Pediatric cancer is the leading cause of disease-related death in children and the need for better therapeutic options remains urgent. Due to the limited number of patients, target and drug development for pediatrics is often supplemented by data from studies focused on adult cancers. Recent evidence shows that pediatric cancers possess different vulnerabilities that should be explored independently from adult cancers.Using the publicly available Genomics of Drug Sensitivity in Cancer database, we explore therapeutic targets and biomarkers specific to the pediatric solid malignancies Ewing sarcoma, medulloblastoma, neuroblastoma, osteosarcoma, and rhabdomyosarcoma. Results are validated using cell viability assays and high-throughput drug screens are used to identify synergistic combinations.Using published drug screening data, PARP is identified as a drug target of interest across multiple different pediatric malignancies. We validate these findings, and we show that efficacy can be improved when combined with conventional chemotherapeutics, namely topoisomerase inhibitors. Additionally, using gene set enrichment analysis, we identify ribosome biogenesis as a potential biomarker for PARP inhibition in pediatric cancer cell lines.Collectively, our results provide evidence to support the further development of PARP inhibition and the combination with TOP1 inhibition as a therapeutic approach in solid pediatric malignancies. Additionally, we propose ribosome biogenesis as a component to PARP inhibitor sensitivity that should be further investigated to help maximize the potential utility of PARP inhibition and combinations across pediatric solid malignancies.The online version contains supplementary material available at 10.1186/s12885-022-10319-7. Worldwide, nearly half a million children aged 0–19 years old will develop cancer each year, and despite technological and treatment advancements over the decades, cancer is the leading cause of disease-related death in children . Not onlTo this end, we sought to explore drug vulnerabilities specific to pediatric solid malignancies, namely Ewing sarcoma (ES), medulloblastoma (MB), neuroblastoma (NB), osteosarcoma (OS) and rhabdomyosarcoma (RMS). While these malignancies all have different etiologies and biology, they are associated with a poor clinical outcome and represent a group of patients in dire need for better therapeutic approaches –8. A druBRCA1/2 mutations—PARP inhibition is synthetically lethal and induces cell death. At present, there are four PARP inhibitors approved for the clinical use in adults , however none have been approved in children yet [PARP is a family of nuclear proteins comprised of 17 members that play an integral role in multiple essential cellular processes, one of which is DNA damage repair. In the presence of single strand DNA breaks, PARP-1 synthesizes the formation of a PAR chain which recruits critical repair proteins to the damage site and facilitates DNA damage repair , 10. Disdren yet .In this study, we explore PARP1/2 as a therapeutic target relevant across a wider range of pediatric solid malignancies and we show that efficacy can be improved when combined with conventional chemotherapeutics, specifically topoisomerase inhibitors. Additionally, we propose ribosome biogenesis as a novel treatment biomarker. Altogether, our results demonstrate the potential of PARP inhibitors and combinations in a broader pediatric context and urge further investigation into PARP as a therapeutic target for pediatric patients.50) and area under the curve (AUC) values are reported. Additionally, this database includes genomic and expression data of the cell lines which we also used in our study.In this study, we use the Genomics of Drug Sensitivity in Cancer version 2 (GDSC2) database to identify potential therapeutic targets in pediatric cancer cell lines . This da2 and regularly tested for mycoplasma infection.A panel of 32 cell lines was used in this study and included six ES cell lines , RD-ES, A-673, POE and sta-et-1), five MB cell lines , seven NB cell lines , seven OS cell lines  and six RMS cell lines . All cell lines used in this study were obtained from the American Type Culture Collection or via historic collaborations and the identity of each cell line was validated by short tandem repeat (STR) analysis. Cells were cultured in appropriate medium with supplementation (outlined in Supplementary Table 2) before being treated with compounds for either high-throughput drug screens or validation screens. Following 72-hour incubation with the compounds at standard culture conditions, cell viability was measured using the 3--2,5-diphenyltetrazolium (MTT) assay [Cells were seeded in black 384-well plates  according to predetermined densities based on cell growth rate of each cell line . Cells were cultured for 16–24 h under standard culturing conditions  assay .High-throughput drug screens were conducted in collaboration with the high-throughput screening facility of the Princess Máxima Center . The scr50 and AUC values were derived from the dose-response curves using the package drc in the R statistical environment (version 4.2.0) [50 and AUC values were derived from the dose-response curves using GraphPad Prism version 9.0 for windows. For all screens, the data was normalized to the DMSO-treated cells (defined as 100% viability) and empty controls (defined as 0% viability). IC50 values at 72-hours were calculated by determining the concentrations of the drug needed to achieve a 50% reduction in cell viability. AUC values were calculated by determining the definite integral of the curve.For the high-throughput screens, ICn 4.2.0) . For valAUC values of the GDSC2 dataset were used to explore differential compound sensitivities of pediatric versus adult cell lines by adapting gene set enrichment analysis . For this we compared AUC values of pediatric and adult cancer cell lines for all 198 compounds using the limma R package and used the resulting t statistics as input for the fgsea R package. We grouped the compounds based on their annotated target or target pathway  as additional input for the GSEA analysis. In addition, we used GSEA to determine which genesets (KEGG subset from MSigDB version 7.5.1)  were corUsing the publicly available GDSC2 database, we investigated differential drug sensitivity of pediatric versus adult cancer cell lines Fig. a. Follown = 19), NB (n = 23), MB (n = 4), RMS (n = 7) and OS (n = 10)) and adult (n = 686) tumor cell lines, we observed a range of sensitivities. Within the pediatric cell lines, Ewing’s sarcoma was the most sensitive and rhabdomyosarcoma was the least sensitive and overall, the pediatric tumor types had lower AUC Z-scores compared to adult cancer cell lines  and found that PARP inhibitors  demonstrated the greatest and most significant efficacy in pediatric cancer cell lines Fig. c. When l50 = 0.12 μM), and rhabdomyosarcoma and osteosarcoma cell lines were the least sensitive overall  combined with a drug library containing a 6-fold concentration range of 198 compounds that are in (pre)clinical development for children, we generated a robust dataset containing nearly twenty thousand combinations in total , 2) fold change in IC50 value (∆IC50) and 3) average synergy score according to the bliss independence model [The clinical use of single compound treatment with PARP inhibitors has been greatly challenged by both inherent and developed resistance to PARP inhibition. An increase in drug efflux, restoration of HR, and stabilization of stalled replication forks are some of the resistance mechanisms observed , 12. As ce model .max or IC50 or had an average bliss score greater than zero were considered. Overall, 47, 50, 43, 52 and 28 of the tested combinations were regarded synergistic in at least one of the three averaged metrics in ES, MB, OS, NB and RMS, respectively. Despite finding numerous synergistic hits in each malignancy type, there was a striking lack of overlapping synergy and only two combinations yielded synergy across the five different pediatric tumor types included in our study: talazoparib combined with KU-60019 or SN-38  and following combination treatment with talazoparib, we observed synergy as evidenced by shifts in dose-response curves Fig. b. Howeve50 range of 2–8.5 nM, the cell lines were very sensitive to SN-38 alone. However, with the addition of talazoparib, greater efficacy was observed and the IC50 range was reduced to 1.1–4.1 nM , on the other hand, demonstrated very promising effects. Having an ICTo investigate our findings further, we conducted combination drug screens using a wider range of concentrations on a panel of pediatric cancer cell lines of each malignancy type which included talazoparib sensitive  and insensitive cell lines  and it demonstrated synergy with the addition of 0.13 nM of SN-38  and HOS (IC50 = 0.89 nM), respectively , was the most sensitive to talazoparib treatment alone . These PARP inhibitors do not possess the same PARP-entrapment properties as talazoparib and had higher ICAs evidenced by our study and other published results demonstrating differential sensitivity to PARP inhibitors and combinations, there exists a need for treatment biomarkers . To explSTAG2 mutations correlated with sensitivity to PARP inhibition with talazoparib, particularly in Ewing’s sarcoma . Being that pediatric cancers account for a very small proportion of cancer diagnoses worldwide, there is a certain degree of hesitancy to fund pediatric-specific (pre)clinical drug development and the execution of robust clinical trials remains difficult. The existence of a drug target across different malignancy types could help mitigate these challenges and aid in the ethical development of safe therapeutics for children with cancer. Furthermore, the poor prognostic outlook for patients with relapsed solid tumors urges the development of more targeted treatment approaches, such as PARP inhibition. Currently, there are no clinically approved PARP inhibitors for children, however the results of a recently published phase 1 clinical study evaluating olaparib in pediatric patients with refractory solid tumors were promising and there are multiple ongoing clinical studies to evaluate the safety, dosing and efficacy of PARP inhibitors in children  .Our study is not the first to identify PARP as a potential target in pediatric solid malignancies. In fact, in our recent systematic review of replication stress as a therapeutic target for pediatric cancers, PARP