Background: Comparative transcriptomic analysis is a powerful approach for investigating the molecular mechanisms underlying various physiological and pathological processes, including liver disease. The liver is a vital organ with diverse functions, including metabolism and detoxification. In vitro models of liver cells, such as HepG2, Huh7, and Hep3B, have been widely used to study liver biology and pathology. However, there is limited information on the heterogeneity of these cell lines at the transcriptomic level. Objective: This study aimed to conduct a comparative transcriptomic analysis of three common liver cell lines  using publicly available RNA-sequencing data. In addition, we compared these cell lines to primary hepatocytes, cells isolated directly from liver tissue and considered the gold standard for studying liver function and disease. Methods: Our study included sequencing data with the following criteria: total number of reads over 20,000,000, average read length of over 60 base pairs, Illumina sequencing, and non-treated cells. The data for the three cell lines were compiled: HepG2 (97 samples), Huh7 (39 samples), and Hep3B (16 samples). We performed differential gene expression analysis using the DESeq2 package, principal component analysis, hierarchical clustering on principal components, and correlation analysis to explore the heterogeneity within each cell line. Results: We identified numerous genes and pathways differentially expressed between HepG2, Huh7, and Hep3B, such as oxidative phosphorylation, cholesterol metabolism, and DNA damage. We report that the expression levels of important genes differ significantly between primary hepatocytes and liver cell lines. Conclusion: Our study provides new insights into the transcriptional heterogeneity of commonly used liver cell lines and highlights the importance of considering specific cell line. Consequently, transferring results without considering the heterogeneity of cell lines is impractical and may lead to inaccurate or distorted conclusions. Human cancer cell lines have been extensively employed as in vitro models for cancer research and drug development. Cell lines can be used to study cell signaling, gene expression, and cell differentiation. For research outcomes to be reliably reproduced between laboratories, it is ideal that these cell lines exhibit genetic stability and cellular profiles. Unfortunately, several studies have highlighted various issues in human cancer cell lines: cell line misidentification, cross-contamination, and poor annotation that could impair the reproducibility of results obtained from these cell lines between laboratories. To authenticate cells, the researchers proposed short tandem repeat and single nucleotide polymorphism profiles. However, genetic changes during culturing and passage can affect gene expression and eventually cellular functions. In 2018, Ben-David et al. investigated 27 strains of the breast cancer cell line MCF7 and reported genetic heterogeneity and widely varied drug responses to 321 anti-cancer compounds [Immortalized liver cell lines are more commonly used in research than liver biopsies because of their low cost, ease of use, and retention of most key enzyme activities. To date, more than 40 hepatic lines, including HepaRG, Huh7, SK-Hep-1, Hep3B, and HepG2, are frequently used [Understanding the degree of heterogeneity within and between different liver cell lines is of utmost importance for researchers using these cells as a model system to study various biological processes, including drug metabolism, gene regulation, and liver disease. Despite their widespread use, the heterogeneity of liver cell lines can present challenges that may impact the reproducibility and interpretability of experimental findings.In this study, we performed a comparative transcriptomic analysis of three commonly used liver cell lines: HepG2, Huh7, and Hep3B. These cell lines were selected due to their popularity in research and the availability of publicly accessible RNA sequencing data. We aimed to explore the heterogeneity within each cell line and compare their transcriptomic profiles with each other. To achieve these goals, we used principal component analysis (PCA), hierarchical component clustering (HCPC), and correlation analysis to identify gene expression patterns that distinguished the cell lines from each other and within samples. Additionally, we utilized the DESeq2 package  to identIn order to investigate the heterogeneity of liver cell lines, we conducted a comprehensive analysis using publicly available data for HepG2, Huh7, and Hep3B cell lines downloaded from the NCBI SRA database. In addition, we incorporated our own data for HepG2 cells sequenced using Illumina NovaSeq 6000 with a read length of 100 base pairs (bp). We focused on control samples that represented the baseline cell lines in their natural, untreated state. This data is fundamental to comprehending cells and is crucial for discerning and evaluating any alterations that may transpire due to treatments, storage, or other influences.The data processing pipeline consisted of several key steps, including quality control, read trimming, alignment to the reference transcriptome, and quantification of gene expression. We performed the final step of the analysis in R, utilizing various statistical and bioinformatics tools such as the PCA, correlation analysis, differential expression (DESeq2 package), and functional enrichment analysis. The overall workflow of the experiment and a summary of data processing are presented in As a result of two transcriptomic profilings at the Institute of Biomedical Chemistry (IBMC), expression data were obtained for the HepG2 cell line in different years . The firAfter performing the quality control of the IBMC data, we defined criteria for published data from the Huh7, Hep3B, and HepG2 cell lines to be comparable in quality. The criteria were as follows: total number of reads >20,000,000; average read length >60 bp; non-treated cells; Illumina sequencing technology.Despite the fact that more than 10,000 RNAseq samples are available for liver cell lines in the NCBI SRA database, the total number of obtained samples matching our criteria makes it possible to compare only three cell lines—HepG2, Hep3B, and Huh7 . For theA quality check of the selected public data revealed that, on average, the number of fragments in cell line samples was 31 million reads, and