation for these RTKs was present in only 11–33% of tumor specimens and UC cell lines suggesting that activation of these RTKs is present in only a small subset of UC cells. Importantly, the absence of RTK pathway activation for PDGFRα, PDGFRβ, VEGFR2 and KIT in the majority of UC tumors and UC cell lines suggests that signaling through these pathways is unlikely to drive the aggressive behavior of UC cells. Prior studies  have demOf note, evidence of RTK phosphorylation was variable in primary UC tumor samples evaluated in this study. This variability may be attributed to tumor tissue heterogeneity as the samples were not microdissected and thus included tumor cells, associated stromal cells within the tumor microenvironment and to a lesser degree, necrosis. The presence of cell types within the tumor microenvironment may confer presence of growth factors and other cytokines that influence the phosphorylation status of RTKs which may otherwise not be activated in UC cells. Additionally, primary tumor tissues were collected from patients that received various chemotherapy treatments prior to sample procurement. It is therefore possible that prior treatment with intravenous chemotherapeutic agents, toceranib phosphate, and/or NSAIDs may have affected the expression and phosphorylation profile of the RTKs of interest in this study population. To further dissect the potential influence of RTK pathway activation on UC cell behavior, we profiled RTK activation in established UC cell lines which represent a model system without the confounding influence of tumor stromal and/or immune cells. Importantly, the cells were serum starved for 2 h in order to eliminate the potential for endogenous growth factor influence on the phosphorylation status of the RTKs. We observed that in the primary tumor samples evaluated, the profile of RTK activation in UC cells lines was similar to that found in tumors with evidence of PDGFRα and PDGFRβ phosphorylation present in only 25% of UC cell lines evaluated. While our results from the phospho-RTK arrays showed moderate phosphorylation of the RTKs of interest, it is important to note the possibility of false negative results. The phospho-RTK array used in the present study was originally designed to detect a wide variety of human phosphorylated RTKs and utilizes optimized human antibodies. The array has not been fully validated in canine tissues and therefore, may underestimate the true phosphorylation status of the primary tumors due to a lack of antibody cross-reactivity.Importantly, we evaluated the direct effect of TOC on cell proliferation in UC cell lines showing variable levels of PDGFR activation and found that treatment with TOC did not result in a dose-dependent decrease in cell viability. Small molecule inhibitors such as TOC frequently demonstrate biological activity in cancer cell lines at low nanomolar concentrations and this correlates with physiologically relevant pharmacokinetic profiles for dogs receiving TOC . We failV595E mutation which is present in 75–87% of canine UC tumors [Clinical benefit has been described in dogs with UC receiving TOC and a non-steroidal anti-inflammatory drug (piroxicam) with the majority of dogs (80%) in this study experiencing stable disease . In lighC tumors  has beenC tumors , 30 has , even at micromolar concentrations. Collectively, these data demonstrate that activation of RTKs targeted by TOC is present in a small subset of UC tumors and UC cell lines and that treatment with TOC at physiologically relevant concentrations has no direct anti-proliferative effect on UC cells.The present study provides an initial characterization of the activation and phosphorylation of RTKs in primary canine UC tumors and UC cell lines. We observed that phosphorylation of several key RTKs targeted by TOC is present in a small subset of primary UC tumor samples and UC cell lines, suggesting that many of these RTKs do not exist in a state of continuous activation in UC tumors. Treatment of UC cell lines with TOC had no significant impact on cell proliferation, including those with evidence of PDGFRβ phosphorylation. Furthermore, UC cell proliferation was not significantly altered following treatment of UC cell lines with TOC in