Breast cancer (BC) is a heterogenous disease classified into four molecular subtypes ) depending on the expression of the estrogen receptor (ER), the progesterone receptor (PR) and the human epidermal receptor 2 (HER2). The development of effective treatments for BC, especially TNBC, remains a challenge. Aminosteroid derivative RM-581 has previously shown an antiproliferative effect in multiple cancers in vitro and in vivo. In this study, we evaluated its effect in BC cell lines representative of BC molecular subtypes, including metastatic TNBC. We found that RM-581 has an antiproliferative effect on all BC molecular subtypes, especially on Luminal A and TNBC, in 2D and 3D cultures. The combination of RM-581 and trastuzumab or trastuzumab-emtansine enhanced the anticancer effect of each drug for HER2-positive BC cell lines, and the combination of RM-581 and taxanes (docetaxel or paclitaxel) improved the antiproliferative effect of RM-581 in TNBC and metastatic TNBC cell lines. We also confirmed that RM-581 is an endoplasmic reticulum (EnR)-stress aggravator by inducing an increase in EnR-stress-induced apoptosis markers such as BIP/GRP78 and CHOP and disrupting lipid homeostasis. This study demonstrates that RM-581 could be effective for the treatment of BC, especially TNBC. Breast cancer (BC) is the second cause of death by cancer in women. The American Cancer Society estimates that 279,100 new cases of invasive BC were diagnosed, and 42,690 deaths were reported among US women in 2020 . Due to Aminosteroid (AM) derivatives are a family of anticancer molecules that have high and selective cytotoxic effects on multiple types of cancers, both in vitro and in vivo ,9,10,11.The endoplasmic reticulum (EnR) is an organelle with a major role in the maintenance of cellular homeostasis . The tumHence, the aim of the present study was to evaluate whether RM-581 can be an effective anticancer molecule on all BC molecular subtypes  and determine if its mechanism of action is via EnR-stress as for pancreatic and prostate cancer. We thus tested RM-581 antiproliferative effects in BC cell lines belonging to different molecular subtypes, alone and in combination with other BC gold standard treatments. We then confirmed these results in 3D BC spheroids and also measured the effect of RM-581 on the expression of EnR-stress markers.2 in water-saturated atmosphere and were shown to be mycoplasma free. We selected BC cell lines representative of luminal A (MCF7), luminal B (BT-474), HER2 (JIMT-1 and MDA-MB-453), TNBC , a non-tumorigenic epithelial cell line (MCF10A), and three metastatic TNBC-derivative cell lines . All the BC cell lines along with their molecular subtype and the culture conditions are listed in −1 µM (after dilution in PBS) only for MDA-MB-453, alone or in combination. MDA-MB-231, MDA-MB-468, BT-549, SUM149PT, MDA-BoM-1833, MDA-BoM-1834, and MDA-MB-231-BR cells received a RM-581 treatment at 0.1, 1, or 5 µM, a docetaxel (DTX)  and a paclitaxel (PTX)  treatment at 10−1 µM, 10−2 µM or 10−3 µM (after dilution in DMSO), alone or in combination. After 72 h of in-cubation, alamarBlue  was added  for 2 h of incubation. Viability rates were determined by fluorescence using a Tecan M-200 microplate reader  with an excitation wavelength at 570 nm and emission wavelength at 585 nm. Percentage viability was calculated for the treated cells compared with the untreated cells receiving only DMSO. All the experiments were performed in triplicate, and mean ± SD was calculated and plotted for each drug concentration. The IC50 values (50% cell growth inhibition) for each cell line were calculated using GraphPad software version 5 with a dose-response model (Y = Bottom + (Top-Bottom)/(1 + 10^(X − LogIC50)). For all the curves, the R2 value was greater than 0.9. All the figures were generated using Prism/Graphpad (version 5). For the combination assays, one-way ANOVA were performed followed by the Tukey test to compare the effects of the drug alone or the combination of the drugs. The significance level was set at p < 0.05.Cells were plated on 96-well plates in triplicate  as described in . After 250 obtained in 2D culture, or with 5 µM for MCF10A as a control. Images of the spheroids were taken before, and 3 and 7 days after the treatment to observe phenotype changes. Viability rates were determined as described in the section above. Nonparametric Mann–Whitney tests were performed. The significance level was set at p < 0.05. BC cells  were plated in 96-well ultra-low attachment plates  in triplicate . After 5 days of incubation to let the spheroids or cellular aggregates form, RM-581 was added at a concentration twice or five times the ICBC cells  were plated in 6-well plates (1.5 × 106 cells per well). After 24 h of incubation, RM-581 was added at specific IC50 concentrations for each cell line in a time-dependent manner . The cells were homogenized with QIazol Lysis Reagent , and total RNA was extracted following the miRNeasy kit’s instructions . RNA concentrations were measured based on their intrinsic absorptive properties (260/280 and 260/230 ratios) using a Nanodrop ND-1000 Spectrophotometer . Reverse transcription was performed using 3 to 5 µg of total RNA. First, total RNA was mixed with 10 mM of dNTP , 50 µM of oligodT  and 50 µM of random hexamers  at 65 °C for 5 min. Then, Reverse Transcriptase  was added for a series of incubations: 10 min at room temperature, 15 min at 50 °C and 10 min at 80 °C. Purification was performed following the QIAquick PCR Purification kit’s instructions . cDNA concentration was measured using Nanodrop (260/280 and 260/230 ratios). Real time PCR quantifica-tion was performed in triplicate using 20 ng of cDNA and SYBr Green mix . HPRT1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as reference genes. To control the genomic DNA contamination in cDNA samples we used a fragment of genomic DNA from the 3β-hydroxysteroid dehydrogenase (3βHSD) gene. The mRNA levels are indicated in percentage terms relative to reference genes. The primer sequences are reported in p < 0.05.A melting curve was created to assess non-specific signals. The relative quantity was calculated using the fit point method and by applying the delta cycle threshold (Ct) method as the amplification efficiencies of the curves were 100% . Hypoxan50. We first tested a normal breast cell line (MCF10A) and BC cell lines depending on their molecular BC subtype. As shown in 50 of RM-581 (50% of cell growth inhibition) for MCF10A was 17.1 µM, which was the highest IC50 found in this study. The graph for each cell line is shown in To assess the antiproliferative capacity of RM-581, we treated each cell line with a range of concentrations of RM-581 to determine the IC50 obtained for all the BC cell lines were significantly different from the IC50 obtained from the normal breast cell line MCF10A, except for MDA-MB-453. This result demonstrates that RM-581 is more effective against cancerous cells than against normal cells in the breast. As shown in 50 values of 2.8, 10.4, 8.7, 8.5, 6.9, and 5.6 µM for MCF7, MDA-MB-231, BT-549, SUM159PT, MDA-MB-468, and SUM149PT, respectively. In contrast, Luminal B (ER+/PR+ and HER2+) and HER2 (ER-/PR- and HER2+) cell lines had IC50 values of 12.0, 13.4 and 12.3 µM for BT-474, MDA-MB-453 and JIMT-1, respectively. This suggests that the overexpression of HER2 leads to a decrease in the effectiveness of RM-581 against these cell lines. Even though the IC50 of MDA-MB-453 and MCF10A are not different, RM-581 could still be effective against HER2 cell lines as the IC50 of the other HER2 cell line, JIMT-1, is significantly lower than that of MCF10A. The ICTaken together, these results demonstrate that RM-581 was effective on all tested BC cell lines. However, each molecular BC subtype cell line had different sensitivity to RM-581. The luminal A and TNBC cell lines were the most sensitive, while the luminal B and HER2 cell lines were less sensitive.50 (MDA-MB-468 and SUM149PT) and the cell line with the highest IC50 (MDA-MB-231) to compare the effect of RM-581 within this molecular subtype. Each cell line was treated with two or five times the concentration of IC50 determined in the 2D culture. As shown in 50 is sufficient to significantly decrease BC cell line spheroid proliferation, except in MDA-MB-468. This could be explained by the fact that the spheroids formed for the MDA-MB-468 cell line were larger in size than in the other BC cell lines, which implies that this cell line proliferates faster , BT-474 , MDA-MB-453 (HER2), MDA-MB-468 (TNBC), MDA-MB-231 (TNBC), and SUM149PT (TNBC)) to perform spheroid assays A. We sels faster A. Howeveere dead B. Moreovere dead C. These One of the treatment strategies for BC is to target one of the molecular markers in order to have a personalized treatment for each molecular subtype. Trastuzumab, a well-established BC treatment, is a monoclonal antibody that targets one of the extracellular subdomains of HER2 . A derivAs shown in Taken together, these results show a benefit of using RM-581 in combination with anti-HER2 therapies to treat HER2+ BC patients effectively.One of the major strategies of treatment for BC is chemotherapy, which is administered regardless of BC subtypes along with personalized therapy. However, for TNBC, no personalized therapy is currently available, and chemotherapy remains the only pharmaceutical treatment strategy . We evalThese results demonstrate that RM-581 could be administered in combination with taxanes as it does not compete with the effects of DTX or PTX on TNBC cell lines. 