presented as the most robustly investigated target within these pathways . HoweverFollowing extensive high-throughput drug screens using a library of compounds that are in (pre)clinical development for pediatrics, we found numerous potential synergistic combinations within each of the different malignancy types, including doxorubicin and cyclophosphamide which have previously demonstrated synergy when combined with PARP inhibition in some pediatric tumor types . HoweverOne possible mechanism that could be underlying the observed synergism between PARP and TOP1 inhibitors is related to ribosomes. In a recent study, it was demonstrated that TOP1 inhibition combined with a ribosomal gene transcription inhibitor caused global replication stress independent of DNA damage and thereby induced cell growth inhibition . PARP itBRCA1/2 mutations [STAG2 mutations. STAG2 is a component of the cohesion complex which is essential to multiple cellular functions, including HR repair, and multiple studies have suggested it as a biomarker for PARP inhibition [STAG2 mutations and the expression of EWS-FLI1 fusion are frequently encountered in Ewing’s sarcoma and are likely an explanation to why this tumor type demonstrates an overall greater sensitivity to PARP inhibition compared to other pediatric tumor types. However, in the broader pediatric context, this is an incomplete explanation. In our study, we show that pediatric cancer tumor types other than Ewing’s sarcoma also demonstrate sensitivity to PARP inhibition, independent of STAG2 mutation or EWS-FLI1 fusion. Altogether, our study encourages further investigation of alternative biomarkers for PARP inhibition, such as ribosomal pathways.In conjunction with the strong correlation we observed between PARP inhibitor sensitivity and ribosomal pathways, we believe this is a mechanism that should be explored further, especially in the context of treatment biomarkers , 34. In utations –37. Howeutations . In the hibition , 38, 39.hibition –43. BothCollectively, our study highlights the potential of PARP as a therapeutic target across different pediatric tumor types and suggests combination with TOP1 inhibition to improve efficacy. However, the understanding of the mechanisms driving PARP inhibitor sensitivity and associated biomarkers remain incomplete. Being supported by decades of literature, it is clear that DNA damage repair deficiencies are a driver of PARP inhibitor sensitivity, but certainly considered in the context of pediatric cancers, this is evidently an incomplete explanation. In our study, we propose ribosome biogenesis as an additional component to PARP inhibitor sensitivity that should be further investigated to help broaden utility of PARP inhibition and combinations across pediatric solid malignancies.Additional file 1: Supplementary Table 1. Summary of cell lines, medium and supplements used in this study. Supplementary Table 2. Compounds and associated targets included in the hight-throughput drug library.Additional file 2: Supplementary Fig. 1. Box plot of lnIC50 values for 32 different pediatric cancer cell lines following 72-hour treatment with talazoparib. Supplementary Fig. 2. A) Dose-response curves following 72-hour treatment with talazoparib in MB (red), ES (orange), OS (purple), RMS (green) and NB (blue) cell lines. All curves represent the average of two replicates and error bares indicate the standard error of the mean (SEM). B) Schematic outlining the high-throughput drug screening approach used in our study. Supplementary Fig. 3. Dose response curves of high-throughput screening combining KU- 60019 with the IC15, IC25, and IC50 of talazoparib for each cell line in A673 , D341 , HOS , NGP  and RMS-YM . Curves are normalized to talazoparib monotherapy and represent singlicate data. Supplementary Fig. 4. Dose response curves of high-throughput screening combining SN-38 with the IC15, IC25, and IC50 of talazoparib for each cell line in A673 , D341 , HOS , NGP  and RMS-YM . Curves are normalized to talazoparib monotherapy and represent singlicate data. Supplementary Fig. 5. Dose-response curves following 72-hour treatment with talazoparib and SN-38. All curves represent the average of two replicates and error bares indicate the standard error of the mean (SEM). Supplementary Fig. 6. Dose-response curves following 72-hour treatment with olaparib and SN-38. All curves represent the average of two replicates and error bares indicate the standard error of the mean (SEM). Supplementary Fig. 7. Dose-response curves following 72-hour treatment with pamiparib and SN-38. All curves represent the average of two replicates and error bares indicate the standard error of the mean (SEM). Supplementary Fig. 8. Dose-response curves following 72-hour treatment with talazoparib and topotecan. All curves represent the average of two replicates and error bares indicate the standard error of the mean (SEM). Supplementary Fig. 9. Dose-response curves following 72-hour treatment with olaparib and topotecan. All curves represent the average of two replicates and error bares indicate the standard error of the mean (SEM). Supplementary Fig. 10. Dose-response curves following 72-hour treatment with pamiparib and topotecan. All curves represent the average of two replicates and error bares indicate the standard error of the mean (SEM). Supplementary Fig. 11. GSEA showing biological pathways (KEGG) that are positively associated with talazoparib sensitivity in adult cell lines included in the GDSC2 dataset .


Small cell lung cancer (SCLC) is frequently disseminated and has a dismal prognosis with survival times of approximately two years. This cancer responds well to initial chemotherapy but recurs within a short time as a globally chemoresistant tumor. Circulating tumor cells (CTCs) are held responsible for metastasis, the extremely high numbers of these cells in advanced SCLC allowed us to establish several permanent CTC cell lines. These CTCs are distinguished by the spontaneous formation of large spheroids, termed tumorospheres, in regular tissue culture. These contain quiescent and hypoxic cells in their interior and are associated with high chemoresistance compared to single cell cultures. Nine CTC lines were compared for their expression of 84 proteins associated with cancer either as single cells or in the form of tumorospheres in Western blot arrays. With the exception of the UHGc5 line, all other CTC lines express EpCAM and lack a complete EpCAM-negative, vimentin-positive epithelial-mesenchymal transition (EMT) phenotype. Upon formation of tumorospheres the expression of EpCAM, that mediates cell-cell adhesion is markedly upregulated. Proteins such as E-Cadherin, p27 KIP1, Progranulin, BXclx, Galectin-3, and Survivin showed variable changes for the distinct CTC cell lines. In conclusion, EpCAM presents the most critical marker for individual SCLC CTCs and the assembly of highly chemoresistant tumorospheres. Small cell lung cancer (SCLC) is a neuroendocrine lung tumor representing 15% of lung cancers and shows metastases in most patients at first presentation . Smokersin vitro that grow continuously in tissue culture [The typical overall 5-year survival of 5–10% for extended SCLC requires new treatment modalities and characterization of the mechanism of drug resistance. The dissemination of SCLC seems to be related to the excessively high counts of circulating tumor cells (CTCs) compared to other malignancies ,9. A min culture ,12. ThesAll SCLC CTC lines exhibit the spontaneous formation of large multicellular aggregates under normal cell culture conditions, which grow to diameters of 1–2 mm, designated tumorospheres ,12. Normin vitro as spheroids represent exactly the chemoresistance phenotype of native solid tumors and display pathways of resistance linked to hypoxia, altered chromatin structure, impairment of apoptosis, cell cycle alterations, and decreased drug perfusion [in vivo growing tumors [Cancer cells grown erfusion . Ki-67-eerfusion ,24. Comperfusion . MCTSs cg tumors ,16,26. Tg tumors .Despite high investments to develop new cancer therapeutics, about 90% of drugs finally tested in clinical trials fail ,29. SpheThe mechanisms leading to the spontaneous formation of SCLC CTC tumorospheres are not clear, and therefore, we searched for differences in protein expression between single cells and corresponding spheroids using Western blot arrays. The Oncology XL array assesses the relative expression levels of 84 human cancer-related proteins comprising growth factor receptors, signaling mediators, growth factors, adhesion proteins, proteases, chemokines, hormones, and others. The CTCs in form of single cells or tumorospheres were checked for their chemosensitivity using Topotecan and Epirubicin, which are administered for second-line treatment of SCLC. These spheroids contain quiescent and hypoxic cells and seem to constitute a vital component of the global drug resistance of SCLC tumors.The SCLC CTC cell lines BHGc7, 10, 16, 26, 27, 71, 500, 590, and UHGc5 were established from blood samples of extensive disease-SCLC patients following disease progression under second-line therapy at our institution as described previously . Blood c4 cells in the form of single cells or tumorospheres (TOS) in 100 μl medium were distributed to wells of 96-well microtiter plates . Therafter, ten 2-fold dilutions of the test compounds were added from stock dilutions. Assays were at least performed in triplicate. Calculating CTC cell size and the surface area of the globular tumorospheres, 1 × 104 suspension cells correspond to the same number of cells accessible at the surface of the spheroids. The mean size of the clusters used for the cytotoxicity tests was determined, and the number of clusters/well adjusted accordingly. The plates were incubated for four days under tissue culture conditions, and viable cells were detected using a modified MTT assay . The respective dilutions of the compounds tested are present for the whole incubation period. IC50 values were determined from dose-response curves using Origin Pro, Version 9.9 .1 × 10We determined the relative expression levels of 84 cancer-related proteins in the investigated cell lines by performing a Proteome Profiler Human XL Oncology Array ). The kit consists of 4 nitrocellulose membranes spotted in duplicate with antibodies for cancer-related proteins. Membranes were treated following the enclosed protocol ). Cell culture lysates of 9 cell lines in the form of single cells and their