vitroKIT ITD mutation in the JM domain, generously provided by Dr. Warren Gold, Cardiovascular Research Institute, University of California – San Francisco, CA, USA) served as a positive control cell line for toceranib phosphate drug treatment assays and was maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco™ Life Technologies) supplemented with 10% fetal bovine serum, non-essential amino acids, sodium pyruvate, antibiotic–antimycotic, GlutaMAX™, and HEPES. All cell lines were cultured in a humidified incubator containing 5% CO2 at 37 °C. The cells were routinely tested for mycoplasma and treated with Plasmocin  as necessary.The canine urothelial carcinoma (UC) cell lines K9TCC-PU-AXA, K9TCC-PU-AXC, K9TCC-PU-NK, K9TCC-PU-Original, and K9TCC-PU-SH were generously provided by Dr. Deborah Knapp . All cell lines were previously established and validated , 32. CelN = 9) were collected from clinical cases treated at The Ohio State University Veterinary Medical Center (OSU-VMC). Owner consent for tissue collection was obtained in all cases in accordance with established hospital protocols and approved Institutional Animal Care and Use Committee protocols (IACUC #2010A0015). Tissue collections were performed by the OSU-VMC Blue Buffalo Clinical Trials Office and Veterinary Clinical Research Shared Resource. Fresh tumors collected at the time of surgery or euthanasia were flash frozen in liquid nitrogen and placed in formalin and processed for routine paraffin embedding for histopathology. Briefly, sections were routinely sectioned at 4–5 µm thickness and stained with hematoxylin and eosin (H&E) on a Leica ST5020 autostainer  using a routine and quality-controlled protocol. All tumor samples were confirmed to be urothelial carcinoma by board certified veterinary anatomic pathologists at OSU-VMC.Primary canine UC tumor samples  and amplification efficiency. All primer sets demonstrated high amplification efficiency (96–98%) with a correlation coefficient (R2) ≥ 0.93  was used to assess relative phosphorylation of 42 different RTKs in primary UC tumor tissue samples and UC cell lines. Briefly, flash frozen tumor samples were pulverized in a frozen mortar and the resulting powder was resuspended in liquid nitrogen and transferred to a 1.5 mL microcentrifuge tube. Upon evaporation of the liquid nitrogen, samples were resuspended in 100–200 μL of complete tissue lysis buffer. Complete tissue lysis buffer was prepared per manufacturer’s instructions using 2.5 mL of Lysis Buffer-17 supplemented with phosphatase and protease inhibitors . Samples were rocked at 4 °C for 1 h, centrifuged for 15 min at 15,000 rpm at 4 °C, and the supernatants collected. Prior to collection, primary UC cell lines were serum starved for 2 h, washed twice with 1X Dulbecco’s Phosphate Buffered Saline (Gibco™ Life Technologies), pelleted, and protein lysates were prepared as described above. Extracted protein concentration was quantified using the Bradford assay BioRad Reagent  and 100 µg total protein lysate was added to each RTK array and developed according to manufacturer’s directions.Canine UC cell lines  were serum starved for 2 h and protein lysates were generated for western blotting, as described previously . 100 µg 3 cells were seeded in 96-well plates and incubated in media overnight. Cells were treated with 0.1% DMSO (control) or increasing concentrations of toceranib phosphate (0.005 µM – 1 µM) for 48 h. The canine C2 mastocytoma cell line which exhibits a predictable dose–response curve following treatment with toceranib phosphate was included in all assays as a positive control for cell proliferation assays. Briefly, 1 × 104 C2 cells were seeded in 96-well plates, incubated overnight in complete medium overnight and treated with 0.1% DMSO (control) or increasing concentrations of toceranib phosphate (0.005 µM – 1 µM) for 48 h at 37 °C. Media was removed at 48 h and plates were frozen overnight at -80 °C. Fluorescence was measured with the UV Spectromax M2 plate reader  with excitation at 485 nm and emission detection at 530 nm. Cell proliferation was calculated as a percentage of the control DMSO wells. All treatments were evaluated in triplicate in three independent experiments.The effect of toceranib phosphate on canine UC cell line viability was assessed with the CyQUANT® Cell Proliferation Assay Kit , as has been described previously . 