50 concentration established with the proliferation assay to observe the early effects of exposure to RM-581. The mRNA expression levels of intrinsic apoptosis markers (BCL2 and CYCS), EnR-stress marker (BIP), and EnR-stress apoptosis marker (CHOP) were analyzed. For each of the BC cell lines tested, exposure to RM-581 did not affect the expression of CYCS ; however, this increase seemed to be transitory as from 12 h, BCL2 expression significantly decreased for MCF7 and BT-474. On the other hand, the expression of BIP and CHOP significantly increased in parallel with the exposure time to RM-581 for all BC cell lines  and prostate cancer (PC-3) cells [The mechanism of action of RM-581 has been previously investigated in pancreatic and prostate cancers as an EnR-stress aggravator ,14. Henc of CYCS A. The exll lines A. Moreov3) cells ,14. SCD transcripts was, therefore, measured after 3, 6, and 12 h of RM-581 exposure in the MCF7, BT-474, MDA-MB-453, and SUM149PT BC cell lines. As shown in SCD expression was observed at the three time points for BT-474 and MDA-MB-453. The SCD expression decreased at 3 h and 12 h of RM-581 exposure for MCF7 and for SUM149PT, at 6 h and 12 h of RM-581 exposure. In addition, EnR is also involved in lipid biosynthesis by producing enzymes of this pathway. Among these, SCD (Stearoyl-CoA desaturase 1) is the rate-limiting enzyme of fatty acids synthesis . The levTaken together, these results support the fact that RM-581 disrupts lipid homeostasis by inducing EnR-stress-induced apoptosis.Metastatic TNBC has the poorest survival rate among the molecular subtypes . Therefo50 values for the three TNBC metastatic cell lines were 9.4 µM, 8.6 µM, and 11.1 µM for MDA-BoM-1833, MDA-BoM-1834, and MDA-MB-231-BR, respectively  seemed to improve the anticancer effect of each drug alone C. IndeedBCL2 and CYCS transcripts for any of the three TNBC metastatic cell lines. In contrast, the level of both BIP and CHOP transcripts were higher than the control (0 h) for all times of exposure  for all three TNBC metastatic cell lines  BC cell line . We foun50 was around 8 µM. RM-581 is particularly effective against MDA-MB-468 (6.9 µM) and SUM149PT (5.6 µM) compared with MDA-MB-231 (10.4 µM), BT-549 (8.7 µM), and SUM159PT (8.5 µM). This difference in sensitivity could be explained by different TNBC subtypes; MDA-MB-468 and SUM149PT are basal, and MDA-MB-231, BT-549 and SUM159PT are of the mesenchymal TNBC subtype, according to the classification of Lehmann et al. [We have shown that RM-581 displays the potential to treat patients with TNBC. For the TNBC cell lines, the mean ICn et al. . It is kn et al. . RM-581 50 for RM-581 compared with the other molecular subtypes. Thus, the combination of trastuzumab or T-DM1 and RM-581 could lead to a reduction in the RM-581 concentration administered to patients with HER2-positive BC. Drug combination is an important part of BC treatments and regimens as it is a very heterogeneous disease. Combination treatments can prevent BC patients from side effects, drug resistance and recurrence . TrastuzDue to the lack of ER and PR expression as well as HER2 overexpression, patients with TNBC cannot receive targeted therapies such as hormonal therapy or anti-HER2 therapy, and chemotherapy remains the only treatment for these patients . Long-teAlthough the survival rate of BC patients is encouraging with a 5 year relative cancer-specific survival rate of 90.3%, the survival rate of metastatic BC patients remains low, especially for patients with metastatic TNBC, with a 5 year relative cancer-specific survival rate of 12% . Thus, iThe EnR is the organelle responsible for the production and folding of cellular proteins and contributes to cell homeostasis. Due to extracellular environmental disturbances, unfolded proteins can accumulate in the EnR lumen, causing the activation of a specific stress pathway named the unfolded protein response (UPR). The UPR leads to two signaling pathways: pro-survival to restore the cell homeostasis or pro-death if EnR-stress is prolonged . The UPRBIP was observed for all the molecular subtype BC cell lines. BIP  is one of the EnR chaperones which are essential to maintain the normal function of the EnR and the activation of the UPR [CHOP with the highest peak at 12 h, was observed for MCF7, BT-474, MDA-MB-231, and SUM149PT and at 24 h for MDA-MB-453 and MDA-MD-468. CHOP is one the UPR downstream effectors and is the principal activator of the apoptosis induced by EnR-stress [Our results support that EnR-stress-induced apoptosis could be involved in the RM-581 mechanism of action. In fact, a gradual increase in  the UPR ,45. ThisR-stress ,14. ThisSCD transcript for all molecular subtype BC cell lines. This suggests that RM-581 could also decrease BC cell proliferation by modulating lipid biosynthesis. Further investigations are needed to decipher the potential mechanism of action of RM-581. The EnR is the site of lipid biogenesis, such as fatty acids. Moreover, in cancer cells, the lipid metabolism is affected, which promotes tumor cell survival . Thus, w50 = 12.3 µM), a well characterized trastuzumab-resistant cell line [EnR-stress and UPR signaling are important in tumorigenesis and lead to treatment resistance. This is one of the major challenges for BC therapy as many patients acquire resistance to their treatment .ThereforIn this study, we demonstrated that RM-581 is particularly effective against TNBC cell lines, and especially basal-like subtypes (MDA-MB-468 and SUM149PT). Here we tested four of the six TNBC subtypes: BL1 (MDA-MB-468), BL2 (SUM149PT), M (BT-549), and MSL (SUM159PT and MDA-MB-231) subtypes . It woulSpheroids are aggregates of cells grown in suspension that are used as 3D culture models. Multicellular tumor spheroids (MCTS) represent avascular tumor nodules with a transcript profile closer to in vivo tumor gene expression profiles than tumor cells in 2D culture. MCTS are widely used to assess tumor response to new synthetized drugs . We demo


Whilst the peptide ligandome presented by cell surface human leucocyte antigen class I (HLA‐I) molecules on cancer cells has been studied extensively, the ligandome of extracellular vesicles (EVs) remains poorly defined. Here, we report the HLA‐I ligandome of both the cell surface and EVs from eight breast cancer cell lines , and additionally the melanoma cell line ESTDAB‐056 and the multiple myeloma line RPMI 8226. Utilizing HLA‐I immunoisolation and mass spectrometry, we detected a total of 6574 peptides from the cell surface and 2461 peptides from the EVs of the cell lines studied. Within the EV HLA‐I ligandome, we identified 150 peptides derived from tumour associated antigenic proteins, of which 19 peptides have been shown to elicit T‐cell responses in previous studies. Our data thus show the prevalence of clinically relevant tumour‐associated antigenic peptides in the HLA‐I ligandome presented on EV.The recent success of monoclonal antibody checkpoint inhibitor therapies that enhance the ability of CD8 + T‐cell epitopes.The HLA‐I peptidomes of both EV and cell were identified from 10 cancer cell lines and 150 peptides derived from tumour‐associated antigens were identified in HLA‐I ligandome of EVs. Our data, show, for the first time the prevalence of clinically relevant tumour‐associated antigenic peptides in the HLA‐I ligandome presented on EV and the potential of EV ligandomes for identification of CD8 CLcell lysatesEVsextracellular vesiclesHLA‐Ihuman leucocyte antigen class ITAAtumour‐associated antigenic proteinsTAApeppeptides derived from tumour‐associated antigenic proteins+ T cells which permits the detection of intracellular pathogens such as viruses, and also up‐regulated or mutated proteins relevant in cancer [+ T cells through the expression of immune checkpoint proteins such as PD‐L1. In recent years, checkpoint inhibitor therapies in the form of monoclonal antibodies directed against PD‐L1 and PD1 have displayed significant clinical success by allowing CD8+ T cells to more readily detect the array of antigenic peptides on cancer cells [Human leucocyte antigen class I molecules (HLA‐I) display short peptides, typically 9–11 amino acids in length, primarily derived from the cellular proteome, to CD8er cells , 3, 4. Ter cells . Such efExtracellular vesicles, including exosomes, are typically 50–200 nm membrane bound vesicles secreted by cells into extracellular environment . EVs relHER2/neu and melan‐A [It has been known for many years that tumour‐derived EVs are present in malignant effusions and that these EVs are enriched in HLA molecules as well as tumour antigens such as  melan‐A . Additio melan‐A , 17. How melan‐A . Here, wg for 10 min at 4° celsius, then resuspended in PFN buffer . Cells were stained with primary mouse IgG anti‐HLA‐A, ‐B, and ‐C monoclonal antibody W6/32 [The breast cancer cell lines, MDA‐MB‐231, MDA‐MB‐361, MDA‐MB‐415, MDA‐MB‐453, MCF7, HCC 1395, HCC 1806, and HCC 1954, were obtained from the ATCC Breast Cancer Cell panel . Multiple myeloma cell line RPMI 8226 was obtained from LGC Standards and melanoma cell line ESTDAB‐056 was a gift from Prof Federico Garrido, . All cell lines were cultured in DMEM or RPMI‐1640 supplemented with 5% fetal bovine serum (FBS) and 50 μg/ml kanamycin . For flow cytometry, cells were harvested from culture flasks with a 1–2 min incubation with trypsin (0.025%)‐EDTA (0.01%) solution at 37° celsius. Cells were then resuspended in culture medium to inactivate trypsin and centrifuged at 300g for 10 min at 4° celsius to remove cells and large debris. EV‐containing supernatants were then filtered with 0.22‐μm Millex‐GP syringe filters (Merck). NTA was performed using an LM‐10 unit  equipped with a 638‐nm laser. A minimum of three videos of 30 s were recorded for each sample using settings of shutter speeds of 17 or 30 ms. Data analysis was performed using NTA 2.3 software with detection thresholds of 2 or 3 and blur, min track length, and min expected size on auto settings. One representative data plot is shown for each cell line.For the initial characterization of EVs, cell lines were grown in serum‐free medium  to exclude the possibility of EV contamination from serum. For nanoparticle tracking analysis (NTA), cells were cultured for 24 hr, conditioned media were then collected and centrifuged at 3002 flasks for 48 hr in serum‐free medium as above. After 48 hr, conditioned media were collected, spun, and filtered through 0.22‐μm filters as above, and culture supernatants were then spun at 100 000g for 2 hr using a Sw32Ti swing‐out rotor in a Beckman L100 ultracentrifuge. EV pellets were resuspended in lysis buffer . Cell lysates were prepared concomitantly by lysis of the cells remaining in the culture flasks with the same lysis buffer, with an additional spin at 20 000g for 10 min at 4° celsius to remove insoluble debris. Protein estimations were performed by Bradford assay (ThermoFisher Scientific). Approximately, 3 μg of cell and EV lysates were electrophoresed on 4%–20% gradient sodium dodecyl sulphate‐polyacrylamide gel electrophoresis gels (ThermoFisher Scientific) and transferred to nitrocellulose filters . Membranes were probed with anti‐CD9, CD63, and CD81 antibodies  at 1:5000 dilution, anti‐HLA‐B and ‐C mouse monoclonal antibody HC10 [For immunoblotting, cells were grown in 175 cmg for 4 hr at 4° celsius using a SW32Ti rotor in a Beckman L100 ultracentrifuge, followed by 0.2‐μm filtration. All cell lines were either grown in DMEM (breast cancer cell lines) or in RPMI‐1640 (myeloma and melanoma cell lines) supplemented with 2.5% EV‐depleted FBS and 50 μg/ml kanamycin. Suspension cell line RPMI 8226 was cultured in 175‐cm2 flasks and cultured to approximately 2 million cells per ml. Conditioned medium containing cells and EVs was collected and centrifuged at 300g for 10 min at 4° celsius. The supernatant containing EVs was filtered with 0.2‐μm syringe filter and stored at −20° celsius and RPMI 8226 cells were re‐plated in EV‐depleted medium. Conditioned medium was collected again after 48 hr and processed as described above.Large‐scale cultures for EV and cell isolation were maintained in EV‐depleted FBS. EV‐depleted FBS was prepared by ultracentrifugation of FBS at 100 0002 flasks or in Falcon 5‐layer multi‐flasks with a surface area of 875 cm2 (ThermoFisher Scientific). At around 80% cell confluence, fresh culture medium was added to flasks and conditioned medium was harvested every 48 hr and processed as above, with new medium then added to the flasks. In total 800 ml conditioned media were harvested from each breast cancer cell line and 250 ml conditioned medium was harvested from ESTDAB‐056 and suspension cell line RPMI 8226. The EV harvests were thawed and spun at 100 000g for 2 hr using a Sw32Ti rotor and the EV pellet lysed in lysis buffer as above, and stored at −20° celsius until