the average read length was 128 bp. In contrast, the length of reads in the IBMC data averaged 100 bp, and the number of reads exceeded the average value in the published data and equaled 69 million. Information regarding the average data quality for each cell line is presented in As cell lines vary between laboratories, we performed statistical analyses to assess potential “laboratory effects”. This involved conducting correlation and PCA analyses and examining differentially expressed genes between the resulting clusters.Correlation analysis indicated a strong positive correlation between the expression levels of transcripts and genes in the HepG2 cell line samples. The average Spearman correlation coefficient between samples was 0.70 at the transcript level and more than 0.86 at the gene level . This suAdditionally, to identify the distribution of samples in the HepG2 dataset, we performed hierarchical clustering on principal components (HCPC) analysis. In this approach, gene expression data are first pre-processed and normalized, followed by the calculation of the principal components representing the main sources of variation in the data. A hierarchical clustering algorithm was then applied to the principal components.The analysis revealed that the HepG2 cell line data were divided into three clusters based on their transcript  and geneFinally, we used differential gene expression analysis to identify genes that were differentially expressed in the third cluster identified by HCPC analysis. The comparison results for each cluster pair are presented in The pathways that show differences between the clusters #1 and #2 in HepG2 cells are essential for energy production and proper mitochondrial function. These pathways include “The Electron Transport Chain (ETC) Oxidative Phosphorylation (OXPHOS) system in mitochondria” ”  between clusters #2 and #3. Differential expression analysis showed that in the #3 cluster, compared to the #1 and #2 clusters, there was a reduced expression of genes associated with pathways involved in drug metabolism and detoxification, such as CYP3A5, CYP20A1, SULT1A4, and SULT1A2.As a result of comparing clusters #1 and #3, we observed differences in the pathways—“The oxidation by cytochrome P450” (FDR = 0.01) and “Metapathway biotransformation phase I and II” (FDR < 0.001). The stability of these pathways is vital for cell lines because the liver is the main site of drug metabolism, and HepG2 cells are commonly used as a model system for studying the metabolism and toxicity of drugs and other xenobiotics [AHI1 and TMEM67 expression in the cluster #3 compared with that in the #1 and #2 clusters. These genes are involved in ciliary assembly, maintenance, and signaling pathways, and mutations in these genes are known to cause Joubert syndrome.Interestingly, in the analysis of cluster #1 vs #3 (FDR = 0.009) and #2 vs #3 (FDR = 0.03), changes in the pathway associated with Joubert syndrome were observed. Differential gene expression analysis showed increased The analysis of HepG2 cell line showed heterogeneity in several critical pathways, including the electron transport chain, oxidative phosphorylation, assembly of mitochondrial complexes I, IV, and III, cytoplasmic ribosomal proteins, retinoblastoma gene, and cytochrome P450 pathways. These pathways play a critical role in ATP production, maintenance of mitochondrial function, regulation of the cell cycle and proliferation, formation of ribosomal subunits, and drug metabolism. Changes in these pathways highlight the importance of careful transferring the results from one cell line to another.We observed a significant average Spearman coefficient between samples: at the transcript level, 0.7, and higher at the gene level, 0.85 . The resHCPC analysis showed that for the Huh7 cell line, five clusters were observed at the transcript level . At the In order to examine the affected pathways between the four clusters in more detail, we performed a differential gene expression analysis. The comparison of the results for each pair of clusters is presented in CDKN2A gene encoding the p16Ink4a protein and RB, are often altered in sporadic human cancer, contributing to the dysregulation of cell proliferation [RB1 gene, which encodes the RB protein, did not change between the clusters. At the same time, CDKN2A expression increased in cluster #2 compared to cluster #1 . The cytoplasmic ribosomal protein pathway (FDR = 0.02), which is responsible for cellular protein synthesis, is involved in ribosome production. DNA damage can potentially affect this pathway [Between the clusters #1 and #2, there were changes in the DNA damage-related pathway—“DNA IR-damage and cellular response via ATR” (FDR = 0.009) as DNA damage sensors in Huh7 cells [feration . The RB1 pathway .DHCR7 , DHCR24  have been downregulated. The HMGCS1 .The “Electron transport chain OXPHOS system in mitochondria” and “Mitochondrial complex I assembly model OXPHOS system” pathways associated with energy production in mitochondria were disrupted between the clusters. The disruption of these processes can lead to mitochondrial dysfunction and cell death. Thus, in the cluster #4, compared to the cluster #1 expression of the following genes was upregulated: In conclusion, our analysis revealed significant changes in various biological pathways in Huh7 cell clusters. The pathways associated with cholesterol metabolism, synthesis, and DNA damage are affected. In addition, changes in the pathways associated with mitochondrial energy production have been observed, which can lead to mitochondrial dysfunction and cell death. These results provide important information regarding the molecular mechanisms underlying the functional diversity of Huh7 cells and highlight the importance of accurately transferring results from one study to another.A strong and significant correlation was observed between the samples in the Hep3B cell line. The Spearman values at the gene expression level had an average of 0.88, whereas at the transcript level it was on average of 0.70. The correlation matrix is presented in We identified three clusters of Hep3B cells based on their transcripts  and geneFurthermore, to gain deeper insight into the affected pathways across the three clusters, we conducted an analysis of differential gene expression. The results of the comparison for each pair of clusters are displayed in CDK4 gene expression increases in cluster #2 compared to cluster #1  