tumorospheres were obtained by removing particulates in samples by centrifugation. Afterward, samples were diluted and incubated with the Human XL Oncology Array overnight, followed by removal of unbound proteins by washing. Subsequently, the array was set with a detection antibody cocktail, followed by applying Streptavidin-HRP and chemiluminescent detection reagents. These reagents produce a signal corresponding to the amount of protein-bound, which was visualized using a ChemiDoc™ Touch Imaging System .Heatmaps were plotted for nine cell lines in the form of single cells and their tumorospheres using protein expression data of 43 cancer related proteins in the application heat map with dendrogram in Origin Version 9.9 . Thereby a hierarchical cluster analysis was performed. Firstly, clustering rows of 43 genes by group average and Pearson correlation to reveal differences in gene expression via z-score of these genes in 9 cell lines in the form of single cells and their tumorospheres. Secondly, to show the differences in expression of these proteins via z-score within tumorospheres and uncover similarities between the different cell lines by clustering rows and columns by group average and Pearson correlation. Missing values were filled up with column means, while standardization was performed for columns.The Reactome database (Version 3.7) organizes signaling and metabolic molecules and their relations into biological pathways and processes . The resp-value < 0.05 was regarded as statistically significant. The significance of the heatmap data (single cells versus tumorospheres) is presented in Analysis of the data from cytotoxicity assays and heatmap analyses were calculated using the Origin 9.9 software, in particular, the heatmap option with dendrograms. A 50 values of the cell lines for single cells are presented in 2 = 0.11).In p = 3.35E−5), downregulation of ERBB2 signaling (p = 3.35E−5), ERBB2 regulates cell motility (p = 5.03E−6), signaling by ERBB2 KD mutants (p = 3.09E−5), signaling by ERBB2 TMD/JMD mutants (p = 2E−5), ERBB2 activates PTK6 signaling (p = 4.28E−6), GRB2 events in ERBB2 signaling (p = 6.77E−6), SHC1 events in ERBB2 signaling (p = 3.35E−5). Furthermore, pathways involving ERBB4, e.g., signaling by ERBB4 (p = 5.29E−9), nuclear signaling by ERBB4 (p = 1.95E−10), SHC1 events in ERBB4 signaling (p = 6.77E−6).The Reactome pathway analysis of the 14 proteins with reduced expression in tumorospheres  revealed a connection of these genes to signal transduction. In detail, there were several pathways involving ERBB2, e.g., signaling by ERBB2 in cancer (p = 7.01E−9), Interleukin-18 signaling (p = 9.84E−7), Inteleukin-4 and Interleukin-13 signaling (p = 1.33E−6). Furthermore, they are associated with Receptor Tyrosine Kinase signaling (p = 2.36E−9), long-term potentiation (p = 2.15E−5), PI3K/AKT signaling in cancer (p = 5.82E−5), and regulation of PI3K/AKT signaling by PI5P, PP2A, and IER3 (p = 6.78E−5). In summary, these alterations affect cellular signaling, proliferation, and interleukin pathways that are effective in the three CTC cell lines exhibiting the highest proliferative activity.These 14 proteins further influence the signaling of interleukins, which are known for their importance in the function of the immune system, e.g., Interleukin-10 signaling . 3D cultures of tumor cell lines resulting in the formation of different spheroids have been propagated as models of actual tumors that are more representative than traditional 2D cultures. However, as opposed to SCLC CTCs, this buildup of spheroids has to be enforced by nonadherent tissue culture conditions such as hanging drops, agitation, or various scaffolds. The so-called MCTS closely mimics otherapy ,34. Beyootherapy ,36. MCTSotherapy ,38. Compotherapy ,40. The otherapy .The proteins participating in the spontaneous formation of tumorospheres in SCLC CTCs have not been characterized. Chemosensitivity tests demonstrated again that the tumorospheres are more resistant to Topotecan and Epirubicin compared to the corresponding single-cell suspensions . The CTCversus single cells. The formation of spheroids by Caco-2, SW480, and HCT116 colorectal adenocarcinoma cell lines under low-adhesion culture conditions were reported to show a 3-fold higher expression of EpCAM compared to monolayers, but no changes in 19 other markers [For array analysis, the cells and spheroids are lysed, and in the latter case, proteins from proliferative and quiescent cells are mixed in the lysate. Among the proteins analyzed, EpCAM was the single factor that showed consistently higher expression in tumorospheres  markers . The pro markers . Cell cl markers ,45,46. L markers . Drugs t markers .in vivo. Detection of disseminated tumor cells (DTC) in bone marrow (BM) of patients with early-stage NSCLC has been associated with poor outcomes [EpCAM is frequently upregulated in primary tumors and metastases, particularly in adenocarcinomas, certain squamous cell carcinomas, and others . Increasoutcomes . BM aspioutcomes . Antibodoutcomes .In conclusion, the formation of highly chemoresistant tumorospheres by SCLC CTCs is accompanied by elevated expression of EpCAM, a protein involved in cell-cell adhesion. Therefore, EpCAM-positive CTCs seem to be the cell population destined to survive in circulation and lead to chemoresistance and metastasis.


This study reports the incidence of chronic graft versus host disease (GvHD) in allogeneic hematopoietic stem cell transplant  recipients who received SARS-CoV2 vaccination. The overall rates of new and worsening chronic GvHD combined were 14%, with median time from vaccination to GVHD being approximately three to four weeks. A majority of the cases were of mild to moderate severity and primarily localized to either the skin, mouth, or joints. Prior chronic GVHD and recent transplant were associated with higher GVHD rates following COVID-19 vaccination. More prospective studies are needed to provide a definitive mechanism for the impact of SARS-CoV2 vaccination on alloHCT patients. In the United States, there have been two vaccines approved by the Food and Drug Administration for severe acute respiratory syndrome coronavirus-2(SARS-CoV2): Pfizer-BioNTech  and Moderna . Both vaccines have displayed high efficacy and safety in the general population, but there are doubts regarding the efficacy of both these vaccines in patients with cancer as this population was excluded from major clinical trials , 5. WithP values of < 0.05 were considered statistically significant. A multivariate logistic regression was performed to identify patient characteristics and secondary endpoints associated with the incidence of GVHD. All statistical analyses were performed on RStudio, version 1.2  and p values < 0.05 were considered significant. Baseline characteristics are summarized in Table We retrospectively reviewed 298 adult patients who received either the Pfizer-BioNTech mRNA COVID-19 vaccine (BNT162b2) or Moderna mRNA COVID-vaccine (mRNA-1273) from December 2020 and July 2021. Inclusion criteria included patients 18 years of age or older and who had received at least two doses of vaccine in the post-HCT setting. Primary endpoint was the incidence of new and worsening GvHD based on physician documentation in the electronic medical record. New GvHD was defined as development of GvHD in a location that was not previously documented and worsening GvHD was defined as either increased severity of GvHD or requiring adjustment in GvHD regimen to address symptoms. Incidence of GvHD for the first dose was based on any reports of GvHD prior to the second dose. For subsequent doses, incidence of GvHD was reported up to 42 days after the dose was received. The severity of GvHD was independently assessed by a physician using the National Institutes of Health consensus criteria for classification of chronic GvHD [p = 0.491). Median time to GvHD from vaccination was around three to four weeks. Approximately 75% of patients who experienced new chronic GvHD were mild to moderate in symptoms and localized to the skin/joints and mouth. Worsening of chronic GvHD did occur at a slightly lower rate than new chronic GvHD. Half of the patients had worsened to severe symptoms of GvHD mainly in the skin/joints and mouth. Five patients were considered to have no change in the severity of GvHD symptoms, but physicians had to make modifications to their regimen to try to improve their GvHD. At the time of our analysis, there were only 54 patients who had received their booster dose at a median of 186 days (range 53–266) from when they received their first dose. Physician follow-up after the booster only occurred in 81.4% of the patients at the time of our analysis with a median follow-up time 34.5 days (range 4–87) after their booster dose. When analyzing the secondary endpoints, timing of vaccine relative to transplant date did have an impact on whether the vaccine caused chronic GvHD, as the further out from transplant the less likely a GvHD flare would occur. Patients who received their SARS-CoV2 vaccine within the first 180 days of transplant had an incidence of 30.3% compared to 11.6% of patients who received their vaccine past one year after their transplant. Those who had chronic GvHD prior to receiving their vaccine had a 10% higher incidence of developing new or worsening GvHD than those who did not have chronic GvHD prior to their vaccine. The presence of prior systemic immunosuppressants did not have an impact on development or worsening of GvHD symptoms with SARS-CoV2 vaccination. A multivariate logistic regression analysis (Table p = 0.003). Given the small sample size, the generalization of the statistical significance remains in question and should be further validated in larger studies.All our results are shown in Tables Our study showed that the rates of new and exacerbation of cGVHD were different in patients with a recent transplant compared to patients who had the vaccine later in the course of the alloHCT. This may help clinicians determine the risk and perhaps consider delaying immunosuppression tapering by 4–8 weeks after the vaccine if clinically feasible to prevent new or exacerbation of chronic GVHD. In conclusion, providers should continue to recommend that alloHCT patients get their SAR-CoV2 vaccination to help protect against infection but should be aware that increased monitoring or more conservative management of the GvHD regimen is necessary to ensure early recognition of new or worsening chronic GvHD.