immunoprecipitation. At culture closedown, cells were harvested from culture flasks with a 1–2 min incubation with trypsin (0.025%)‐EDTA (0.01%) solution at 37° celsius and spun at 300g for 10 min at 4° celsius. Cell pellets were lysed in lysis buffer as above, spun at 20 000g for 10 min at 4° celsius to remove debris and the cell lysates were stored at −20° celsius until immunoprecipitation. EV and cell isolation experiments were independently repeated two to three times for breast cancer cell lines and six times for myeloma and melanoma cell lines.All adherent cells were grown in either multiple 175‐cmEV and cell lysates (CL) were thawed and incubated with 0.5 ml anti‐HLA‐A, ‐B, and ‐C antibody, W6/32, coupled to Protein G‐Sepharose beads  for 1–2 hr. The beads were then washed extensively with wash buffer , and resuspended in 1 ml of 1% TFA for 10 min at room temperature to release HLA‐I and bound peptides. Eluted HLA‐I and peptides were then bound to Pierce C18 100‐μl tips  based on the manufacturer's instructions. The peptide fraction was eluted in 30% acetonitrile and 0.1% TFA and dried down by speedvac for mass spectrometry.m/z 400–1250) and MS2 (m/z 95–1800) scans, respectively, and 15‐s dynamic exclusion. Rolling collision energy was used for fragmentation.Peptides were then analysed on an AB Sciex TripleTOF 5600+ system mass spectrometer (Sciex) coupled to an Eksigent nanoLC AS‐2/2Dplus system. The samples were loaded in loading buffer (2% acetonitrile and 0.05% trifluoroacetic acid) and bound to an Aclaim pepmap 100 μm × 2 cm trap (ThermoFisher Scientific), and washed for 10 min to waste after which the trap was turned in‐line with the analytical column (Aclaim pepmap RSLC 75 μm × 15 cm). The analytical solvent system consisted of buffer A (2% acetonitrile and 0.1% formic acid in water) and buffer B (2% water with 0.1% formic acid in acetonitrile) at a flow rate of 300 nl/min with the following gradient: linear 1%–20% of buffer B over 90 min, linear 20%–40% of buffer B for 30 min, linear 40%–99% of buffer B for 10 min, isocratic 99% of buffer B for 5 min, linear 99–1% of buffer B for 2.5 min, and isocratic 1% solvent buffer B for 12.5 min. The mass spectrometer was operated in the DDA top 20 positive ion mode, triggering on +2 to +5 charge states, with 120 and 80 ms acquisition time for the MS1 (https://www.bioinfor.com/peaks-studio). Following mass spectrometry data acquisition, data files from the AB Sciex Triple TOF 5600+ were converted into mzML format using MSConvert software of proteowizard. Converted data files were imported into PEAKS Studio X for peptide identification. Data files were subjected to default data refinement followed by PEAKS de novo and PEAKS DB (database) searches to identify peptide sequences. PEAKS de novo and PEAKS DB searches were carried out by setting parent mass error tolerance to 15 parts per million (ppm), the fragment mass error tolerance to 0.1 Da, no enzyme selection, unspecific digestion mode, and filtering charge between 2 and 5. No post‐translational modification (PTM) was selected in PEAKS de novo and PEAKS DB searches. A 0.1% FDR cut off was applied during PEAK DB searches to select high confidence peptides and peptides only identified by PEAKS DB were selected for further analysis.LC–MS/MS data were searched against the human proteome  with an additional contaminant list from the global proteome machine (cRAP protein sequences). We used decoy fusion method of PEAKS DB search to estimate false discovery rate during peptide identification . PeptideMass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via the PRoteomics IDEntifications (PRIDE) repository with the dataset identifier PXD025345.https://github.com/peterthorpe5/Cancer_cell_line_RNAseq_assemblies. RNA‐seq data of breast cancer cell lines were obtained from the Gene Expression Omnibus (GEO) (accession GSE73526). The HLA‐I genotypes of breast cancer cell line, MCF7, was obtained from previously reported HLA‐I genotypes of cancer lines [https://www.ebi.ac.uk/cgi-bin/ipd/estdab/print_cell.cgi?ESTDAB-056) and RPMI 8226 HLA‐type was determined from previous report [The HLA‐I genotypes of breast cancer cell lines, MDA‐MB‐231, MDA‐MB‐361, MDA‐MB‐415, MDA‐MB‐453, HCC 1806, HCC 1395, and HCC 1954, were identified by using paired end reads in fastq format as input to the Optitype version 1.3.1 . Scriptss report . HLA‐I thttp://www.cbs.dtu.dk/services/NetMHCpan/) [50 up to 1000 nM were tabulated and plotted using Prism 8  software. For identification of immunogenic peptides reported in previous studies and the peptides derived from TAA proteins, peptides with 8–15 length were searched against IEDB database  and Tantigen database (http://projects.met-hilab.org/tadb/index.php).Eluted peptides identified from each replicate were combined, duplicate peptides were removed and filtered to select 8–15 amino acid long peptides. The total number of 8‐15‐mer peptides was defined as HLA‐I ligandome of cell and EVs from each cell line. Peptides present in HLA‐I ligandome were assessed for their predicted allele‐binding and affinity using algorithm netMHCpan 4.0 (MHCpan/)  followinWe first characterized the expression of HLA‐I molecules on our chosen cell lines. Cell surface flow cytometry was performed on the breast cancer cells lines MCF7, MDA‐MB‐231, MDA‐MB‐361, MDA‐MB‐415, MDA‐MB‐453, HCC 1806, HCC 1395, and HCC 1954, the melanoma cell line ESTDAB‐056, and the myeloma cell line RPMI 8226, using the HLA‐A, B, and C specific monoclonal antibody W6/32 Figure . HLA‐I eTo characterize the EVs released by each of these cell lines and to determine HLA‐I expression on EVs, cell lines were cultured in serum‐free media to eliminate potential contamination from FBS‐derived EVs. We performed nanoparticle tracking analysis (NTA) on conditioned medium collected from cells grown for 24 hr in these serum‐free conditions Figure . All celg to remove debris, filtered at 0.2 μm and then ultracentrifuged at 100 000g for 2 hr to isolate EV, followed by detergent lysis and immunoisolation with antibody W6/32 coupled to Sepharose beads. CLs were processed concomitantly. After release of the peptides by acidification in trifluoracetic acid and clean up on C18 matrix tips, peptides were analysed by mass spectrometry. HLA‐I immunoisolated and eluted peptides were identified by PEAKS DB software (hereinafter defined as eluted peptides). Eluted peptides from independent experiments of cell and EV lysates of each cell line were combined together and duplicate peptides (peptides identified in more than one experiment) were removed to obtain a complete list of unique eluted peptides from cell or EVs. Peptides which are 8–15 amino acids long have been reported to bind HLA‐I molecules and elicit immune responses. Therefore, eluted peptides identified by PEAKS DB, were filtered to select 8‐15‐mer peptides for further analysis. The total number of 8‐15‐mer peptides identified from cell and EV lysates is hereinafter defined as cell or EV ligandome, respectively.Based on our previous study , we usedThe details of eluted peptides and HLA‐I ligandomes from each cell lines are presented in File Peptides present in HLA‐I ligandomes were further processed through the HLA‐I binding affinity prediction algorithm netMHCpan 4.0. Details of HLA‐I binding peptides from each cell line, for each expressed HAL‐I allele, and their respective predicted binding affinities (BA) are presented in Figures Owing to the presence of HLA‐I molecules, EVs have long been suspected to carry tumour antigens. A detailed description of the peptidome and the full extent and identity of tumour antigens has, however, not been reported for EVs. Therefore, EV ligandomes of each cell line were searched for the presence of tumour antigens. For clarity, herein we define TAA as tumour‐associated antigenic proteins and TAApep as the peptides derived from tumour‐associated antigenic proteins that are presented on HLA‐I molecules.First, we determined if EVs can carry immunogenic peptides which have been identified in previous studies. The cell and EV ligandomes were searched for the presence of peptides showing sequence match to known T‐cell epitopes using IEDB and Tantigen databases since IEDB and Tantigen databases are currently amongst the most comprehensive tumour antigen databases. The number of identified peptides showing sequence match with known T‐cell epitopes from EV and cell ligandomes are presented in Tables Additionally, the list of known T‐cell epitope sequences identified in the EV ligandome includes peptides derived from antigens which are either associated with cancer or used as diagnostic and prognostic biomarkers. For example, peptides KTINPQVSKTEY and TYLPTNASLSF detected in the EV ligandomes of MDA‐MB‐415 and HCC 1954, respectively, are known T‐cell epitopes from Mammaglobin‐A and Her‐2, and thus relevant to breast cancer. Similarly, the peptide STAPPAHGV identified in the EV ligandome of myeloma cell line RPMI 8226, is a T‐cell epitope of MUC1 which has been identified as a tumour antigen in several multiple myeloma cell lines , 29, 30.The cell and EV ligandomes of all cell lines were also searched for the presence of additional peptides (TAApep) derived from TAA proteins. We predicted these would be present in our ligandomes because of the extended HLA‐I allele cohort present in our study, such that peptides derived by antigen processing of other parts of the TAA would likely be detected. We used Tantigen 2.0 database to identify peptides derived from the full‐length protein sequences of TAA. A total of 277 additional TAApep derived from 107 TAA proteins were identified in cell ligandomes of all lines File . Out of In summary, our data indicate that the EV ligandomes of common cancer cell lines spanning breast, melanoma, and myeloma carry peptides from known TAAs, potential T‐cell epitopes, and additional TAApep derived from the protein sequences of known and established TAA.In this study, we report the first direct evidence of known tumour‐associated antigens and T‐cell epitopes presented on HLA‐I molecules on extracellular vesicles released by cancer cell lines. We have analysed the EV HLA‐I ligandomes of 10 cell lines, predominately representing breast cancer, but also including melanoma and myeloma to indicate that the presence of HLA‐I associated TAA on EV is likely to be a widespread observation and not exclusive to just one cancer type.In recent years, EVs have emerged as promising tools in both cancer diagnosis and cancer immunotherapy . EVs repDespite the clinical relevance and therapeutic potential of EVs, the EV HLA‐I ligandome of tumour cells has not been established in detail. To date, only two reports of the HLA‐I EV ligandome of cell lines have been published . This loWe have identified peptides in HLA‐I ligandomes of 10 cancer cell line. The peptide repertoire reported here may be used as database to identify multiple markers for diagnosis, prognosis of tumour as well as targets for immunotherapy. Interestingly, 26% of peptides present in EV HLA‐I ligandome were not found in HLA‐I ligandome of cells. The presence of unique peptides in HLA‐I ligandomes was also detected in previous two studies of Jesthom and JY cells , 35. The+ T