gene in cancer”, both involved in the regulation of the cell cycle and the DNA damage response, and their dysregulation can contribute to the development of cancer. The TP53  and “Retinoblastoma (RB) gene in cancer pathway” (FDR = 0.001). The key genes of the latter pathway, TCN2 gene, involved in the vitamin B12 pathway, was downregulated in cluster #3 compared to cluster #2  and “Vitamin B12 metabolism” (FDR < 0.001). The Hep3B cells originate from liver tissue, which is the main site for the synthesis of blood clotting factors. Between clusters #3 and #2, the “Blood clotting cascade pathway” changed (FDR < 0.001). The Hep3B cell line was used to evaluate the effects of coagulation factors on autophagy .Changes in the “Network map of the SARS-CoV-2 signaling pathway” (FDR = 0.001) between the clusters #3 and #2 can be explained by changes in the expression of genes associated with the metabolism of folic acid, vitamin B12, or selenium in these cells since SARS-CoV-2 may influence the activities of these pathways. Therefore, folic acid may be an antagonist of SARS-CoV-2; however, its effect on viruses remains unclear ,16.As a result of transcriptomic profiling of HepG2 cell line, on average, 14,609 protein-coding genes and 37,844 transcripts (TPM > 0.1) were expressed. The Huh7 cell line has 14,879 genes and 38,662 transcripts, while Hep3B has 13,940 genes and 35,864 transcripts (TPM > 0.1). We performed a correlation analysis to study the similarity between the liver cell lines. Spearman correlation values were high for both gene and transcript expression. At the gene level, the average levels of Spearman correlation for Huh7 and HepG2 samples were 0.95, while when comparing transcript expression, it was 0.92 . Similarhttps://www.ncbi.nlm.nih.gov/sra, accessed on 5 September 2022) by criteria: total first/second fragment count >20,000,000; average read length >60 base pairs (bp); non-treated cells; Illumina sequencing technology. The accession numbers for the experiments are provided in Liver cell lines were also compared with cell lines that originated from other tissue types: A549 (lung cancer), HeLa , and HEK293T (kidney cancer) as outgroups. The transcriptomic data for these cell lines were also searched in the NCBI SRA database  are liver cells that are isolated directly from tissue. In contrast, liver cell lines such as HepG2, Huh7, and Hep3B are derived from hepatoblastoma and hepatocellular carcinoma cells, respectively. Thus, comparison of hepatic cell lines with primary hepatocytes can provide insight into similarities and differences in gene expression and pathway activation between these cell types.https://www.ncbi.nlm.nih.gov/sra, accessed on 3 May 2023) according to the criteria specified above. For the primary hepatocytes, 31 samples were found. The accession numbers for the experiments are provided in The transcriptomic data for primary hepatocytes was searched in the NCBI SRA database  compared to primary hepatocytes (PHP). Dysregulation of the RB pathway is strongly associated with the development of cancer. Notably, key genes involved in this pathway, such as PPARA, is downregulated in all liver cell lines compared to PHP [Additionally, in all cell lines, the “PPAR signaling” pathway, which is involved in the regulation of lipid metabolism and glucose homeostasis, is affected. This pathway was found to play a crucial role in liver diseases, including non-alcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma . One of d to PHP . PPARs aFurthermore, to assess the differences between three liver cell lines and primary hepatocytes (PHP), we identified 752 common genes by combining all statistically significant differentially expressed genes between hepatic cell lines and PHP . Of notehttp://pantherdb.org/, accessed on 5 May 2023). Our analysis revealed that the genes were involved in several biological processes, including “Cell cycle”  by criteria: total first/second fragment count >20,000,000; average read length >60 base pairs (bp); non-treated cells; Illumina sequencing technology. Accession numbers for the experiments found are provided in The search of public RNAseq data was performed in the NCBI SRA database . We limited our analysis to protein-coding genes and did not include transcripts assigned to the non-standard chromosomes, resulting in 60,740 protein-coding transcripts and 19,670 genes.https://github.com/kevinblighe/EnhancedVolcano, accessed on 1 February 2023), and ClusterProfiler [The R software environment was used for computations and visualization (ver. 4.1) . We usedProfiler  for geneUpon performing PCA analysis, we observed that samples from one study were separated into distinct clusters. To address this issue, we utilized the Combat method to correct for the batch effect . HoweverOur study aimed to analyze the heterogeneity among liver cell lines—HepG2, Huh7, and Hep3B. In brief, we found that HepG2, Huh7, and Hep3B cell lines differed in oxidative phosphorylation, cholesterol metabolism, and DNA damage between samples and cell lines. In addition, despite the high correlation of gene expression between samples of the same cells lines, a batch effect was noted, which is likely due to the cultivation conditions. Thus, in order to transfer the results of experiments to other cell line samples, many factors must be considered, including growing conditions such as nutrient and oxygen concentrations, pH of the medium, and other parameters, which may lead to inaccurate or distorted data.A notable fact is that when comparing cell lines of different origins based on gene expression, the difference between them is not obvious, while the use of transcript expression gives a clear separation by cell source. Together with our previous results on the difference in the metabolomic profile between the passages of HepG2 cells, it indicates that the closer the omics layer is to the phenotype, the greater the differences between the cells will be observed.Comparative transcriptomic analysis of liver cell lines and primary hepatocytes shows that these cells have a common difference in their cell cycle processes. Interestingly, there are the most differentially expressed genes in the Hep3B cell line compared to primary hepatocytes. Thus, using liver cell lines as a model for normal hepatocytes may not be appropriate in some experiments, as key pathways involved in cancer development and liver disease are affected in HepG2, Huh7, and Hep3B cell lines.