5 × 103p < 0.05 were considered statistically significant.All experiments were performed in triplicate and repeated three independent times for UC cell lines. Data were presented as mean plus or minus standard deviation. All qRT-PCR data was normalized to endogenous control (GAPDH) and the ∆∆ Ct method  was usedAdditional file 1: Semi- quantitative Assessment of Percentage of Tumor Necrosis. Supplemental Table 1. summarizes the findings and degree of necrosis observed in the primary UC tumor samples. A single tumor sample was comprised of markedly more necrosis than the remaining samples, with >50% of examined tissue consisting of necrotic debris.Additional file 2: Figure S1. Uncropped RTK arrays and western blots. (Fig. ts. Fig. A Phosphots. Fig. A Phosphots. Fig. B Canine


ALK, MYC, and MET, were prioritized as having a likely role in canine OSA. Mutations in eight genes, previously described as human OSA drivers and including TP53, PTCH1, MED12, and PI3KCA, were retrieved in our cell lines. When variants were cross-referenced with human OSA driver mutations, the E273K mutation of TP53 was identified in the Wall cell line and tumor sample. The transcriptome profiling detected two possible p53 inactivation mechanisms in the Wall cell line on the one hand, and in D17 and D22 on the other. Moreover, MET overexpression, potentially leading to MAPK/ERK pathway activation, was observed in D17 and D22 cell lines. In conclusion, our data provide the molecular characterization of a large number of canine OSA cell lines, allowing future investigations on potential therapeutic targets and associated biomarkers. Notably, the Wall cell line represents a valuable model to empower prospective in vitro studies both in human and in dogs, since the TP53 driver mutation was maintained during cell line establishment and was widely reported as a mutation hotspot in several human cancers.Osteosarcoma (OSA) represents the most common primary bone tumor in dogs and is characterized by a highly aggressive behavior. Cell lines represent one of the most suitable and reproducible pre-clinical models, and therefore the knowledge of their molecular landscape is mandatory to investigate oncogenic mechanisms and drug response. The present study aims at determining variants, putative driver genes, and gene expression aberrations by integrating whole-exome and RNA sequencing. For this purpose, eight canine OSA cell lines and one matched pair of primary tumor and normal tissue were analyzed. Overall, cell lines revealed a mean tumor mutational burden of 9.6 mutations/Mb (range 3.9–16.8). Several known oncogenes and tumor suppressor genes, such as  TP53, RB1, DLG2, PTEN, MYC, and MET, were equally mutated in both cOSA and its human counterpart (TP53 pathway were detected in non-sensitive cell lines (Canine osteosarcoma (cOSA) represents the most common primary malignant bone tumor in dogs , 2 and interpart , 8. Morenterpart , 9 and pnterpart –12. Two nterpart , 14. Notll lines .in vitro models will allow the identification of new targets and offer valuable tools in translational medicine  and D22 (ATCC® CRL-6250™) were obtained from American Type Culture Collection.2. The FFPE samples were obtained from the same OSA from which the Wall cell line was established.These were cultured in Dulbecco's modified Eagle's medium  and Iscove's standard medium, supplemented with 10% fetal bovine serum (FBS), 1% glutamine, 100 μg/mL penicillin, and 100 μg/mL streptomycin. Cells were cultured at 37°C in a humidified atmosphere of 5% COGenomic DNA (gDNA) was isolated and purified from cell lines and the FFPE samples  using DNThe isolated DNA and RNA were stored at −20 and −80°C, respectively, until further use.High-quality whole-genome libraries from 10 samples (eight cells lines and two FFPE samples) were prepared using the KAPA HyperPlus Library Preparation Kit . Exome capture was executed using Roche's SeqCap EZ Share Prime Developer Kit  for non-human genomes following the SeqCap EZ HyperCap Workflow User's Guide. Probes for the exome capture were designed on the target enrichment design