cells approaching a tumour site could theoretically encounter a ‘cloud’ or gradient of EV containing HLA‐I presented TAA that could activate the T cell off‐target. There have, to date, been very few studies on the ability of HLA‐I on EV to activate CD8+ T cells, but EV loaded in vitro with common antigenic viral peptides can stimulate IFNγ secretion in purified CD8+ T cells, thus indicating that EV can indeed directly activate T cells [The presence of cell surface antigenic peptides presented on HLA‐I derived from TAA proteins, be it from differentiation antigens, over‐expressed cellular antigens, cancer/testis antigens or cancer related viral antigens allows the immune system the opportunity to mount responses that can impact on the progression of cancer growth . Their p T cells .Studies of patients undergoing monoclonal antibody checkpoint inhibitor therapy, which unleashes wider T cell activity, reveal that the best responses occur against tumours that typically have increased numbers of mutations, that is, which have the potential to generate large numbers of neo‐epitopes . In receThe authors declare no conflicts of interest.Simon J. Powis and Sally Shirran conceived the research plan. Pankaj Kumar, Simon J. Powis, and Sally Shirran designed the experiments. Pankaj Kumar, Simon J. Powis, Caitlin Boyne, Sydney Brown, and Ayesha Qureshi performed experiments. Sally Shirran and Silvia A. Synowsky performed mass spectrometry analysis of samples. PT identified HLA type of cell lines. Pankaj Kumar, Caitlin Boyne, Sydney Brown, and Simon J. Powis analysed data. Pankaj Kumar, Simon J. Powis, and Caitlin Boyne wrote manuscript.Supporting informationClick here for additional data file.TFile S1Containing details of HLA genotypes of cell lines used in the study.Click here for additional data file.TFile S2Containing details of HLA‐I peptides identified by PEAKS DB search from cell and EV lysates of each cell lineClick here for additional data file.File S3Containing details of cell and EV ligandomes and their comparisonsClick here for additional data file.File S4TAApep identified in EVs of breast cancer, melanoma and myeloma cell linesClick here for additional data file.


The dirhodium complexes show moderate cytotoxic activity in the tested tumor cell lines, with acetate and methyl-substituted formamidinate compounds displaying increased cytotoxicity that is relative to cisplatin in the A2780cis cisplatin resistant cell line. Additionally, methyl- and fluoro-substituted formamidinate complexes showed comparable and increased cytotoxic activity in the OVCAR-3 cell line when compared to cisplatin. The low-valent metallodendrimers show some activity, but a general decrease in cytotoxicity was observed when compared to the precursor complexes in all but one case, which is where the more active acetate-derived metallodendrimer showed a lower IC50 value in the OVCAR-3 cell line in comparison with the dirhodium tetraacetate.Two diphenyl formamidine ligands, four dirhodium complexes, and three axially modified low-valent dirhodium metallodendrimers were synthesized and evaluated as anticancer agents against the A2780, A2780 The success of cisplatin and related platinum-based chemotherapies in the treatment of cancer has pioneered the development and study of various inorganic and organometallic compounds, such as cytotoxic agents . Metal-cConsequently, potential drug leads containing metals, such as rhenium, ruthenium and rhodium, have been at the forefront for development as anti-tumor agents with some success ,10,11,12Dirhodium complexes are of interest in this field of research and possess unique physicochemical properties, such as a metal–metal bonded binuclear core, the presence of vacant axial sites for further ligand occupancy, and the accessibility of a wider range of oxidation states when compared to mononuclear rhodium complexes . In the cis human ovarian cancer cell line, and the highly prolific OVCAR-3 human ovarian cancer cell line. These were then compared to the toxicity profiles of ligands ; the precursor complexes containing acetate and formamidinate bridging ligands (3–6); and the platinum-based drugs, cisplatin and carboplatin. The results reported in this work suggest that structural modifications of the dendritic molecules are required to improve their solubility in the biological medium. This would allow for accurate selectivity studies against healthy non-tumorigenic cell lines.Herein, we report the synthesis of three new axially modified low-valent dirhodium  metallodendrimers containing dirhodium complexes, bound via a single axial coordination site. The dendritic structure may allow for increased selective cellular uptake, alongside a multivalent drug delivery approach. The synthesized compounds were evaluated as potential antitumor agents against the A2780 human ovarian cancer cell line, the cisplatin resistant A27801, 2) and bimetallic dirhodium complexes (3–6) used in this study  evaluated in this study were obtained in good to excellent yields (76–84%). This was achieved by reacting the synthesized dirhodium complexes  with 1 equivalent of the pyridyl dendritic scaffold (7) in dichloromethane at ambient temperature (20–25 °C) for 6 h (8–10) were found to be air stable and sparingly soluble in DCM, chloroform, acetone, and DMSO.The known trivalent dendritic scaffold 7,  that is  for 6 h . Single 3, 7, and 8 in order to corroborate the axial site binding of the scaffold (7). The electronic spectra were collected in coordinating  and non-coordinating (DCM) solvents for comparison. The distinctive absorption maxima of both precursor compounds (3 and 7) were noted. They were noted to differ from the absorbances observed in the spectrum obtained for complex 8, which was recorded in DCM , relative to those observed for acetate compound 8, was attributed to the steric influence of the ligated diphenylformamidinate moeities that was present on the conformational arrangements of the dendritic arms, which was coupled with the enhanced solubility exhibited by complexes 9 and 10. In spite of the challenges regarding solubility, a 13C-NMR spectrum could be obtained for 9 in benzene-6d, and displays the expected signals pertaining to the interior scaffold and terminally bound dirhodium units (The formamidinate complexes 9 and 10 . The lacI) units .8–10 shows a shift in the pyridyl C=N absorption band from the 1610 cm−1 to 1586 cm−1 region, further supporting the coordination of the pyridyl nitrogen atom to the dirhodium core in each case  for complexes 8–10 reveals the base peaks corresponding at m/z = 1768.4221, m/z = 2072.4199, and m/z = 1294.4554, thereby corresponding to the [M + Na]+, ([M + 4(CH2Cl2) + 2K]2+) and ([M + 3Na]3+) ions for complexes 8, 9, and 10, respectively. The complex fragmentation patterns observed in the mass spectra of complexes 8 and 10 were attributed to the macromolecular monodispersed nature of the dendritic structures, allowing for multiple points of fragmentation upon ionization  over a 48 h period. No observable change in the electronic spectra  was notehe study .cis) and OVCAR-3 ovarian cancer cell lines , dirhodium complexes (3–6), and metallodendrimers (8–10). These results are presented in 50 values . The solubility of the compounds did not allow a testing of over 100 µM, and the calculation of the quantitative IC50 value was not possible for the selected compounds. The testing of 7 was not carried out due to a negligible solubility in the medium, which is required for biological evaluation. Nonetheless, the absorbance values and, hence, the cell survival showed a continuous leaning decrease. Therefore, the linear regression was applied in order to analyze the dose–effect relationship. Furthermore, the statistical significance  of the calculated hillslope clearly demonstrated a cell growth inhibitory tendency in the studied concentration range; this was observed for all compounds in the A2780 cell line . Ligand 1 showed no appreciable activity in the tested concentration range in the A2780 cell line, and the IC50 values for ligands 1 and 2 in the A2780cis and OVCAR-3 cell lines could not be determined in the tested concentration range of 0.1–100 µM, which serves as a confirmation by the linear regression (see The compounds show a similar outcome in A2780sion see .3 and 4)—with IC50 values of 45.65 and 52.57 µM, respectively—were found to favor the methyl-substituted complex (3). Interestingly, the application of complex 3 to the A2780cis cell line shows a marginally enhanced activity (43.25 µM) when compared to the A2780 cell line, as well as showed a greater cytotoxicity when compared to cisplatin (80.49 µM) or carboplatin (89.37 µM). Complex 4 showed no appreciable activity in the A2780cis cell line at the tested concentration range; however, its cytotoxicity was comparable to that of cisplatin, which was observed in the OVCAR-3 cell line. These results agree with previous literature findings, in which the interaction between dirhodium complexes and DNA may allow for a growth inhibition through multiple pathways 2+, calcd. 2072.4528). Elemental analysis for C204H213N31Rh6·DCM: Found C, 64.65, H, 5.94, N, 10.95%; calcd. C, 64.79, H, 5.68, N, 11.19%.Yield: 0.051 g, 76%. Melting point: Darkening onset at 120 °C, full decomposition without melting at 400 °C. FTIR  =3267 , 1590 , 1581 , 1195 . 1H NMR : δ (ppm) = 8.52 , 7.68 , 7.24 , 6.82 , 6.55 , 3.69 , 2.68 . MS(HR-ESI)(m/z): 1294.4554 . Elemental analysis for C180H141F24N31Rh6·3H2O: Found C, 55.96, H, 3.72, N, 10.78%; calcd. C, 55.93, H, 3.83, N, 11.05%.Yield: 0.046 g, 84%. Melting point: 397.7–398.5 °C. FTIR . The A2780 and A2780-cis cells were cultured in RPMI-1640 cell culture media, which were supplemented with 10% fetal bovine serum; OVCAR-3 and 20% fetal bovine serum; and 0.01 mg/mL insulin from a porcine pancreas . For cytotoxicity testing, the cells were seeded on 96-well Nunclon Delta surface assay plates ; in addition to in wells containing 190 µL cell suspensions. The compounds 1–6 and 8–10 were diluted in dimethyl sulfoxide  to obtain 10 mM stock solutions, and serial dilutions were prepared using a 0.01M PBS solution in order to obtain concentrations in the range of 2–2000 µM. Cisplatin and carboplatin were received from Actavis, through Sindan-Pharma Srl, Bucharest, Romania. The platinum-based drugs were diluted in a physiological serum, and serial dilutions were made in PBS to obtain the same concentration range as for Rh2 compounds at 2–2000 µM. For testing, each well containing cells was treated with the respective compound in a proportion of 1:20 (compound: cell culture media) in order to obtain a final concentration range of 0.1–100 µM for the compounds in the cell culture media. In every well, a 10µL compound was pipetted and the plates were incubated for 24 h, in triplicate. Untreated cells were used as a reference for 100% cell growth. In addition, the cell culture media without cells were used as a blank, and wells filled with cell culture media and the serial dilutions of each compound were used as color control; three independent measurements were performed. To assess the cytotoxicity of the compounds, the colorimetric MTT -2,5-diphenyltetrazolium bromide) reduction assay was performed [The in vitro study was performed on three ovarian cell lines: the A2780 platinum sensitive ovary carcinoma, the A2780erformed . Briefly2–6 show a cytotoxicity that was greater than or comparable to carboplatin in the A2780 cell line; none of which, however, exceeded the cytotoxicity of cisplatin in this cell line. Compounds 3 and 5 showed an approximate two-fold increase in cytotoxicity in the A2780cis cell line when compared to cisplatin and carboplatin, whereas compounds 4 and 5 show the highest antiproliferative effect of the synthesized compounds in the OVCAR-3 cell line. Generally, the results for dendritic complexes (8–10) show a decrease in cytotoxicity when compared to the bimetallic precursor complexes, with acetate-containing metallodendrimer (8) displaying the greatest antiproliferative effect for the macromolecular series of compounds. Compound 5 shows promising cytotoxicity against highly proliferative human ovarian tumor cell lines in vitro, having a comparable to, or greater, activity in comparison to the tested platinum-based drugs, with similar efficacy observed across all tested cell lines. Further investigations are underway to improve on the design strategy and fine-tuning of the physicochemical properties toward the investigation of the mechanisms of action on which these complexes exert their influence, thus their selectivity for cancer cells over healthy cells may then be determined.In summary, new trivalent metallodendrimers bearing dirhodium complexes bound on the periphery were prepared in good yields (76–84%); they were characterized by various spectroscopic and analytical techniques. The dirhodium compounds