This review summarizes the significant advancements in prophylaxis against graft-versus-host disease (GvHD) presented at the 2022 ASH Annual Meeting. The use of innovative agents and regimens, along with the conventional prophylactic approach of combining post-transplant cyclophosphamide and anti-thymocyte globulin, were discussed. The innovative agents and regimens highlighted in this review include abatacept, the first FDA-approved drug for acute GvHD prophylaxis; RGI-2001, which promotes the expansion of regulatory T cells; and cell therapies such as Orca-T and Orca-Q. These advancements provide promising strategies and options for GvHD prevention, offering hope for improved post-transplant patient outcomes in terms of survival rates. Hematopoietic stem cell transplantation (HSCT) can lead to a severe multisystem disorder known as graft-versus-host disease (GvHD), resulting in considerable morbidity and non-relapse mortality (NRM). We reviewed several latest reports on GvHD prophylaxis from the 2022 ASH Annual Meeting (ASH2022).Abatacept (ABA), a CD28:CD80/86 co-stimulation blockade, has shown potential in controlling early T cell allo-proliferative escape, a major contributor to breakthrough acute GvHD (aGvHD) following calcineurin inhibitor/methotrexate (CNI/MTX) prophylaxis . ABA + CThe augmentation of regulatory T cells (Tregs) has gained significant attention as an effective strategy for preventing GvHD while preserving the graft-versus-leukemia (GvL) effect. RGI-2001, a liposomal glycolipid, expands Tregs by activating invariant natural killer cells through CD1d receptor on antigen-presenting cells, thereby modulating GvHD pathogenesis. RGI-2001 administered intravenously with tacrolimus/MTX was effective in preventing aGvHD in individuals receiving grafts from peripheral blood stem cell (PBSC) and bone marrow sources, with a favorable safety profile. Promising results justify the planning of a phase 3 clinical trial to assess the efficacy of the regimen . Orca-T Combining anti-thymocyte globulin (ATG) and PTCy as prophylaxis against GvHD is a promising strategy, surpassing the limitations of individual administration. ATG-based regimens pose viral infection risks, while PTCy-based prophylaxis is less effective with PBSC grafts. A retrospective study  showed tSalas et al. conducted trials investigating the use of reduced-dose ATG in various transplantation settings. In Abstract 2127 , a compaLow-dose PTCy has been investigated in elderly patients and those with cardiac comorbidities undergoing haplo-PBSCT . The stuThe emergence of novel agents and regimens has expanded the donor pool and allowed the use of different graft sources, including mismatched donors and PBSC, to address patient needs while minimizing the risk of GvHD. For PTCy and ATG combination prophylaxis, future endeavors could focus on investigating lower-dose combination of these agents to achieve the dual goals of minimizing adverse events and maintaining prophylactic efficacy.