of 150 megabases developed by Broeckx et al. . The devA single-end sequencing (75 SE) was carried out on a NextSeq 500 platform .https://www.bioinformatics.babraham.ac.uk/projects/download.html) software. Trimmomatic was used to select high-quality reads and remove adapter sequences.Quality control prior to alignment was performed on output from Illumina software and was processed by FastQC v.0.11.9 (https://gatk.broadinstitute.org/hc/en-us/articles/360035894731-Somatic-short-variant-discovery-SNVs-Indels). Briefly, single-nucleotide variants (SNVs) and small insertion and deletions (indels) were identified with the GATK Mutect2 tool (http://dogsd.big.ac.cn/)  using BWA software . To verict2 tool  and filtct2 tool  13, 24), 24https.ac.cn/) . Distrib.ac.cn/) .The detailed WES workflow applied to both canine OSA cell lines and the FPPE samples is summarized in All RNA-seq analyses were performed using conventional RNA-seq analysis tools . DetaileAnnotated variants were subjected to three filtering levels with increasing stringency and designated as follows .Level 1: variants included the totality of exonic-only SNV and indels retrieved from the variant call described above. These were further filtered for number of reads (min. 2), alternate allele frequency (min. 0.05), and each variant's depth of coverage (min. 10). The resulting variants were analyzed to describe the mutational profile of cOSA cell lines.https://cancer.sanger.ac.uk/census) (https://www.intogen.org/search?cancer=OS) . Lev. Lev7). /census) . 5′ UTR ncer=OS)  to identTP53 identified in the Wall cell line and FFPE tumor sample was validated by Sanger dideoxy sequencing on Wall samples gDNA and Penny cell line (negative control). Briefly, two primers (sense 3′-ATGAGGGTGGCTAGGAGTCA-5′) and (antisense 5′-CAGTGCTGGGAAAGAGAGGA-3′) spanning the mutated region were designed by PRIMER3 Express software, and PCR on gDNA was performed using HotStar Taq (Qiagen) at 58°C  for 35 cycles. After evaluation of agarose gel, amplification products were purified by QIAquick PCR Purification Kit (Qiagen).E273K mutation of DNA extracted from eight canine OSA cell lines underwent WES, and an average of 158 million reads per sample (range 143–164) was obtained.The mean depth of reads mapping to the canine reference genome (CanFam3.1) was 187.7 (range 128–219), with over 99% of the targeted exome uniquely aligned. The optimal coverage was achieved for six out of eight cell lines. For each cell line, all reads passed the quality control  ≥ 30. The FFPE samples  achieved a mean depth of 54.71.The median tumor mutational burden of all Level 1 somatic variants was 9.6 mutations/Mb (range 3.9–16.9); in the Wall FFPE sample, the mutational burden reached 17.7 mutations/Mb . On averIn all our cell lines, missense mutations were the most frequently represented somatic coding mutation type, accounting for an average of 38.7% across all exonic variants. Frameshift insertion and deletions were 15.8 and 12.5% of the variants, respectively . In WallThe most common base change identified in all samples was C > T transition . The anaUsing the FidoSNP pathogenicity prediction tool, 50.5%  of all missense point mutations were categorized as deleterious.PDGFRB, PTCH1, and WRN were retrieved in at least three cell lines, whereas oncogenes and tumor suppressor genes, such as TP53, ALK, MYC, and MET, were retrieved in only one cell line each  to 10 genes (Sky) . Overallhttps://portal.gdc.cancer.gov/), four overlapping genes were identified, namely, PIK3CA, KRAS, APC, and NF1, which ranked 2nd, 6th, 10th, and 13th, respectively. At last, four genes were also identified in the Wall FFPE sample but did not overlap those of the corresponding cell line  in known cancer-causing genes in humans.Level 3 included 438 variants coding for 235 genes, regardless of their incidence across the cell line panel . A totalOverall, 190 genes were indicated as having a relevant and documented activity that promotes oncogenic transformation. In particular, 28 were designated as fusion genes, 74 as tumor suppressors, and 63 as oncogenes, and 25 functioned as both. The distribution of putative driver mutations across all cell lines is depicted in About 88% of the SNVs encoding for these