Embryonic stem cell (ESC) derivation from single blastomeres of 8-cell mouse embryos results in lower derivation rates than that from whole blastocysts, raising a biological question about the developmental potential of sister blastomeres. We aimed to assess the ability of 8-cell blastomeres to produce epiblast cells and ESC lines after isolation, and the properties of the resulting lines. Our results revealed unequal competence among sister blastomeres to produce ESC lines. At least half of the blastomeres possess a lower potential to generate ESCs, although culture conditions and blastomeres plasticity can redirect their non-pluripotent fate towards the epiblast lineage, allowing us to generate up to seven lines from the same embryo. Lines originated from the same embryo segregated into two groups according to their transcriptional signatures. While the expression of genes related to pluripotency and development was higher in one group, no differences were found in their trilineage differentiation ability. These results may help to improve our understanding of the ESC derivation process from single blastomeres and cell fate determination in the preimplantation mouse embryos. Embryonic stem cells (ESC), traditionally derived from the inner cell mass (ICM) of blastocysts , can alsCurrently, the main limitation of using single blastomeres as a source of ESCs is the decreased derivation rate compared to the use of whole embryos, particularly when blastomeres of 8-cell embryos are used. Because ESCs are known to derive from the pluripotent epiblast (EPI) of the preimplantation embryo , this raDuring preimplantation development, blastomeres gradually lose their developmental potential as cleavage progresses, and eventually differentiate into three separate lineages. It is widely accepted that the formation of the ICM and TE in the blastocyst is the first lineage segregation in the mammalian embryo , followeAlthough heterogeneities among blastomeres in cleavage-stage embryos could play a role in cell fate commitment during normal development, it is known that preimplantation embryos are able to adapt to experimental perturbations such as the addition or removal of cells  because The aim of this study was to determine whether, under optimized culture conditions that promote pluripotency maintenance and expansion of the EPI cell population, all blastomeres of the 8-cell stage mouse embryo are able to generate mESCs lines, and whether the lines originating from the same embryo have similar characteristics or whether they differ depending on the blastomere from which they originated. To this aim, we first examined the potential of individual sister blastomeres of 8-cell embryos to develop into mESCs lines and precursor cell aggregates containing pluripotent EPI cells. Next, we compared the transcriptional profile of the mESCs lines derived from blastomeres of the same 8-cell embryos and their ability to differentiate into cells of the three primary germ layers. Our findings will help to improve the generation of ESCs from single blastomeres and to better understand the developmental potential of single blastomeres of 8-cell stage embryos and the determination of cell fate during preimplantation embryo development.Departament d’Agricultura, Ramaderia, Pesca i Alimentació of the Generalitat de Catalunya .Mouse care and procedures were conducted according to the protocols approved by the Ethics Committee on Animal and Human Research of the Universitat Autònoma de Barcelona and by the 2 at −196°C. Cryopreservation allowed us to optimize the use of donor females and to work with the exact number of embryos required for each experimental replicate regardless of superovulation success.Mouse embryos were obtained from 6 to 12 weeks old B6CBAF1/J females (Charles River Laboratories). Females were induced to superovulate by intraperitoneal injection of 5 IU Pregnant Mare’s Serum Gonadotropin  followed by the injection of 5 IU Human Chorionic Gonatropin (Divasa-Farmavic) 48 h later and mated with B6CBAF1/J males. Oviducts were flushed with HEPES-buffered KSOM medium (H-KSOM)  48 h lat® KSOM Medium (Merck Millipore) under mineral oil (Sigma) at 37°C and 5% CO2, and biopsied when they reached the 8-cell stage, as previously reported , as previously reported .3 U/mL leukemia inhibitory factor  and R2i inhibitor cocktail, a combination of MEK inhibitor PD0325901  and TGFβ inhibitor SB431542 . During the first week of culture, 0.1 mg/mL adrenocorticotropic hormone  was also added to the medium.Mouse ESC derivation medium (KSR + R2i) consisted of DMEM high glucose (BioWest) supplemented with 100 μM 2-β mercaptoethanol (Gibco), 1x non-essential amino acids (Gibco), 50 U/mL penicillin and 50 μg/mL streptomycin (Biowest), 20% KSR (Gibco ThermoFisher), 102 for 7–10 days, and the medium was changed every 48 h until outgrowths were observed. After the first passage, the outgrowths were cultured in 4-well plates without ACTH. Putative mESCs lines were passaged once or twice a week using trypsin-EDTA (BioWest) and maintained in culture for at least six passages.Blastomeres were cultured at 37°C and 5% COThe stemness of all putative mESCs lines was assessed at the seventh passage by Alkaline Phosphatase Activity (ALP) staining and immunodetection of pluripotency and differentiation markers.ALP activity was determined using a commercial kit (Sigma AB0300) and a previously reported protocol . Brieflyin vitro differentiation of the putative mESCs lines to confirm their pluripotency and differentiation potential, respectively. Spontaneous differentiation was achieved by culturing s lines in DMEM high glucose supplemented with 10% fetal bovine serum  under feeder-free conditions for 10 days. Cells were fixed in 4% PFA for 20 min at RT, and blocked and permeabilized with a PBS solution containing 0.2% sodium azide (Sigma), 0.5% Triton X-100 (Sigma) and 3% goat serum (BioWest) for 30 min at 37°C. The fixed cells were incubated with primary antibodies overnight at 4°C, washed, and incubated with secondary antibodies for 2 h at RT.Immunofluorescence staining was performed both before and after spontaneous α smooth muscle actin  and mouse monoclonal anti-alpha-fetoprotein  antibodies were used to detect ectoderm, mesoderm, and endoderm differentiation markers, respectively. The secondary antibodies used were anti-mouse IgG Alexa Fluor 488  for OCT4, TUJ1, αSMA and AFP, and anti-rabbit IgG Alexa Fluor 594  for SOX2 and NANOG.Mouse monoclonal anti-OCT4 , rabbit polyclonal anti-NANOG  and rabbit polyclonal anti-SOX2  antibodies were used to detect the pluripotency markers. Mouse monoclonal anti-Tubulin β3 , mouse monoclonal anti-Finally, the cells were mounted with 10 μg/mL Hoechst 33258  diluted in Vectashield (Vector Laboratories) and analyzed using an epifluorescence microscope (Olympus BX61) and Cytovision software .2 for 72 h to allow the formation of cell aggregates. Whole 8-cell embryos previously incubated in acidic Tyrode’s solution to remove the zona pellucida were also cultured under the same conditions and used as controls.Single blastomeres were seeded on a monolayer of inactivated HFF (iHFF) in KSR + R2i medium, as detailed for the derivation of mESCs, and incubated at 37°C and 5% COCell aggregates were fixed and immunostained following the same protocols described above for mESCs. Mouse monoclonal anti-OCT4 and rabbit polyclonal anti-NANOG were used as primary antibodies for the detection of ICM and EPI cells, respectively. The secondary antibodies used were anti-mouse IgG Alexa Fluor 488 for OCT4 and anti-rabbit IgG Alexa Fluor 594 for NANOG.Finally, the cell aggregates were mounted with 10 μg/mL Hoechst 33258 diluted in Vectashield. The number of total cells (Hoechst staining), OCT4+ cells and NANOG + cells were counted using an epifluorescence microscope (Olympus BX61) with the Cytovision and ImageJ software.2. After this short incubation, feeder cells attached to the plates whereas mESCs remained in solution.Feeder cells were removed from mESCs cultures using the protocol described by Total RNA was extracted from mESCs using the Maxwell RSC SimplyRNA Tissue Kit (Promega). Its concentration and quality were assessed using a NanoDrop spectrophotometer (ThermoFisher Scientific).