Sebaceous neoplasms describe a group of tumors with sebaceous differentiation commonly seen in lesions located primarily in the face and neck. The majority of these lesions are benign, while malignant neoplasms with sebaceous differentiation are uncommon. Sebaceous tumors present a strong association with the Muir–Torre Syndrome. Patients suspected with this syndrome should undergo neoplasm excision, followed by histopathologic and additional immunohistochemistry and genetics examinations. Clinical and dermoscopic features of the sebaceous neoplasms, as well as management procedures collected from the literature analysis regarding sebaceous carcinoma, sebaceoma/sebaceous adenoma, and sebaceous hyperplasia are described in the current review. A special note is made for describing the Muir–Torre Syndrome in patients presenting multiple sebaceous tumors. Sebaceous differentiation of neoplasms is represented by sebaceous carcinoma, sebaceoma, adenoma, and sebaceous hyperplasia. The benign sebaceous gland hyperplasia is very common among the general population, while malignant neoplasms, sebaceous carcinoma, sebaceoma, and adenoma are very rare. Sebaceous gland tumors can appear at any age, but they affect mainly elderly people. The group of sebaceous tumors have been strongly associated with Muir–Torre Syndrome (MTS)—when multiple lesions are present—while sebaceous hyperplasia has been correlated with chronic sun exposure. In this review, an effort was made to describe the clinical and dermoscopic features of sebaceous neoplasms, along with the management of such lesions, regarding tumor excision and the presence of MTS.Clinically, sebaceous tumors are often mistaken for more common malignant skin neoplasms, such as basal cell carcinoma and squamous cell carcinoma. In these cases, the diagnosis can be assisted by the use of dermoscopy and confirmed with the help of histopathology. Sebaceous tumors are mainly located on the head and neck area, but they may be traced to any region of the body. The clinical presentation can vary from small, skin-colored, umbilicated papules to ulcerated lesions, subcutaneous nodules, or exophytic tumors. The lesions are solitary except in patients with MTS. In this case, numerous sebaceous tumors may be located outside the head and neck area. Moreover, patients affected from the syndrome can present multiple keratoacanthomas, or manifest gland neoplasms at a young age.For this review, we searched the following databases: PubMed, MEDLINE, Scopus, OVID, Embase, Science Direct, Cochrane library, Research Gate, and Google Scholar. The search terms were “sebaceous neoplasms” OR “sebaceous carcinoma” OR “sebaceoma” OR “adenoma” OR “sebaceous hyperplasia” OR “Muir–Torre Syndrome”. Studies were included from inception to February 2023. Data extraction was performed individually for each neoplasm. The pertinent information obtained from each study was: the first author; year of publication; type of study; type of sebaceous neoplasm; epidemiologic data; clinical features; dermoscopic features; and management. The search included exclusively English-language academic papers. The reference list of the shortlisted articles was also examined for supplementary studies. The study has been designed, and the results have been described as per the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Our literature search yielded 494 studies. After excluding case reports, systematic reviews, duplicate studies, and studies that did not present any relevant data, we ended up with 52 studies.Sebaceous carcinoma is an uncommon cutaneous neoplasm. The prevalence reported in two studies, based on the US and Texas registries of skin malignancies, varies between 0.05% and 0.7% among other skin tumors ,2. AlthoIn a 9-year-study about sebaceous neoplasms, from a total of 8819 biopsies, 207 sebaceous neoplasms were isolated. Among them, 1.6% were identified as sebaceomas and 1.1% as adenomas . Wh. Wh62]. Once the diagnosis has been established, MTS patients should undergo annual surveillance for visceral and cutaneous malignancy. Gastrointestinal endoscopy should be performed annually, with colonoscopy examination beginning from the 18th year of age, and upper endoscopy from the 25th . Men shoThe management of cutaneous lesions in the context of MTS does not differ from that of solitary neoplasms and was described earlier in the current review.Neoplasms with sebaceous differentiation present an everyday challenge for the clinician. In cases of benign neoplasms that clinically present relevant difficulty regarding the differential diagnosis, the use of dermoscopy could be of a great help. In order to establish the diagnosis, histopathology should be performed as neoplasm differentiation variations could lead to misdiagnosis and mistreatment. Moreover, since the differential diagnosis should include the sebaceous carcinoma, the excision management could differ significantly. The therapeutical approach in sebaceous carcinomas should include histological margin control excision techniques, as the rate of relapse or metastasis is significantly high. When this is not applicable, wide margin excision limits should be granted. Association with Muir–Torre Syndrome should also be considered in all cases with multiple tumors with sebaceous differentiation. Immunohistochemistry may provide useful information in cases where histologic distinction is difficult, mainly between sebaceous adenoma and sebaceous hyperplasia. This dilemma should be confronted since sebaceous hyperplasia is not associated with MTS, whereas sebaceous adenoma is the most common sebaceous tumor in the syndrome. The use of genetics is the gold standard in order to detect the germline mutation and achieve the diagnosis of MTS. In MTS patients, a multidisciplinary approach is essential, while close follow-up should be performed.Understanding the nature and the behavior of sebaceous tumors, defying the overlap between the various neoplasms with sebaceous differentiation, describing and improving the clinical and dermoscopic criteria for the diagnosis, and investigating the possible presence of immunohistochemistry germline mutation markers should be subject to further studies. Since clinical or dermoscopic definitive diagnosis is not achievable, all sebaceous tumors should be treated with caution, and excised totally.