Background: Chronic rhinosinusitis (CRS) is a heterogeneous disease. The pathogenesis of chronic sinusitis is still unclear; however, the nasal cavity and paranasal sinuses are commonly affected by type 2 inflammation, which is caused by Th2 cytokines such as interleukin (IL)-5, IL-4, and IL-13. Previous studies have shown that pendrin promotes local infiltration of neutrophils through the production of human neutrophil elastase (HNE), which is essential for the secretion of mucin 5AC (MUC5AC) in chronic inflammatory diseases of the lower respiratory tract. This study investigated pendrin expression and its relationship to mucin in type 2 inflammation. Methods: A total of 40 patients  were included in this study and were divided into the CRS group and the NC group. A normal nasal mucosa tissue culture model was established in vitro. IL-13 was used to stimulate primary cultures of human nasal epithelial cells (HNECs). Western blot (WB), enzyme-linked immunosorbent assay (ELISA), and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of pendrin, MUC5AC, and MUC5B. After transfecting HNECs with siRNA pendrin or negative control (NC), EGF receptor (EGFR), HNE, MUC5AC, and MUC5B expression were analyzed using qRT-PCR, WB, or ELISA in terms of their relationships with pendrin. Pendrin expression in the tissue was also analyzed. Results: After IL-13 stimulation, pendrin, MUC5AC, and MUC5B expression levels were upregulated; the optimal concentration of IL-13 was 50 ng/mL. The expression levels of HNE, EGFR, MUC5AC, and MUC5B were downregulated after transfection with siRNA pendrin-1650. Pendrin expression in the NC group was lower than in the CRS group. Conclusion: IL-13 is implicated in the inflammation of nasal mucosa, and pendrin is closely related to the excessive secretion of mucin. The expression of mucin is downregulated after transfection with siRNA pendrin. There is a positive relationship between pendrin and EFGR/HNE. Moreover, pendrin plays an important role in type 2 inflammation. Chronic rhinosinusitis (CRS) is a common disease classified as primary chronic rhinosinusitis and secondary chronic rhinosinusitis, which affects 5–12% of the general population . InflammPendrin protein is a soluble transporter. An important part of the family 26A (SLC26A) member, encoded by the SLC26A4 gene, can mediate cell chloride ion, iodine dissociation, and the exchange of various anions such as zinc and bicarbonate ions to maintain a steady state of medium ions in the tissue. Pendrin is primarily localized in the inner ear, thyroid, and kidney ,16,17; hCombining the results of the above studies, we explored that with the stimulation of some inflammatory factors and other pathogenic factors, pendrin may enhance the infiltration of NEU in the nasal cavity and paranasal sinus mucosa, and then promote the secretion and expression of mucin in epithelial and glandular tissues through EGFR signal, which led to the occurrence and development of CRS.In this study, through interleukin (IL)-13 stimulation, and pendrin siRNA in HNECs, we analyzed the expression of HNE, EGF receptor (EGFR), MUC5AC, and MUC5B, and investigated the pendrin–EGFR–MUC pathway in CRS.Samples of nasal mucosa were collected from patients undergoing functional endoscopic surgery. A total of 40 patients  were included in this study from The First Affiliated Hospital of Nanchang University. The 40 patients were divided into the CRS group and the NC (negative control) group. The CRS diagnoses were based on the patients’ clinical histories and anterior rhinoscopy/nasal endoscopy/computed tomography results, in accordance with EPOS 2020 guidelines [The study was approved by the Medical Research Ethics Committee of The First Affiliated Hospital of Nanchang University, and each participant has signed the informed consent form.2), centrifuged, and cultured in bronchial epithelial cell growth medium (BEGM) .HNECs were cultured using a method described in a previous study . Brieflyg, centrifuge for 10 min, and take the supernatant.After the cells reached 80–90% confluence, they were stimulated with different concentrations of recombinant IL-13  over a 24 h period. The cell lysates for protein are as follows. Remove the culture medium and drugs, gently rinse the adherent cells with precooled PBS for 3 times, pay attention to gentleness to prevent the cells from falling off, suck the remaining PBS solution in the culture dish for the last time, and discard it, add protein lysate and phosphorylated protease inhibitor, blow repeatedly, put them on the ice for 10 min, and then use the cell scraper to scrape the cells and lysate, and transfer them to 1.5 mL EP tube, blow repeatedly to avoid gas. Bubble is generated, but attention should be paid to avoid the generation of foam, and it should be placed on ice to continue cracking for 20 min. Put the EP tube into a precooled 4 °C centrifuge, 12,000× The cell lysates for RNA are as follows. The cells were washed by D-hankS solution three times. After trypsin digestion, the cell suspension was collected with a 15 mL centrifuge tube. Total RNA was extracted from cells with Trizol™ reagent .The cell lysates were collected and MUC5AC, MUC5B, and pendrin mRNA expression were determined by qPCR. MUC5AC and MUC5B expression in the cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The protein expression of pendrin was evaluated using Western blot (WB) analysis.FAM NC , NC , Pendrin-homo-373 , Pendrin-homo-920  and Pendrin-homo-1650 .Pendrin (si pendrin) and a non-targeting siRNA (siCTRL) were synthesized by Sangon Biotech  and transfected into target cells using Lipofectamine 2000 . SiNRA are as follows: A qRT-PCR assay was performed as described by Ye . Total R5′-GTAACAGTCCGCCTAGAAGCAC-3′; MUC5AC, forward: 5′-ACCTGTGACAGCAGGATGTG-3′, reverse: 5′-ACTCGCAGTCTCCGTTGAAG-3′; MUC5B, forward: 5′-TCCCACTATTCCACCTTTGACG-3′, reverse: 5′-CCAGGTAGAGGCTGAGATTCCC-3′; HNE, forward: 5′-GCGTGGCGAATGTAAACGTC-3′, reverse: 5′-ACCCGTTGAGCTGGAGAATC-3′; EGFR, forward: 5′-GCCAAGGCACGAGTAACAAGC-3′, reverse: 5′-GGGCAATGAGGACATAACCAGC-3′.Cell supernatants were collected and subjected to ELISA to determine the levels of human MUC5AC and MUC5B secretion using a specific ELISA assay kit  and  according to the manufacturer’s instructions.WB was performed in accordance with the method described by Luo et al. . Total ct-test. In all analyses, p < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS 22.0 . Expression data are presented as dot plots of the median and interquartile range. For the in vitro experiments, cell culture data are presented as means ± standard error of the mean (SEM). Data were analyzed using GraphPad Prism 8 . The differences between the CRS and negative control (NC) groups were examined for significance using Student’s p = 0.0090 and p = 0.0019, respectively); the difference in pendrin expression between the CRSsNP and CRSwNP groups was not statistically significant  stimulation. FAM NC was used to evaluate transfection efficiency. The results showed that after the transfection of siRNA pendrin-1650, both the mRNA and protein levels of pendrin decreased . SiRNA iThe methods of culturing and transfecting HNECs were described earlier. According to qRT-PCR and WB analyses, mRNA and protein levels of HNE and EGFR expression decreased, with pendrin-1650 being the most efficient siRNA . These rCRS is a common and complex disease that affects the quality of life. The main histopathological manifestations of chronic sinusitis are mucosal epithelial thickening, goblet cell proliferation and/or metaplasia, gland hyperplasia, and edema . MUC5AC However, in our previous research, we found that HNE induces the upregulation of MUC5AC in primary HNECs . Zhao etIt has been reported that several inflammatory factors, including IL-4, IL-8, IL-6, IL-13, and IFN-γ, are significantly increased in the mucosa of CRS patients, and that secretion is progressive . Many stPendrin is mainly expressed in the inner ear, thyroid, and kidney; however, recent studies showed that it is also expressed in airway diseases . One stuThe expression of EGFR is low in the normal respiratory epithelium; however, when activated by HNE, EGFR expression is enhanced, which promotes cell metaplasia and mucus secretion, especially the expression of MUC . The secWith IL-13 stimulation, pendrin may promote the secretion and expression of mucin in the epithelium and glands through EGFR signaling, which can lead to the onset of type 2 inflammation.There are still some limitations in our study. First, we did not use the EGFR pathway blocker to explore the relations between EGFR and MUC. Second, we lacked animal models. Experiments in vivo should be carried out to further explore the function of these genes in CRS.In our study, stimulation of HNECs with IL-13 resulted in upregulated MUC5AC, MUC5B, and pendrin expression at the levels of transcription and translation. In addition, the knockdown of pendrin inhibited MUC secretion. Thus, the results of our study indicate that targeting pendrin has the potential as a therapeutic strategy for mucin hypersecretion in CRS patients.