® RNA BR Assay kit (Thermo Fisher Scientific), and RNA integrity was estimated using an Agilent RNA 6000 Pico Bioanalyzer 2,100 Assay (Agilent). All the samples used in the study had RNA integrity numbers (RIN) above 9.Two micrograms of total RNA obtained from each mESC line was sent to the Centre Nacional d’Anàlisi Genòmica (CNAG) in Barcelona (Spain) for RNA sequencing (RNA-seq). The samples were quantified using a QubitRNA-Seq libraries were prepared with the KAPA Stranded mRNA-Seq Illumina Platforms Kit (Roche) following the manufacturer´s recommendations. Briefly, 500 ng of total RNA was used for poly-A fraction enrichment with oligo-dT magnetic beads following mRNA fragmentation. The strand specificity was achieved during the second strand synthesis performed in the presence of dUTP instead of dTTP. The blunt-ended double stranded cDNA was 3′adenylated and Illumina platform compatible adaptors with unique dual indexes and unique molecular identifiers (Integrated DNA Technologies) were ligated. The ligation product was enriched using 15 PCR cycles. The size and quality of the libraries were assessed using a High-Sensitivity DNA Bioanalyzer assay (Agilent).The libraries were sequenced on a NovaSeq 6,000 (Illumina) with a read length of 2 × 51 bp, following the manufacturer’s protocol for dual indexing. Image analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 3.4.4).Mus musculus reference genome (GRCm39) using STAR software version 2.7.8a  enrichment analysis using gProfileR v07.0  plot was generated with the rlog transformed counts using the top 500 most variable genes and the ggplot2 R library . Heatmapin vitro differentiation into cells of the three germ layers, mESCs lines were subjected to embryoid body (EB) formation following the protocol described by 2. Every 2 days, 100 µL of medium was removed and 100 µL of fresh differentiation medium was added to each well.To assess the potential for Oct4, Nanog, and Rex1), endoderm lineage genes , mesoderm lineage genes  and ectoderm lineage genes  by quantitative PCR (qPCR).After 7 days, 20-30 EBs per mESCs line were collected to analyze the expression of pluripotency genes .The qPCR reactions were performed in triplicate on a CFX3846 Real-Time System thermocycler (Bio-Rad) using iTaq universal SYBR Green Supermix (Bio-Rad). The amplification program consisted of an initial denaturation step of 3 min at 95°C, followed by 40 cycles of 10 s at 95°C (denaturing) and 30 s at 60°C . An additional thermal denaturing cycle was performed to obtain the melting curve of the qPCR products.Oct4 , Rex1  and Nanog (qMmuCID0005399) were used to assess pluripotency. Afp (qMmuCID0023288), Foxa2 (qMmuCED0048428) and Ttr (qMmuCID0006861), T-Brachyury (qMmuCID0015169), Nkx2-5 (qMmuCID0020858) and Kdr (qMmuCID0005890), and Nes (qMmuCID0023067), Pax6 (qMmuCID0026137), and Fgf5 (qMmuCID0008576) were used to assess differentiation to endoderm, mesoderm and ectoderm lineages, respectively. A no-template control (NTC) was added for each primer and the results were normalized to Gapdh (qMmuCED0027497) expression. Additionally, the relative expression values in EBs samples were normalized to their respective undifferentiated ESC lines. The cycle quantification value  was determined for each sample using BioRad CFX Maestro™ 195 software, and relative expression was calculated using the ΔΔCq method.Validated commercial PrimePCR SYBR Green Assays (BioRad) for t-test or a Mann-Whitney test. All the statistical analyses were performed using the GraphPad Prism 8 software and p < 0.05 was considered as statistically significant.Aggregate formation rates were analyzed with Fisher’s exact test. The mean numbers of total, OCT4+ and NANOG + cells and the mean ratios of OCT4+/total cells, NANOG+/total cells and NANOG+/OCT4+ were first analyzed using the Shapiro-Wilk test to check the normality of the samples and then compared using either an unpaired Mean values are indicated in the text, tables, and figures as mean ± SEM.in vitro spontaneous differentiation for 10 days) . We obtained a total of 292 mESCs lines (29%), whose stemness was confirmed at the seventh passage by morphological criteria (colonies with rounded morphology and defined edges), pluripotency criteria  activity and OCT4, SOX2 and NANOG markers), and differentiation criteria  .Of the total embryos, 84.1% (106/126) formed at least one mESCs line. Most of the embryos (97/126) gave rise to one to four lines, but two embryos were able to produce seven lines . These rTo assess whether the failure of some blastomeres within each embryo to produce mESCs lines was related to their lack of pluripotent potential, we next examined the ability of single blastomeres to develop into cell aggregates containing pluripotent EPI cells. Cell aggregates are the precursors of outgrowths, and because of their higher formation efficiency and earlier manifestation in the derivation process, their formation could be a better marker of blastomere developmental potential.n = 15) were singly extracted by micromanipulation  were able to develop into cell aggregates containing pluripotent EPI cells after 72 h of culture. This ability was reduced in single blastomeres, as only 62.5% of the initial blastomeres (75/120) developed into aggregates containing OCT4+ and NANOG + cells, even though 72.5% (87/120) of the blastomeres formed aggregates with OCT4+ cells . It mustWhen comparing the mean ratios of OCT4+/Total cells, NANOG+/Total cells and NANOG+/OCT4+ cells between the aggregates derived from single blastomeres  and those derived from control 8-cell embryos , statistically significantly differences were found only in the NANOG+/Total cells ratio .Altogether, these results suggest that single blastomeres isolated from 8-cell embryos have the same potential as whole 8-cell embryos to develop into cell aggregates, but the specification of pluripotent EPI is slightly impaired.Next, we sought to determine how the potential to develop into cell aggregates containing pluripotent cells was distributed among the different blastomeres of the 8-cell embryo. For this purpose, the results obtained from single blastomeres were grouped according to their parental embryos.The number of single blastomeres per embryo that developed into a cell aggregate after 72 h in culture ranged from 3 to 8, with a mean number of 6.5 ± 0.3 . Most emThe sum of aggregates formed by the sister blastomeres of the same parental embryo had an average total number of 190.5 ± 16.8 cells (222.8 ± 15.9 if only embryos forming seven to eight aggregates are considered), a value significantly higher than the average total number of cells in control 8-cell embryo-derived aggregates (95.2 ± 10). These results indicate that when blastomeres from the same embryo are isolated and cultured separately, they can generate a higher number of cells than when they remain together.The mean number of aggregates per embryo that were positive for both OCT4 and NANOG was 5.0 ± 0.4, ranging from 3 to 7 . Thus, tThe number of OCT4+ and NANOG + cells in the cell aggregates was heterogeneous and showed large variation both among sister blastomeres and among parental embryos . The numFinally, given the variation in the number of pluripotent EPI cells among sister blastomeres and that only 29% of the blastomeres were able to produce an mESCs line in the first set of experiments, we tried to estimate the minimum number of NANOG + cells required for the successful establishment of an mESCs line from single blastomeres. To do this, we classified the cell aggregates formed by all the individual blastomeres according to the number of NANOG + cells they contained and selected the 29% upper range, assuming that mESCs lines would most probably be formed from cell aggregates possessing a higher number of NANOG + cells. This 29% upper range contained 5–18 NANOG + cells, indicating that single blastomeres should generate cell aggregates containing at least five NANOG + cells after 3 days in culture for them to be able to subsequently establish an mESCs line. Considering that in the first set of experiments the majority of the embryos  generated one to four mESCs lines, we next calculated the percentage of embryos in the second set of experiments that were able to form one to four aggregates containing the estimated minimal number of NANOG + cells (≥5). Interestingly, the percentage was very similar .To characterize and compare the molecular features of mESCs lines produced from different blastomeres of the same parental embryo in terms of their gene expression patterns, we selected the lines produced by embryos that had given rise to the largest number of mESCs lines in the first set of experiments. In particular, five mESCs lines established from one embryo and seven mESCs lines established from two other embryos  were selected and subjected to RNA sequencing (RNA-seq) . Two intTo reveal possible transcriptional variations among blastomeres-derived mESCs other than those related with the parental embryo of the blastomeres, between-embryo variability was corrected and a new PCA was performed. This new analysis revealed that lines segregated into two clusters (Groups I and II) and, interestingly, half of the lines from each embryo were allocated to each cluster , except Next, a WGCNA was performed on the embryo-adjusted gene counts, in which mESCs line 2.2 was shown to cluster together with lines in group II. Using this analysis, we identified five clusters of highly correlated genes  , which aFinally, to visualize how the two groups of mESCs lines differed in their pluripotency potential, we produced a heatmap of the differential expression of several core, naïve and primed pluripotency genes . Lines iin vitro differentiation for EB formation, and the expression of pluripotency , endoderm lineage , mesoderm lineage  and ectoderm lineage  marker genes was measured by qPCR on the EBs formed. All but one of the mESCs lines  were able to form EBs. Heterogeneity in the expression of these markers was evident among mESCs lines, regardless of the group they belonged to or the parental embryo they originated from  upon differentiation, when compared with mESCs lines from group II, no biased differentiation towards a specific lineage was found among the mESCs lines of the two groups.Finally, we sought to determine whether the transcriptional differences observed between the mESCs lines generated from single blastomeres (groups I and II) would create a bias in their ability to form cells of the three developmental germ layers. To this end, the same blastomere-derived mESCs lines used in the RNA-seq analyses were subjected to spontaneous ted from . Althougn = 126) and blastomeres (n = 1.008) and by keeping track of the parental embryo of each blastomere. We first analyzed the potential of sister blastomeres of 8-cell embryos to produce mESCs lines and cell aggregates containing pluripotent cells (as precursors of mESCs lines). To maximize the development of blastomeres into cell aggregates, outgrowths and mESCs lines, isolated blastomeres were cultured on feeder cells and in the presence of LIF and R2i in the culture medium  was similar to the 85.3% derivation rate obtained by our group from whole blastocysts of the same mouse strain . NonetheThe large variation in the number of mESCs lines obtained per embryo (0-7) suggests that blastomeres from 8-cell stage embryos are not equally competent at establishing mESCs lines and that the number of competent blastomeres is not uniformly distributed among embryos. Nonetheless, among the embryos that produced at least one mESCs line, those in which the mESCs generation potential was restricted to half or less of the blastomeres were much more abundant (91.5%) than those in which more than 50% of the blastomeres produced an mESCs line (8.5%). To the best of our knowledge, no similar studies have been performed with 8-cell embryos, but One of the limiting factors during mESCs establishment from whole blastocysts is the rapid loss of pluripotent EPI cells, the progenitors of mESCs, during the first few days of culture . Therefon = 72) compared with our study (n = 1.008), rather than to the use of ideal experimental conditions for the maintenance of blastomere developmental potential.Several studies have shown that blastomeres isolated from most precompaction embryos differ in their ability to form TE, EPI, and PE cells, indicating their unequal developmental potential . A develIn addition to this biological constraint, the plasticity