Renal proximal tubule epithelial cells (RPTECs) are a primary site for kidney injury. We created two RPTEC lines from CD-1 mice immortalized with hTERT (human telomerase reverse transcriptase) or SV40 LgT antigen (Simian Virus 40 Large T antigen). Our hypothesis was that low-level, repeated exposure to subcytotoxic levels of 0.25–2.5 μM cisplatin (CisPt) or 12.5–100 μM aflatoxin B1 (AFB1) would activate distinctive genes and pathways in these two differently immortalized cell lines. RNA-seq showed only LgT cells responded to AFB1 with 1139 differentially expressed genes (DEGs) at 72 h. The data suggested that AFB1 had direct nephrotoxic properties on the LgT cells. However, both the cell lines responded to 2.5 μM CisPt from 3 to 96 h expressing 2000–5000 total DEGs. For CisPt, the findings indicated a coordinated transcriptional program of injury signals and repair from the expression of immune receptors with cytokine and chemokine secretion for leukocyte recruitment; robust expression of synaptic and substrate adhesion molecules (SAMs) facilitating the expression of neural and hormonal receptors, ion channels/transporters, and trophic factors; and the expression of nephrogenesis transcription factors. Pathway analysis supported the concept of a renal repair transcriptome. In summary, these cell lines provide in vitro models for the improved understanding of repeated renal injury and repair mechanisms. High-throughput screening against toxicant libraries should provide a wider perspective of their capabilities in nephrotoxicity. Proximal tubule cells of the kidney are primary sites for reabsorption of ions and intermediary metabolites driven by energy-driven transporters. Renal proximal tubule epithelial cells (RPTECs) express multiple transporter systems that have broad substrate specificity and special functions including the disposition of many xenobiotics ,2. WhileCKD can occur after long-term renal injury as a comorbidity from diabetes and hypertension , by enviHuman RPTECs immortalized by hTERT (telomerase reverse transcriptase) have proven an extremely useful model for in vitro studies ,17,18. OWe have been intrigued by the experimental approaches using low-level, repeated exposure ,13,15 thp values. Using DESeq2 for differential expression analysis to CisPt and AFB1, we filtered for two-fold expression differences at an adjusted p ≤ 0.05 value to diminish false positives. However, for PCA analysis all differentially expressed genes were used to optimally visualize the separation among the treatment groups. In the absence of cell death, we observed conventional biomarkers of renal injury, such as Havcr1 (Kim-1) and Il6, were mildly increased, over concentration and time with AFB1 and CisPt. For example, Havcr1 ranged from a 2- to 4-fold increase in either cell line with either chemical treatment at 72–96 h. In addition, we often found increased expression of Mmp9, Mmp10, Mmp13, and Mmp25 that was consistent with their recognized roles in renal damage Hoxc12 an [Nlrp12 . It was  failure . Similarre [Lhx2 , Scn7a [2 [Scn7a , Negr1 [a [Negr1 , Ntrik1  [Ntrik1 , and Grmk1 [Grm1  are incrSlc22a2 (Oct2), is well recognized as highly responsible for CisPt bioaccumulation over the course of chemotherapeutic treatment resulting in nephrotoxicity. Since Slc22a2 is poorly expressed in human and rodent immortalized RPTEC lines, many studies have used Oct2-overexpression systems to study its transporter functions with various therapeutics [Slc31a1) are highly expressed in our cell lines and have a demonstrated function in CisPt uptake in other studies [Slc31a1, Slc31a2 , and Slc47a1 .Another aspect to CisPt toxicity is its pharmacokinetic entry into and removal from proximal tubule cells. Entry can occur by passive diffusion as well as by facilitated movement from specific membrane transporters ,65. Clinapeutics . In muri studies . AlthougOtof, ↑ Scn4b, ↓ Fhit, ↓ Pacrg) as well as observing some unique high-expression and low-expression transcripts seen either in the hTERT  or in the LgT cells . These observations reflect the diversity of biological responses at the individual gene level as cells adjust to repeated CisPt exposure over time. In addition, pathway-level interrogation is helpful for interpreting differential expression of large gene sets [2O2 stress from 25 µM CisPt for up to 24 h exposure in mouse proximal tubule cells [Trp53 signaling, Nrf2 and mitochondria-mediated oxidative stress, as well as mTOR, AMPK, and EIF2 signaling pathways [Fold changes of the top 20 DEGs reflect response intensity of individual transcripts to CisPt, though just a small proportion of the same DEGs were conserved in the top DEG group over 24–96 h. We also found several transcripts that were shared between the two cell lines  from Nanostring data agreed with the RNA-seq findings but were statistically significant due to reproducibility of high fluorescence counts. Similarly, the Nanostring data showed modest fold increases in Cdkn1a (p21) at 1.3X and 1.4X fold in the hTERT cells and 1.7X and 2.4X fold in the LgT cells at 72 h and 96 h, respectively. However, Gadd45 (p53-dependent gene) was down-regulated. Mdm2 levels shown by Nanostring were slightly increased in expression and ranged from 2 to 4 fold in both types. Although Mdm2 and Cdkn1a are generally controlled by Trp53 in primary cells, Trp53-independent roles for these transcripts are being increasingly recognized [Trp53-mediated cell death did not play a role in the response of our cell lines to nephrotoxicants under our exposure conditions. However, we did note large fold changes in the