Co-pyrolysis is one possible method to handle different biomass leftovers. The success of the implementation depends on several factors, of which the quality of the produced bio-oil is of the highest importance, together with the throughput and constraints of the feedstock. In this study, the fast co-pyrolysis of palm kernel shell (PKS) and woody biomass was conducted in a micro-pyrolyser connected to a Gas Chromatograph–Mass Spectrometer/Flame Ionisation Detector (GC–MS/FID) at 600 °C and 5 s. Different blend ratios were studied to reveal interactions on the primary products formed from the co-pyrolysis, specifically PKS and two woody biomasses. A comparison of the experimental and predicted yields showed that the co-pyrolysis of the binary blends in equal proportions, PKS with mahogany (MAH) or iroko (IRO) sawdust, resulted in a decrease in the relative yield of the phenols by 19%, while HAA was promoted by 43% for the PKS:IRO-1:1 pyrolysis blend, and the saccharides were strongly inhibited for the PKS:MAH-1:1 pyrolysis blend. However, no difference was observed in the yields for the different groups of compounds when the two woody biomasses (MAH:IRO-1:1) were co-pyrolysed. In contrast to the binary blend, the pyrolysis of the ternary blends showed that the yield of the saccharides was promoted to a large extent, while the acids were inhibited for the PKS:MAH:IRO-1:1:1 pyrolysis blend. However, the relative yield of the saccharides was inhibited to a large extent for the PKS:MAH:IRO-1:2:2 pyrolysis blend, while no major difference was observed in the yields across the different groups of compounds when PKS and the woody biomass were blended in equal amounts and pyrolysed (PKS:MAH:IRO-2:1:1). This study showed evidence of a synergistic interaction when co-pyrolysing different biomasses. It also shows that it is possible to enhance the production of a valuable group of compounds with the right biomass composition and blend ratio. Fast pyrolysis can be used to convert biomass into bio-oil and chemicals. The pyrolysis oil  is composed of compounds such as anhydrosugars, furans, alcohols, ketones, aldehydes, acids, and phenols ,2. HowevThe co-pyrolysis of biomasses is described as the pyrolysis of blends, including two or more different biomasses. Previous studies have shown this to improve the overall quality of the pyrolysis oil, such as by increasing the calorific value and promoting the yield of volatile compounds ,13,14,15A review of most of the previous studies shows that the information is scarce about the primary products’ characteristics and interactions during the fast co-pyrolysis of different native biomass blends comprising an agricultural residue and a woody biomass residue. Moreover, most of the studies were carried out with the use of a fixed-bed and/or thermogravimetric analyser (TGA) ,20,22,23A summary of the proximate analysis in terms of the moisture content, volatile matter, fixed carbon, and ash content, as well as the biomass characterisations for all three biomasses used as blends in this study, are shown in The product distribution from the pyrolysis of the individual biomasses and their blends are found in To investigate the synergistic effects on the product yield of the co-pyrolysed biomass blends, the yield based on pure biomass according to the blend ratio was added  and compared to the measured value . An analysis of the volatile compounds formed for the two biomass blends during pyrolysis, as shown in The relative yields of phenol, 1-hydroxy-2-propanone, and furfural for PKS co-pyrolysed with either MAH or IRO in equal proportions showed no difference. However, the relative content of levoglucosan was strongly inhibited for the pyrolysis of PKS:MAH-1:1. The inhibition of levoglucosan for the PKS:MAH-1:1 blend and the promotion of AA, and HAA when PKS is co-pyrolysed with MAH or IRO, as well as the enhancement of 2,6-dimethoxyphenol and furfural for the co-pyrolysis of the woody biomass, are evidence of a synergistic interaction of the biomass components.2O and CaO decreased, and the decreased yield was most pronounced for the guaiacol and 4-allyl-2-6-dimethoxyphenol compounds. Chang et al. [Nannochloropsis sp. (NC) were co-pyrolysed at 600 °C with a blend ratio of 1:1. The inhibition of the phenols is important for improving the quality of the bio-oil, as they contribute to the instability of the bio-oil during storage and transport [The experimental (Exp.) and predicted values (Pred.) for the different classes of compounds are presented in g et al.  also obsransport . No diffransport . The majransport ,44. Richransport  observedransport . Their rransport , who invransport .Additionally, a comparison of the experimental and predicted values for the woody biomasses (MAH and IRO), co-pyrolysed in equal proportions, indicates that no difference was observed in the relative yield across the different classes of compounds. This could be due to the woody biomasses being composed of similar polysaccharide structures. The result is similar to that presented by Edmunds et al. , who obs4  to 7.53 × 104  for PKS:MAH-1:1, from 3.57 × 104  to 7.36 × 104  for PKS:IRO 1:1, and, finally, from 3.60 × 104  to 6.59 × 104  for MAH:IRO 1:1. This shows that co-pyrolysis could lead to an increase in the amount of volatile compounds formed.Considering the total yields of the volatile compounds formed (count/µg sample) from the pyrolysis of the biomass blends PKS:MAH-1:1, PKS:IRO-1:1, and MAH:IRO-1:1, it can be seen that the experimental values were about two times higher than the predicted values . The yieThe pyrolysis of the three biomass blends was achieved by first blending them in equal proportions (PKS:MAH:IRO-1:1:1 (0.33:0.33:0.33)), and then, secondly, mixing one part of the PKS with two parts each of the woody biomass (PKS:MAH:IRO-1:2:2 (0.20:0.40:0.40)), and thirdly, using two parts of the PKS and one part each of the woody biomass (PKS:MAH:IRO-2:1:1 (0.50:0.25:0.25)).Similar to the two biomass blends, the main chemical compounds formed for each class of the three-biomass blend were as follows: phenol (phenols), 1-hydroxy-2-propanone (ketones), HAA , AA (acids), levoglucosan (saccharides), and furfural (furans) see . A compaThe results from the three biomass blends showed an impact on the primary pyrolysis product. While the yield of the saccharides was promoted when the three biomasses were blended in equal promotion and co-pyrolysed, an increase in the woody biomass composition resulted in a decreased yield of levoglucosan. Additionally, the synergistic interaction was more pronounced when the blend ratio of PKS was lower in the blend, as seen in the PKS:MAH:IRO_1:1:1 and PKS:MAH:IRO-1:2:2  blends, than when they were blended and co-pyrolysed in equal proportions, as seen in the PKS:MAH:IRO-2:1:1  for the ternary blend. An analysis of the volatile compounds formed in response (count/µg sample), shown in Elaeis guineensis), mahogany (Khaya ivorensis) sawdust, and iroko (Chlorophora excelsa) sawdust. These biomasses were selected because of the environmental concerns associated with their disposal, the volume of the produced waste, and their suitability as feedstock for thermochemical conversion. The woody biomasses (MAH and iroko (IRO) sawdust) were collected from sawmills, while the PKS was obtained from a palm oil site, all in the southern parts of Nigeria. The three biomass samples were milled in a cutting mill (Retsch SM 100) with a screen size of 6 mm and sieved to a particle size of 125–250 µm by a Haver (EML 200 pure) test sieve shaker. The samples were then dried in an oven at 105 °C overnight to a constant weight and stored for further use. The biomasses were then blended into binary and tertiary blends. The binary blends were made from equal amounts of two samples, while the tertiary blends were made from all three samples, but in different proportions. The binary blends correspond to the following weight proportions: PKS:MAH_1:1 (0.50:0.50), PKS:IRO_1:1 (0.50:0.50), and MAH:IRO_1:1 (0.50:0.50), while the tertiary blends correspond to the following weight ratios: PKS:MAH:IRO_1:1:1 (0.33:0.33:0.33), PKS:MAH:IRO_1:2:2 (0.20:0.40:0.40), and PKS:MAH:IRO_2:1:1 (0.50:0.25:0.25).The materials used for this study were palm kernel shell  [0-42618 [The calorific values of the biomass samples were determined with a bomb calorimeter ) by placing 1.0 g of each biomass sample inside a steel vessel containing 500 µL of deionised water. Then, the vessel was filled with oxygen at 30 bar pressure before it was placed inside the equipment in which the sample was ignited.Pyrolysis and analysis of the gaseous product from the raw and blended biomass samples were obtained with a micro-pyrolyser ) connected to a Gas Chromatograph (GC), Mass Spectrometer (MS), and Flame Ionisation Detector (FID), also known as a Py-GC-MS/FID. The biomass samples were blended in different proportions, and close to 500 µg was weighed on an analytical balance (KERN ABT 320-4M) and placed on the platinum filament in the micro-pyrolyser. The platinum filament enables the investigation of primary reactions under isothermal conditions due to its high heating ramp of about 100,000 °C/s and good temperature control. Fast pyrolysis of the raw and blended biomasses was performed at a temperature of 600 °C with a residence time of 5 s. The operating condition was selected in order to obtain the maximum product yield of the released volatile compounds, as reported in previous studies ,29.