of embryonic development and the presence of signaling modulators in the derivation medium should also be considered when interpreting the results of our study. Blastomeres of 8-cell embryos show impaired ability to form pluripotent EPI cells after isolation and culture in standard embryo culture medium, and they tend to develop as trophoblastic vesicles instead of forming blastocysts . This coNext, we were interested in determining whether mESCs lines produced from different blastomeres of the same embryo are equivalent in terms of their transcriptional signature. To the best of our knowledge, only one study has addressed this question, using mESCs derived from single blastomeres of 8-cell human embryos . We founversus the naïve pluripotency of mESCs lines. Moreover, only two of the four 8-cell embryos used in this previous study had produced more than two lines, and the other two embryos had resulted in only one line.When we removed between-embryo variability, differences at the transcriptional level between mESCs lines derived from the same embryos became apparent, and interestingly, the lines consistently segregated into two distinct groups. When compared with the lines in group II, the lines in group I displayed upregulation of genes involved in developmental and morphogenesis processes and in signaling pathways associated with stem cell pluripotency, and of core and naïve pluripotency genes. This suggests that half of the mESCs lines generated from each embryo possess a higher pluripotency potential than the other half. Because the derivation of mESCs lines is a long process, it is not possible to completely rule out that differences in the lines’ transcriptomes were acquired during the generation of the line and extended culture. However, the fact that the same pattern was observed in all three parental embryos and that a high number of lines were analyzed for each embryo (5-7), rather suggests that these differences stem from the blastomere that the lines originated from and were maintained during derivation and culture. It is tempting to speculate that lines in group I could have been established from the blastomeres that in the intact embryo possessed the highest developmental potential, whereas lines in group II could derive from those blastomeres with lower developmental potential in which redirection of cell fate towards the EPI lineage after isolation and culture successfully occurred. Because most of the lines used in the transcriptome analysis were obtained from two embryos in which 7 of the 8 blastomeres produced mESCs lines, this reinforces the idea that in the intact 8-cell stage embryo about half of the blastomeres would belong to each of these two categories. It would be interesting to investigate transcriptional differences among sister blastomeres of the 8-cell embryo in future studies to corroborate this idea. Finally, despite the higher pluripotency of mESCs lines belonging to group I and the upregulated expression of genes related to nervous system development in lines belonging to group II, the two groups of lines did not show biased differentiation into any of the three primary germ layers. Terminal differentiation into derivatives of the three germ layers could be performed in future experiments for a more definitive assessment of whether a correlation exists between the transcriptional differences between the two groups of lines and their differentiation potential.In summary, we have demonstrated that it is possible to obtain multiple mESCs lines from the blastomeres of a single 8-cell embryo, but that blastomeres at this developmental stage are unequally competent at producing mESCs lines. We estimated that this competence is related to their ability to produce at least five EPI cells during the first 3 days of culture. Our results also suggest that the current derivation rates probably reflect a combination of three factors: biological constraints, developmental plasticity, and culture conditions. Although in most embryos up to half of the blastomeres would have a higher developmental potential , isolation and culture of the blastomeres could reduce this potential. This could be prevented by culture conditions, such as the use of R2i, which could also allow to “rescue” those blastomeres with reduced developmental potential, by redirecting their cell fate to the EPI lineage. It is possible that the two distinct types of transcriptional signatures identified in the mESCs lines produced from the same embryos reflect the intrinsic developmental potential of the parental blastomeres from which they originated. However, these two different transcriptional signatures did not affect the ability of the lines to differentiate into cells of the primary germ layers. Our findings may help improve the generation of mESCs from single blastomeres and the understanding of cell fate determination in the preimplantation mouse embryo.


O-lkyl naringenin derivatives and their oximes exhibit antimicrobial activity against antibiotic-resistant pathogens. The current study was aimed at determining the modulatory effect of these compounds on the adhesion of selected representatives of the intestinal microbiota: Escherichia coli, a commensal representative of the intestinal microbiota, and Enterococcus faecalis, a bacterium that naturally colonizes the intestines but has disease-promoting potential. To better reflect the variety of real-life scenarios, we performed these studies using two different intestinal cell lines: the physiologically functioning  3T3-L1 cell line and the disease-mimicking, cancerous HT-29 line. The study was performed in vitro under static and microfluidic conditions generated by the Bioflux system. We detected the modulatory effect of the tested O-alkyl naringenin derivatives on bacterial adhesion, which was dependent on the cell line studied and was more significant for E. coli than for E. faecalis. In addition, it was noticed that this activity was affected by the concentration of the tested compound and its structure (length of the carbon chain). In summary, O-alkyl naringenin derivatives and their oximes possess a promising modulatory effect on the adhesion of selected representatives of the intestinal microbiota.Because of the close connection between adhesion and many vital cellular functions, the search for new compounds modulating the adhesion of bacteria belonging to the intestinal microbiota is a great challenge and a clinical need. Based on our previous studies, we discovered that  Cell adhesion is a phenomenon of paramount importance in various physiological processes. It affects a plethora of physiological functions of cells, e.g., growth, differentiation, and migration. Interactions during adhesion can occur either on the cell-to-cell or cell-to-extracellular matrix  pathway, which for eukaryotic cells is essential for the development of tissues, organs, and even whole organisms . Non-spe13–1014 cells , being a concentration able to induce 50% inhibition of cell proliferation. The obtained data are presented in NG) and naringenin oxime (NGOX).In the next step, we decided to determine whether the tested naringenin derivatives negatively affect the viability of the examined intestinal cell lines. The cytotoxic effect of 50 value for these two model compounds was >100 µg/mL.Naringenin and its oxime showed no proliferative effects against both cell lines, 3T3-L1 and HT-29, over the concentration range tested. The ICO-alkyl derivatives of naringenin, e.g., 1a, 3a, 5a, 7a, and 9a. The same compounds, with the exception of 9a, also showed antiproliferative activity against the HT-29 line. It is interesting to note that the cytotoxic effect was observed only for compounds having one carbon chain attached at the C-7 position of the naringenin ring. Furthermore, a correlation between the number of carbon atoms and the level of cytotoxic activity was observed. Indeed, the shorter the attached carbon chain, the greater the effect of the compound on the proliferation of the adenocarcinoma cells examined. In fact, the IC50 value for compound 1a, which contains a hexyl group attached to the C-7 position of naringenin, was 25.39 µg/mL, which was 1.8 times lower than compound 7a (IC50 = 46.72 µg/mL), which possesses a nonyl moiety.Among the tested naringenin derivatives lacking an oxime group, cytotoxic activity against the 3T3-L1 line was demonstrated for monosubstituted 9a having 10 carbon atoms). Naringenin derivatives with two alkyl chains attached (the second at the C-4’ position) with different numbers of carbon atoms  showed no effect on the proliferation of the tested cell lines in the tested concentration range.In contrast, against the 3T3-L1 line, compounds having chains with 6 to 9 carbon atoms showed an effect on proliferation at a level 3–4 times higher than compounds with longer chains , an effect on 3T3-L1 cell proliferation was also noted only for monosubstituted compounds, e.g., 1b, 3b, 5b, and 7b. For them, IC50 values were in the range of 32.12 µg/mL to 49.93 µg/mL. A similar relationship was observed for the human adenocarcinoma cell line because the same compounds  showed cytotoxic effects against this cell line. The IC50 value depended on the length of the attached chain and was higher for the oxime derivatives with a longer alkyl chain attached to the naringenin ring. On the other hand, both 4b and 10b, oximes of O-alkyl naringenin derivatives with attached alkyl chains at the C-7 and C-4’ positions, affected cell differentiation of the HT-29 line with comparable IC50 values of 43.35 and 48.23 µg/mL, respectively. In contrast, the other di-substituted compounds, e.g., 2b, 6b, and 8b, showed no harmful effects against human colon cancer cells in the concentration range tested (IC50 > 100 µg/mL).In the case of naringenin derivatives bearing an additional oxime group  as well as our detailed review of the literature indicating the highest biological activity of flavonoids with a C8–C10 side chain ,31,32,338b at 50 µg/mL had a very strong, pro-adhesive effect on E. coli with respect to both cell lines tested. Here, for the physiological 3T3-L1 cell line, the ratio of the area of bacterial biomass (BB) adhered to the surface of the eukaryotic cells (EC) [the BB/EC ratio] was 0.6 ± 0.12 vs. 0.37 ± 0.13 for compound-exposed and control samples, respectively   without a negative, dysbiotic effect on other representatives of the microbiota .Applying microfluidic conditions, we observed that the use of ectively . For theectively . Interesll line) . RepreseNaringenin is an organic flavonoid compound that is widely distributed in nature. The largest source of naringenin is citrus fruits , although it is also present in smaller amounts in grapes, cherries, fenugreek, and Greek oregano, as well as in coffee, tea, and red wine. The literature review confirms the broad-spectrum, health-promoting effect of this flavonoid on the human body. It has been shown that such activity is closely related to the affinity of naringenin for scavenging reactive oxygen species and increasing the antioxidant defense of the host. This strictly translates into its anti-inflammatory effect and determines its anti-atherosclerotic and neuroprotective