Trp53-related family members, Trp63 and Trp73, in both the cell types by RNA-seq. Trp63 and Trp73 have roles in the differentiation and development of squamous epithelial and neural cells, respectively [Trp63 and Trp73 in proximal tubule cell recovery after injury merits further investigation.Interestingly in our studies, we did not observe significant fold changes (≤1.1X) in the pressors . Nominalcognized . Furtherectively , and recectively ,76. An iIl6 and Il18 pathway engagement that was consistent with transcriptional increases over concentration and time in both the cell types. However, other critical immune transcripts and pathways were also highly upregulated including the S100a8/S100a9 pathway noted in AKI [Il17f, Il24, Il33, and Tnf pathways in kidney injury [Ltb4r1 and Ltb4r2 pathway [Lilrb4a,b receptor pathway, both involved in leukocyte recruitment [Cxcr4 [Hc (C5a) [Ptgs2 transcript (Cox2) pathway [Wnt11 [Fgf3 [Mmp12 [Csf2, secreted from renal proximal tubules cells during AKI, can promote the transition of proinflammatory M1 macrophages to an anti-inflammatory M2 phenotype in a dose and time-dependent manner [Csf2 pathway activation and increased expression over 24–96 h in both the cell lines. In addition, the GPCR-pathways were widely activated in this study since many GPCR ligands were likely elevated during injury that could include hormones, peptides, proteins such as chemokines, ions like Ca++, synaptic transmitters such as glutamine, and intermediary metabolites ranging from fatty acids, ATP, ADP, to many others [Pathway activation marked by the presence of DEGs are controlled by upstream regulators and compiling these cellular events could give broader insights into CisPt-induced effects. The number of activated pathways outnumbered the inhibitory pathways as they both increased over time from 24 to 96 h. We focused our attention on the upstream regulators that showed significant differential expression with the potential to activate or inhibit pathways. In our study, immune, neurogenic, and tissue remodeling and repair pathways were the primary cellular responses to subcytotoxic CisPt exposure in these two new cell lines. We observed d in AKI ; cytokiny injury ,79,80; t pathway  and the ruitment ,83; the t [Cxcr4  and pathHc (C5a) ; and the pathway . Upreguly [Wnt11 , Fgf3 [811 [Fgf3 , and Mmp3 [Mmp12  have beet manner , contriby others .The molecular pathogenesis of acute renal injury is highly complex but in vitro systems can help identify specific genes and orthologs for unraveling the multiple pathways involved for early detection, therapeutic intervention, and prevention. Instead of a single nephrotoxicant challenge, we feel advanced understanding in chronic kidney disease can be gained from repeated subcytotoxic exposures. We observed most expression changes at 72 h or 96 h but recognize some genes and pathways were activated as early as 24 or 48 h. We interpreted these temporal transcription differences in genes and pathways for each cell type as early molecular adjustments (3–48 h) based on their unique genomic structure. However, in the case of CisPt exposure we found each cell type eventually converged on similar pathways responses at later time points (72–96 h). We speculate that the two different immortalization processes could affect early transcriptional variations in the hTERT and LgT responses to CisPt. AFB1 toxicity in the LgT cells, compared to the absence of response in the hTERT cells, does suggest some important genomic differences to be further defined. We further expect that nephrotoxicant screening libraries will be valuable tools to uncover the unique capabilities of LgT and hTERT moRPTEC lines in various cellular formats including microfluidic RPTEC chips  and, 2D Tnf, Il17, and family members of CXC- and CC-motifs as found in our study.Single or repeated acute kidney injuries may lead to chronic kidney failure exacerbated by comorbidities, contributions by distal organs, therapeutics, and environmental exposures ,94,95. NCell Immortalization and Culture: Mouse renal proximal tubule epithelial cells (moRPTECs) isolated from CD-1 mice were obtained commercially  and were transduced with lentivirus vectors containing either human TERT (hTERT) or SV40 Large T Ag (LgT) with antibiotic selection modules. The Ef1a_Large T-antigen Puro lentivirus was a gift from Linzhao Cheng [g centrifugation for 5 min to form a loose pellet, followed by resuspension in culture medium and plating.RPTECs were grown on Biocoat collagen-1 coated 6-well plates  in a commercial culture medium  containing 2% FCS, Penicillin/Streptomycin antibiotics and a growth supplement. hTERT immortalized cells were selected and maintained by supplementing culture medium with 100 ng/mL G418-Geneticin™  while LgT immortalized cells were selected and maintained at a concentration of 0.5 ug/mL of puromycin . Cells that had been passaged for over 50 cell divisions under continuous antibiotic selection with consistent cobblestone appearance and contact inhibition were considered stable cell lines. We observed that moRPTECs strongly adhered to culture plates surfaces and notably possessed cell self-adhesion properties in suspension. We found that successfully passaging moRPTECs required 1–2 PBS washes and 5–10 min incubation in PBS at 37 °C prior to trypsinization and 120× Chemical Treatment: Aflatoxin B1  and Cisplatin  were selected as representative nephrotoxicants to characterize transcriptional responses. Experiments were conducted in confluent cultures in 6-well plates. AFB1 was solubilized in DMSO (control) and CisPt was solubilized