−1) and a split ratio of 1:7, while the GC oven programme was set at a temperature ramp of 1 min at 60 °C, followed by a ramp of 8 °C/min to 265 °C, and was then held at 265 °C for 20 min. The MS was operated with an ionisation energy of 70 eV, while the MS transfer line and the FID base temperatures were set to 250 °C. Furthermore, the ion-source temperature was kept at 200 °C, and the MS scan was obtained from 25 to 250 m/z. More than 50 chromatography peaks were extracted by the GC-MS Xcalibur software (Version 2.1.0–2.3.0) and identified by the NIST library (NIST MS Search 2.0) and based on previous research [Both the micro-pyrolyser chamber temperature and the transfer tube were set to 150 °C. The low chamber temperature minimises secondary reactions, while it is high enough to keep many of the products in the gas phase. The released pyrolysis vapour was directly transferred to the GC  via a continuous flow of helium gas with a purity of 99.9%. The volatile compounds were identified via the MS  and quantified via an FID (Thermo Scientific). A Zebron™ ZB-5MS (30 m × 0.25 mm × 0.25 µm) capillary column was used with helium carrier gas  decreased the relative yield of phenolic compounds by 19% compared to the pyrolysis of each material individually;The saccharides, mainly levoglucosan, were inhibited to a large extent, while HAA was promoted by 43% for the PKS:IRO-1:1 pyrolysis blend;The relative yields of 2,6-dimethoxyphenol and furfural were also promoted by 21 and 37%, respectively, for the pyrolysis of the MAH:IRO-1:1 blend;No major difference in the relative yield was observed across the different classes of compounds when the woody biomasses were co-pyrolysed together, which is due to their similar chemical structures;The ternary blends showed that the pyrolysis of PKS, MAH, and IRO in equal proportions (PKS:MAH:IRO_1:1:1) led to an increase in the relative yield of the saccharides to a large extent, while an increase in the proportion of the woody biomass in the pyrolysis blend (PKS:MAH:IRO-1:2:2) led to a strong inhibition in the relative yield of the saccharides;Analysis of the individual volatile compounds formed shows that the pyrolysis of PKS:MAH:IRO-1:2:2 resulted in a decreased yield of phenols by 25%, while the relative yields of HAA and levoglucosan were promoted by 34 and 24%, respectively, for PKS:MAH:IRO_1:1:1.


When comparing clinically approved drugs and POMs, better results of POMs in relation to drugs were observed in many cases, since the dose required to have an inhibitory concentration of 50% is 2 to 200 times less, depending on the POMs, highlighting that these compounds could become in the future an alternative to existing drugs in cancer therapy.Polyoxometalates (POMs) are oxoanions of transition metal ions, such as V, Mo, W, Nb, and Pd, forming a variety of structures with a wide range of applications. Herein, we analyzed recent studies on the effects of polyoxometalates as anticancer agents, particularly their effects on the cell cycle. To this end, a literature search was carried out between March and June 2022, using the keywords “polyoxometalates” and “cell cycle”. The effects of POMs on selected cell lines can be diverse, such as their effects in the cell cycle, protein expression, mitochondrial effects, reactive oxygen species (ROS) production, cell death and cell viability. The present study focused on cell viability and cell cycle arrest. Cell viability was analyzed by dividing the POMs into sections according to the constituent compound, namely polyoxovanadates (POVs), polyoxomolybdates (POMos), polyoxopaladates (POPds) and polyoxotungstates (POTs). When comparing and sorting the IC The application of metals such as platin (Pt), lithium (Li), tungsten (W), gold (Au), and vanadium (V), among others, within chemical species, complexes, compounds and/or nanoparticles in biology has been a rapidly growing branch of science ,4,5,6,7.10) is perhaps the most widely studied POV in biology, affecting several biochemical and cellular processes ·6H2O (abbreviation V18) being a vanadium ·6H2O, also designated in the article by compound 1, in order to facilitate [2Na[AsIIIMo6O21(O2CCH2NH3)3]·6H2O (abbreviation POM@SiO2) [2Na2[γ-Mo8O26(O2CCH2NH3)2]·6H2O, designated as compound 2, which are heteropolymolybdates .5H2O [2)2(Mo8O26)]·8H2O .5H2O ; and fin6)]·8H2O .8[Pd13As8O34(OH)6]·42H2O (abbreviation Pd13), Na4[SrPd12O6(OH)3(PhAsO3)6(OAc)3]2NaOAc·32H2O (abbreviation SrPd12), Na6[Pd13(PhAsO3)8]·23H2O (Pd13L), Na12[SnIVO8Pd12(PO4)8]·43H2O (abbreviation SnPd12), Na12[PbIVO8Pd12(PO4)8]·38H2O (abbreviation PbPd12) 20H2O (abbreviation PW9Cu) which has a Dawson structure [6O4(H2O)3[Co(hmta)SbW8O31]3}15− [3GeW9O34]4−·26H2O PAC-320 that has been shown to be an inhibitor of histone deacetylase (HDACi) [2O)4][Na0.7Ni5.3(imi)2(Himi)(H2O)2(SbW9O33)2]} 10H2O, designated as compound 1 and H3[(CH3)4N]4[Na0.7Co5.3(imi)2(Himi)(H2O)2(SbW9O33)2] 12H2O, also known as compound 2 .8H2O, where it was shown to be quite effective with a minimum inhibitory concentration of approximately 135 μg/mL, which is the lowest value reported so far for any octamolybdate-based POM ·8H2O (2Na[AsIIIMo6O21(O2CCH2NH3)3]·6H2O (POM@SiO2) with the same structure as the previous one, but inserted in silica nanoparticles  at 72 h ·8H2O . This sa at 72 h , however13  3}]13− which presents a homochiral, sandwich-like structure 6{CoSb6O4(H2O)3[Co(hmta)SbW8O31]3}]13− being 1.78 µM [2O)6{CoSb6O4(H2O)3[Co(hmta)SbW8O31]3}]13− with a value of 12.75 µM 6H2O 6H2O , represeIn order to analyze in detail which type of POM affected the different phases of the cell cycle in the respectively studied lines, a table was prepared, with the division of the phases of the cell cycle that were blocked by corresponding POMs and respective cell lines .50 previously determined and are described in It should be noted that in the articles where several POMs were studied, cell cycle arrest was only tested for the compound that showed the greatest efficacy. Likewise, of the cell lines analyzed, only those that had presented the best efficacy values were focused on, to verify cell arrest. Therefore, in 2Na[AsIIIMo6O21(O2CCH2NH3 )3]·6H2O (POM@SiO2)), stopping in the S phase [As can be seen, the S phase is predominant under the action of the POMs with 14 of them, followed by the G2/M phase with nine polyoxometalates, and two in the G1 phase. It is also verified that VMOP-31 is the only one that has the ability to stop the cell cycle of SMMC-77221 cells in two distinct cell phases, namely S and G2/M . On the  S phase .Most of the proposed modes of action for antitumor POMs were recently reviewed . Among tIn this review paper, bibliographic research was carried out with the keywords “polyoxometalates” AND “cell cycle”. Thirteen articles were selected on the effect of POMs with anticancer activities, namely in gastric, colon, liver, lung, ovary, breast, prostate, leukemia, osteosarcoma, neuroblastoma and human hepatocellular carcinoma. The types of cancer in which the articles most focused on were lung and breast cancer, with six articles each, followed by liver cancer, covered in four articles. The effects of POMs on cancer cells can be diverse, such as their interaction in the cell cycle, protein expression, mitochondrial effects, ROS production and cell viability. In the present study, we focused mainly on cell viability and cell cycle arrest, since all selected articles analyzed these two effects.50 values were compared and sorted in ascending order, it was found that the POV (VMOP-31) at 24, 48 and 72 h presented, respectively, the lowest values of 1.52, 0.63, and 0.53 µM, in the MCF-7 (human breast cancer cell line). In clinically approved drugs, the lowest IC50 value was found to be 5.78 µM in the BCG-823 lineage at 48h using cisplatin. When comparing drugs and POMs, better results of POMs were observed. In many cases, the POMs dose required to have an inhibitory concentration of 50% is 2- to 200-times lower than the dose that would be necessary with a clinically approved drug. Therefore, POMs are future potential candidates for cancer therapeutic applications.Cell viability was analyzed by dividing the POMs into sections according to the constituent compound, namely POVs, POMos, POPds and POTs. When the ICIn addition to the cell viability, the effect of POMs on cell cycle arrest is highlighted. For the majority of the POMs, cell cycle arrest occurs mostly in the S phase (56%), where DNA synthesis occurs, whereas 36% blocks the G2/M phase. Fewer POMs interfere with the G1 phase (8%), that is, at the beginning of the cell cycle. Although the mechanism of action, directly and/or indirectly, responsible for the POMs cell cycle arrest is still to be deduced and clarified, the scientific evidence described above strengthens the potential use of such metallodrugs in anticancer therapy in the near future.