genes were categorized as pathogenic.TP53, PTCH1, MED12, and PI3KCA, were identified.When compared to the top 20 cancer driver genes involved in human OSA (COSMIC Cancer Browser), eight genes, encoded by 21 variants, were retrieved in Level 3 genes . Among tTP53E273K mutation (c.818C>T) corresponding to the human TP53E285K mutation hotspot.All putative driver variants were cross-referenced with human OSA driver mutations available on IntOgen. Only the Wall cell line and FFPE tumor sample harbored a putative driver mutation, namely, the This putative driver mutation was further validated in homozygosis in the Wall cell line and tumor sample by Sanger sequencing .RNA sequencing generated over 10 million reads per sample. Quality control and trimming procedures retained the vast majority of the sequences, and unique alignment to the canine reference genome was successful for 86% of the cleaned reads .The normalized gene expression of the osteosarcoma-associated genes described above was then profiled within the same gene across all samples.TP53 gene expression was increased in the Wall cell line, which harbored a hotspot mutation, as well as in D17 and D22, which retained a wild-type gene status. Interestingly, D17 and D22 showed a 9-fold and 5.5-fold increase of MDM2 and MDM4 transcript levels, respectively, compared to the other cell lines. Conversely, the MDM2 transcript level was decreased by 2 to 20 times in the Wall cell line compared to the rest of the cell lines.In particular, MET by 19-fold was observed in the D17 cell line, which harbored a frameshift insertion on this gene, and to the same extent in D22 which retained a wild-type gene status. Notably, genes involved in the downstream MAPK/ERK pathway, such as MAPK1, MEK, and MYC, showed increased transcript levels, although no mutations were detected in WES analysis.Transcriptional upregulation of as PTCH1, MED12, PDGFRB, and PIK3CA, did not show any transcript level change.Conversely, downstream signaling components of mutated cancer-associated genes, such Cancer cell lines are considered valuable models in basic cancer research, drug discovery, and translational medicine , 33, 34.The recent characterization of a large panel of human cancer cell lines with omics technologies has empowered data-driven precision medicine , 35. Desin vivo cOSA pathogenesis and identifying suitable targets for drug discovery. To date, this dataset represents one of the largest explored for a single tumor in dogs.In the present study, we integrated data from whole-exome and RNA sequencing of eight cOSA cell lines to obtain genomic and molecular data recapitulating Specifically, two commercial and six primary cOSA cell lines established and validated in our laboratory were analyzed. The assorted cell lines were previously used in several studies to investigate cOSA pathogenetic mechanisms and response to therapies , 36–38. About exome sequencing, variants were analyzed using three levels of increased stringency: first, describing the mutational profile of each cell line; second, identifying recurrently mutated genes; and third, unraveling putative driver genes having a likely role in cOSA pathogenesis.Overall, the mutational burden in our cOSA cell lines ranged between 3.9 and 16.9 mutations/Mb but was lower than the one described by Das et al. . HoweverConsistent with previous reports in cOSA cell lines and hOSA, the mutational type distribution showed a prevalence of missense mutations, and C>T transitions dominated this mutational spectrum , 39.PDGFRB and MET were retrieved. Both genes are known to play an important role in the development and progression of many canine cancers and were thoroughly investigated in cOSA as well  can be found here: RD and LA contributed conception and design of the study. PB and EM enrolled the canine osteosarcoma cases used for cell line establishment. CG, PM, SI, and RD cultured the cell lines and extracted DNA and RNA from canine osteosarcoma cell lines and FFPE samples. AR performed the NGS sequencing. DG performed the computational analyses. CG performed data post-processing. CG and SI performed data visualization. CG and DG wrote the manuscript. RD edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.