in phosphate buffered saline . Concentrations were chosen for minimal cytotoxicity at the highest level of chemical exposure in commercial growth media at 2 mL/well. Immortalized LgT and hTERT moRPTEC lines were grown to near confluence in 6-well tissue culture plates before treatment. To examine concentration-dependent transcriptional responses to CisPt or AFB1, cells were treated for 72 h with 0.25–2.5 μM CisPt, or with 12.5–100 μM AFB1. A second experiment determined transcriptional changes in LgT, and hTERT cells that were exposed to 2.5 μM CisPt over a 96 h time period. Samples at each time point were conducted in triplicate for CisPt and a triplicate time-matched control. At 0 h, untreated cells were nearly confluent and were harvested for RNA. After a single exposure to 2.5 μM CisPt, cells were harvested for RNA at 3 h, 6 h, or 24 h. For the remaining time points, media was changed daily containing either CisPt treatment or PBS at each 24 h period and then harvested for RNA 24 h later. Chemical treatments are summarized in ® 3500 xl Genetic Analyzer . Authentication data are presented in Mouse Cell Authentication: hTERT and LgT moRPTEC lines were submitted for mouse cell authentication using a short tandem repeat (STR) profiling  performed according to published protocols . BrieflyRNA and DNA isolation; RNA-seq: Cell viscosity was reduced by use of cell column shredders prior to nucleic acid isolation . For RNA isolation , cells were washed with PBS prior to addition of lysis buffer and spin columns with on-column DNAase-1 digestion followed by washes, elution, and storage at −80 °C. RNA integrity was measured on an Agilent Model 5300 Fragment Analyzer  with RQN values of 9–10. RNA samples were prepared for RNA-seq by rRNA depletion, fragmentation , bar coding, and library construction as previously described . BrieflyActb; Gapdh, Hprt, Rpl32) that were determined by RNA-seq to be treatment stable. Samples were analyzed by nCounter at 150 ng RNA input. Data were analyzed by ANOVA and Newman–Keuls testing.Nanostring Analysis: NanoString nCounter  was used as a multiplexed gene expression platform to test for specific transcripts as previously described . Brieflyin vivo testing in both animal models or more complex multicellular experimental constructs such as organoids or microphysiological systems. The development of interconnected, pulsatile culture media systems carrying signaling molecules and hormones from different cell types represent promising new technologies for combining different immortalized cell types to better understand the causes of toxicity and disease.There are several considerations for future directions of the current work. Functional data for ion transporter activity and xenobiotic metabolic activation of these two cell lines would be important for placing context to the transcriptional changes that we reported. This could include functional assays for Oct  and Oat  transporters that are of clinical importance in the disposition of many drugs or xenobiotics. The creation of additional immortalized cell lines by human papilloma virus (HPV) or Epstein–Barr virus could provide additional breadth to the biological responses possible in moRPTEC lines. Further, the inclusion of different mouse strains of both sexes could add important gender representation and complexity of response to these two in vitro cell line models. A more comprehensive view of RPTEC responses to toxicant challenge could be gained by addition of other omics methodologies including proteomics, metabolomics, and epigenomics that could be strategically applied to critical early time points or more prolonged exposure paradigms to better simulate the conditions leading to chronic renal kidney disease. Finally, the value of in vitro findings and hypotheses generated from these renal cell lines would greatly benefit from This study presents two new immortalized RPTEC lines with similar and some distinct capabilities in their response to two different nephrotoxicants. We believe the findings from these two cell lines can provide in vitro models for improved understanding of renal toxicity and repair mechanisms. First, data in the LgT cells suggest AFB1 may have direct nephrotoxic properties independent of hepatic metabolism and processing. Second, expression from CisPt experiments in both the cell lines support the concept of a ‘coordinated transcriptional program’ of injury signals and repair processes represented by (1) externalization of immune receptors as well as cytokine and chemokine secretion for leukocyte recruitment; (2) a robust expression of both synaptic and SAM molecules to facilitate increased expression of neural and hormonal receptors, ion channels/transporters, and trophic factors; and (3) expression of nephrogenesis transcription factors to assist in regeneration and repair. Many of the neural and cardiac canonical pathways evoked by pathway analysis are not unexpected since these tissues share similar cellular processes to kidneys in synaptic and bioelectric transmission, and intravesicular transport. Third, we anticipate differences in the immortalization processes of the parent RPTECs affect the genomics that shape the kinetics of phenotypic responses in these two cell lines—a research area that is actively being investigated in our laboratory. High throughput screening of LgT and hTERT-derived moRPTECs against chemical toxicant libraries will provide a wider perspective of biological responses and the unique capabilities of these cell lines in nephrotoxicity research.


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