As these three GVHD prevention/treatment modalities continue their path toward clinical application, it is imperative the field understand both the biological advantages and disadvantages of each approach.The development of graft versus host disease (GVHD) represents a long-standing complication of allogeneic hematopoietic cell transplantation . Different approaches have been used to control the development of GVHD with most relying on variations of chemotherapy drugs to eliminate allo-reactive T cells. While these approaches have proven effective, it is generally accepted that safer, and less toxic GVHD prophylaxis drugs are required to reduce the health burden placed on allo-HCT recipients. In this review, we will summarize the emerging concepts revolving around three biologic-based therapies for GVHD using T regulatory cells (Tregs), myeloid-derived-suppressor-cells (MDSCs) and mesenchymal stromal cell (MSC) exosomes. This review will highlight how each specific modality is unique in its mechanism of action, but also share a common theme in their ability to preferentially activate and expand Treg populations  Graft versus-host disease (GVHD) remains a significant problem following an allogeneic hematopoietic cell transplantation  with GVHD patients experiencing a lower quality of life and a higher risk of death. Our understanding of the cellular and molecular mechanisms of GVHD has provided researchers additional clarity on the etiology of its pathogenesis. This has allowed for a growing number of non-biologic and biologic-based GVHD prophylaxis/treatment therapeutics to be investigated as safer modalities for allo-HCT patients. Specifically, the investigation into the use of biologics for GVHD therapies is still in its infancy but there are several exciting possibilities in the pipeline. In this mini-review, we will highlight three emerging biologic-based therapies that are at different stages of conceptualization and development which include T regulatory cells (Tregs), myeloid-derived-suppressor cells (MDSCs) and mesenchymal stromal cell (MSC) exosomes. We will also emphasize the similarities and differences of each modality in terms of biologic activity and possible clinical use.via CD39/CD73 activity (T regulatory cells (Tregs) represent a T cell polarization state with potent immunosuppressive activity –4. Classactivity Figure 1While the activity of Tregs is predicated on the recognition of self-peptides presented by antigen-presenting-cells (APCs) to prevent the development of an autoimmune disease, Tregs can also develop against non-self-peptides in the periphery . Moreovehigh characteristic of all Treg populations  develop during T cell development and are characterized in mice by the expression of neuropilin-1, the transcription factor Helios and in the expression of CD3, CD4, FOXP3 and CD25ulations –13. Mostulations . This Tr17 polarization. Studies have shown that iTregs can be polarized into TH17 cells and vice versa, highlighting the plasticity of this population and how the balance of iTreg/TH17 polarization can influence immune responses  exit the thymus as conventional T cells but are polarized to iTregs in environments with high levels of TGFβ, retinoic acid, IDO, and other mediators , 14, 15.esponses –20.In both pre-clinical mouse models and clinical studies, it has been established that elevated Tregs frequencies in the graft is correlated with lower rates of GVHD –26. AnotThe TCR repertoire of tTregs and iTregs do not overlap, highlighting the different antigens they recognize (germline self-antigens vs microbiota-derived antigens) , 28, 29.in vitro  may be equally important –35. mTORrization , 37. Add17 cells –37.The polarization of naïve CD4 T cells to iTregs by sirolimus makes it an effective therapeutic candidate that is currently used in the clinic. One multicenter study (BMT-CTN-1501) revealed that sirolimus was equivalent to prednisone for initial GVHD treatment and resulted in a shorter time to immune suppression discontinuation . Anotherin vivo should continue to be prioritized.With so many current GVHD drugs connected with an expansion of the iTreg population, it is logical for a growing number of clinical trials to investigate the direct treatment of Tregs. Unfortunately, iTreg adoptive therapies are hindered by high production costs, the inability to generate/store multiple doses and the need for a patient specific product. Thus, the investigation into new therapies preferentially expand/polarize iTregs Myeloid-derived-suppressor-cells (MDSCs) represent a heterogeneous population of cell types with the general function of dampening immune responses –50. MDSC+, CD33high) but also express several M2 markers  and are CD16-/HLA-DR- while both classical and inflammatory monocytes are all HLA-DR+  but also express the same immunosuppressive markers listed above  but maintain expression of CD33 and the immunosuppressive markers ; however, they have also been shown to prevent GVHD –58. One MDSCs populations have been shown to use a variety of molecular pathways to control T cell responses including indoleamine-pyrrole 2,3-dioxygenase (IDO), cyclooxygenases, arginase, TGF-β, IL-10 and PD-L1 Figure 12 by the receptors EP2 or EP4 have been shown to prevent the polarization of T cells into TH1 cells and directly promote the expression of FOXP3 similar to TGF-β , mRNA and possibly even DNA , 82, 83.As an ever-growing number of studies support the role of exosomes in generating MDSC populations, the mechanism(s) utilized by exosomes to generate these MDSC populations remains largely unexplored Figure 1Compared to standard cellular therapies, exosomes have several fundamental advantages including i) long-term storage, ii) low-immunogenicity, iii) no requirement for HLA-matching and iv) the ability to be mass-produced , 81, 89.Despite the lack of approved exosome-based therapies, there are a growing number of pre-clinical studies using exosomes to treat a variety of diseases. Most of these studies have utilized MSC-based exosomes, which builds upon the growing field of MSC-based cellular therapies, as they contain similar immunosuppressive properties. A series of studies by one team of investigators have sequentially shown that the treatment with human MSCs, or exosomes “educated” myeloid cells were able to prevent lethal acute radiation syndrome (ARS) , 82, 90.The use of MSCs for GVHD prevention and treatment has been explored in a large number of trials with most reporting that MSCs are safe and well tolerated. Unfortunately, the response rates among trials varied considerably and FDA approval is still elusive . RecentlThe field of GVHD research is at an exciting inflection point where the investigation into biologics as novel prophylaxis/treatment modalities may soon become standard. While the three modalities discussed in this review are all poised to make significant advances toward clinical use, these biologic modalities are not without their limitations. Similar to the experiences of CAR manufacturing, the use of Tregs or MDSCs as a cellular therapy will always be challenged with their production times, the manufacturing process and capacity to be used in the allogeneic setting. For these reasons, MSC-exosome treatment represents an exciting and promising modality. Exosome treatments have the potential to be used in the allogeneic setting, mass-produced for repeated injections, and stored for immediate use. Their eventual use in the clinic will ultimately be decided by their ease of use and long-term efficacy compared to direct cellular therapies which boast strong short term activity but are limited to a single infusion and long-term activity.Promoting the expansion and activation of Tregs after allo-HCT remains the most validated method of suppressing GVHD though the optimal approach to achieve this remains under investigation. Exploring the use of MDSCs and MSC-exosomes as novel approaches to activate Tregs should be a focus of investigation in the next decade. The development of these and other biologic-based prophylaxis/treatment modalities will offer allo-HCT recipients an improved quality of life by reducing off-target toxicity and improving overall efficacy by directing their new immune system to control the development of GVHD.NH drafted and performed the literature search. NH, JK and PH revised and edited the manuscript. All authors approved the final version of this manuscript. All authors contributed to the article and approved the submitted version.