features. Additionally, it has been documented that naringenin also has anti-microbial and anti-cancer activity ,28.E. coli adhesion to eukaryotic cells and the decrease in cell membrane fluidity of these bacteria. In another study carried out on four selected bacterial species, it was shown that, together with the entry into the biofilm (sedentary) phase, microorganisms accumulate saturated fatty acids in their cell membranes, leading to a decrease in their membranes’ fluidity  containing one of the tested bacterial strains  and a selected flavonoid, 8b (50 µg/mL), was added to the inlet wells. In the control sample, the above compound was not added (the cell line medium [without antibiotics] together with bacteria). In regard to this, in both tested and control samples, the cell line-to-bacteria ratio was maintained at MOI = 50. After this, the medium was set to flow from inlets to outlets at 0.5 dyne/cm2 for 4 h. In the next step, the inlet wells were emptied, and 100 µL of saline solution containing a mix of two fluorescent dyes, SYTO9  to visualize bacterial biomass and DAPI , to detect eukaryotic cells, was added to each well. The inlet-to-outlet flow was turned on at 0.5 dyne/cm2 for 10 min, followed by a 0.5 h incubation to induce the absorption of the dyes by cells. After completing this stage, photographs of the microcapillaries containing the tested biological materials were taken using an inverted fluorescence microscope  and the Bioflux Montage software . When determining the area occupied by bacterial cells adhered to the surface of eukaryotic cells, the entire bacterial biomass attached was taken into account . These calculations did not include bacterial cells adhering only to the surface of the microcapillaries. The tests were performed in three biological repetitions with three technical replications (constituting images from different fragments of the examined microcapillaries).Determination of the impact of a dynamic medium flow on the adhesive capacity of bacteria was performed using the Bioflux 1000Z  with a coupled microscope environmental chamber , enabling the maintenance of 37 °C and microaerophilic conditions. At the very beginning, all channels of dedicated microfluidic plates  were flushed with 100 µL of αMEM or DMEM high-glucose medium with supplements  applying an intensive flow of 10 dyne/cmt-test was further used. The results of statistical analyses were considered significant for values with p < 0.05.Statistical analysis was performed using GraphPad Prism version 9 . The normality of the distribution was checked by the Shapiro–Wilk test. As all values were normally distributed, Student’s O-alkyl derivatives of naringenin was focused not only on different intestinal cell lines  but also on both static and microfluidic conditions. Therefore, the promising results obtained here provide a basis for in vivo animal studies to clarify the effect of these bioflavonoids on the gut environment. This may provide valuable information on the bioavailability and effect of these compounds on the whole intestinal microbiome.The present study showed that the synthesized naringenin derivatives modulate the adhesion of selected intestinal microbiota to the tested cell lines. It was noticed that this activity was affected by the concentration of the tested naringenin derivatives and their structure (length of the carbon chain). To better reflect the variety of real-life scenarios, the current research on


Once validated, these models can aid in the design of experiments that steer evolution with density-dependent selection.Many cancer cell lines are aneuploid and heterogeneous, with multiple karyotypes co-existing within the same cell line. Karyotype heterogeneity has been shown to manifest phenotypically, thus affecting how cells respond to drugs or to minor differences in culture media. Knowing how to interpret karyotype heterogeneity phenotypically would give insights into cellular phenotypes before they unfold temporally. Here, we re-analyzed single cell RNA (scRNA) and scDNA sequencing data from eight stomach cancer cell lines by placing gene expression programs into a phenotypic context. Using live cell imaging, we quantified differences in the growth rate and contact inhibition between the eight cell lines and used these differences to prioritize the transcriptomic biomarkers of the growth rate and carrying capacity. Using these biomarkers, we found significant differences in the predicted growth rate or carrying capacity between multiple karyotypes detected within the same cell line. We used these predictions to simulate how the clonal composition of a cell line would change depending on density conditions during  Cellular heterogeneity is a defining feature of most cancers, and it is critical to tumor progression and treatment failure ,2. AdvanOne contributor to a cancer cell’s phenotype includes large-scale somatic copy number alterations (SCNAs) of 10 mega base pairs or more. Studies show that SCNAs correlate with progression rates and overall survival, with cancer cells grouped by copy number landscape exhibiting the same resistance to chemotherapy ,5,6,7. THere, we aim to characterize a targeted subset of the phenotypic differences between co-existing karyotypes, which are defined by their ability to out-compete each other at low vs. high cell densities. Cell densities naturally vary in the tumor over space and time, thus creating niches with distinct selection pressures. A cell that is in a densely packed environment will be under more pressure to overcome contact inhibition than a cell that finds itself in sparse conditions. This variation in evolutionary pressures likely contributes to the coexistence of cancer cells with heterogeneous phenotypes. Density-dependent selection occurs when fitness is a function of population density . A relatWe present a framework for examining how density-dependent selection acts on coexisting clones defined by karyotypes and apply it to a set of stomach cancer cell lines. Hereby, we focus on carrying capacity as defined by spatial limitations rather than metabolic constraints.By sequencing the DNA and RNA of >36,000 cells from nine stomach cancer cell lines, our prior work classified cells into groups with unique karyotypes , which ain-vitro experiments.We changed the culture media every 3.05 days on average, with media changes becoming more frequent as cells became more confluent. We compared cell counts derived from a Countess (Life Technologies Countess II FL Automated Cell Counter) to cell counts derived from live cell imaging to confirm segmentation accuracy , thus suWe fit the Gompertz growth model and two instances of the generalized logistic growth model (Richards and Verhulst)  to this ll lines  Table A1ll lines . We thusll lines . Therefoll lines  Table A2r) included the ‘Amoebiasis during G2M’ , the ‘PI3K-Akt signaling pathway’  and carrying capacity (K), respectively.Overall, there was no significant correlation between the growth rate and the carrying capacity across cell lines , most phenotypic differences between karyotypes will be latent ,30, thusin-vivo framework. However, an understanding of the spatial distribution of r- and K-selected sub-populations could facilitate the design of therapy regimens, such as adaptive therapy or double bind therapy [Clones that are far from each other in r–K space may also be spatially segregated and have differential treatment sensitivities. For example, the slower growth rate of K-selected cells may render them more resistant to cytotoxic therapies —an effec therapy , aiming Our in silico results add to the evidence accumulating from many other studies ,29,37 th2) in order to allow for the population’s exponential growth and subsequent plateau at the carrying capacity (range: 2). The end-point for each experiment was determined by fitting a generalized logistic growth curve to the in-vitro data at various points during the time in culture. Across 8 cell lines, the time in culture ranged from 6–21 days, with media being refreshed on average every 48 h until the cells reached confluence, whereupon media was refreshed every 24 h. The experiment was stopped once the fold change in the inferred carrying capacity was less than 4% when removing any subset of the last 20% of time points.The identity of each cell line was determined through independent karyotyping and mycoplasma contamination assessment. Cells were cultured in their recommended media conditions at 37°C. For, HGC-27, EMEM ; KATOIII, RPMI-1640, and EMEM (1:1 mix) were used; for NCI-N87, RPMI-1640 (ATCC modified) was used; the remaining five cell lines  used RPMI-1640. All cell lines were grown in the aforementioned media with 10% fetal bovine serum  and 1% penicillin—streptomycin (Gibco). For cell passaging, Trypsin EDTA (0.25%) with phenol red (Gibco) was added, followed by inactivation using the respective growth media. Cells were seeded at a low initial density . Growth curves were generated to examine the carrying capability of each cell line.Cells were seeded in a T25 flask (Fisherbrand), and sets of 4 phase contrast images were taken at 20× magnification for NCI-N87, and 10× magnification for the aforementioned cell lines on an Evos FL. Segmentation using Cellpose was utilized to quantify the cell count and cellular features . Therefore, we applied pre-processing steps to our pipeline, which consisted of the following: (i) gamma correction to reduce darkness; (ii) histogram equalization to improve image contrast, and (iii) Gaussian blurring for smoothing.TV yes it shouldCell segmentation: Fully automated cell segmentation of phase contrast images was performed using Cellpose software.Cellpose is a deep learning approach based on the U-Net architecture for cell segmentation ,39, wherDetermining relative clonal carrying capacities, growth rates, and loss of contact inhibition parameter values is a necessary first step in modeling density-dependent selection in heterogeneous cell lines. To achieve this, we used gene expression signatures as surrogates of growth parameters as previously described ,41.Quantification of single cell pathway activity from gene expression:In order to identify cells with active gene sets, we utilized AUCell version 3.18 for R version 4.3.1 . AUCell Clonal growth parameters:r) and carrying capacity (K). The test takes the form of a linear model:r or K), and p is the pathway activity. The pathways are then ranked by adjusted We tested these 25 KEGG pathways quantified by AUCell as biomarkers of growth rate . However, even when many of these dependencies are held constant, it is difficult to discern between various models of cancer cell growth ,44,45,46dNidt=Ni∗in-vitro growth using the logistic growth model. To model clonal growth dynamics, we used the ODE45 solver for MatLab version 9.7.0.1216025 to solve Equation (In addition to the two models mentioned above, we also fitted cell line population growth with the Gompertz model . These mll lines  Table A1ll lines  and founll lines . Thus, wEquation  after ben clones identified within a given cell line. For each pair t-test to compare the growth rates predicted for cells assigned to clone x (y (p-value r and K, we further calculate Let