BRCA-mutated breast cancer (BC), including triple-negative BC (TNBC) and ovarian cancer (OvCa). A key challenge is to identify the factors associated with PARPi resistance; although, previous studies suggest that platinum-based agents and PARPi share similar resistance mechanisms.Poly (ADP-ribose) polymerase inhibitors (PARPi) are approved for the treatment of BRCA-mutated TNBC cell lines. In-silico analysis were performed using multiple databases including The Cancer Genome Atlas, the Genotype-Tissue Expression, The Cancer Cell Line Encyclopedia, Genomics of Drug Sensitivity in Cancer, and Gene Omnibus Expression.Olaparib-resistant (OlaR) cell lines were analyzed using HTG EdgeSeq miRNA Whole Transcriptomic Analysis (WTA). Functional assays were performed in three p = 0.001) as well as in tumor tissues from TNBC patients (p = 0.001). We hypothesized that miR-181a downregulates the stimulator of interferon genes (STING) and the downstream proinflammatory cytokines to mediate PARPi resistance. BRCA1 mutated TNBC cell lines with miR-181a-overexpression were more resistant to olaparib and showed downregulation in STING and the downstream genes controlled by STING. Extracellular vesicles derived from PARPi-resistant TNBC cell lines horizontally transferred miR-181a to parental cells which conferred PARPi-resistance and targeted STING. In clinical settings, STING levels were positively correlated with interferon gamma (IFNG) response scores (p = 0.01). In addition, low IFNG response scores were associated with worse response to neoadjuvant treatment including PARPi for high-risk HER2 negative BC patients (p = 0.001). OlaR TNBC cell lines showed resistance to platinum-based drugs. OvCa cell lines resistant to platinum showed resistance to olaparib. Knockout of miR-181a significantly improved olaparib sensitivity in OvCa cell lines (p = 0.001).High miR-181a levels were identified in OlaR TNBC cell lines (miR-181a is a key factor controlling the STING pathway and driving PARPi and platinum-based drug resistance in TNBC and OvCa. The miR-181a-STING axis can be used as a potential marker for predicting PARPi responses in TNBC and OvCa tumors.The online version contains supplementary material available at 10.1186/s13578-023-01151-y. BRCA-mutated (gBRCA mt) or HER2-negative metastatic breast cancer (BC) who have been treated with chemotherapy either in the neoadjuvant, adjuvant, or metastatic settings [Poly (ADP‑ribose) polymerase inhibitors (PARPi) promote DNA damage accumulation in tumor cells with homologous recombination (HR) deficiency, leading to the concept of synthetic lethality . As a resettings . PARPi asettings . Severalsettings , 7. Althsettings . TherefoBRCA mt ovarian cancer (OvCa) [Three large clinical studies, using PARPi treatment for patients with r (OvCa) , 10, conr (OvCa) ; althougRecently, we reported that the downregulation of the cytosolic DNA sensor stimulator of interferon genes (STING) leads to cisplatin resistance by decreasing the expression of downstream proinflammatory cytokines in triple-negative breast cancer (TNBC) . STING iHomo sapiens (hsa)-miR-181a-5p (or miR-181a-5p from this point on) downregulates STING and thereby allows fallopian tube secretory epithelial cells (FTSEC) to bypass interferon-mediated cell death leading to cancer cell transformation and development of high-grade serous ovarian cancer (HGSOC) [hypothesized that the factors regulating STING levels modulate the downstream signaling in TNBC and OvCa and correlate with PARPi sensitivity as well as cross-resistance to platinum-based drugs.Previously, we reported that tumor tissue  (HGSOC) . High tu (HGSOC) . Thus, win-vitro findings were validated using clinical specimens from TNBC and OvCa patients. This study describes a novel mechanism of PARPi resistance, as well as platinum-based drug cross-resistance; and suggests that the upregulation of miR-181a is a significant factor predicting PARPi responses in TNBC and OvCa.In this study, we obtained the miR profiles of PARPi resistant cell lines using HTG EdgeSeq miRNA Whole Transcriptome Assay (HTG miR WTA) and identified that miR-181a is significantly upregulated in PARPi-resistant TNBC cell lines. Functional assays were then performed to characterize the role of miR-181a in controlling STING pathways as a mechanism of PARPi resistance. Furthermore, the The study was conducted following the Declaration of Helsinki and was approved by the Ethics Committee at Saint John’s Cancer Institute (SJCI) and WIRB: MORD-RTPCR-0995. Six formalin-fixed paraffin-embedded (FFPE) OvCa tissues surgically resected were obtained from patients at the Department of Obstetrics and Gynecology, Perelman School of Medicine, U. of Pennsylvania, PA. Three tissues were treatment naïve at the time of surgery and three tissues were from a second debulking surgery after patients relapsed to adjuvant chemotherapy (Carboplatin/Paclitaxel). All the patient specimens were de-identified. Furthermore, a clinically annotated tissue microarray (TMA) for TNBC (#BR1301) was obtained from US Biomax .BRCA mt TNBC cell lines MDA-MB-436 (BRCA1 mt), HCC1395 (BRCA1 mt), and HCC1937 (BRCA1 mt) were obtained from the American Type Culture Collection  and were cultured as recommended. The establishment of HCC1937 and HCC1395 cell lines olaparib-resistant (OlaR) are described in Additional file Three established human -436 BRCA mt, HCC1Total RNA from cell lines was extracted by the Direct-zol RNA miniprep kit  according to the manufacturer’s instructions. RNA isolation from EV is described in Additional file The HTG miR WTA  was utilized to assess the difference in human miR transcripts between parental and OlaR cell lines using direct next-generation sequencing (NGS) as previously described . HTG miRPurified lentiviral particles for STING (LPP-E1218-Lv103-050), miR-181a (LPP-HmiR0023-MR03-050-S), and their respective controls   were transduced into TNBC cell lines MDA-MB-436, HCC1395, and HCC1937 as previously described [Knockdown experiments were performed as previously described . TNBC ceOlaparib  was dissolved in molecular-grade water at a concentration of 10 mM. For cell viability assays, the measurements were performed after treatment with different concentrations  of olaparib for 48 h, as previously described , 18. ForBC FFPE TMA sections were stained with STING antibody  by immunohistochemistry (IHC) as previously described [In-situ hybridization (ISH) for miR-181a was performed on a FFPE TNBC TMA section and FFPE OvCa sections using the miRNAscope Assay  according to the manufacturer’s instructions and as previously described [escribed . MiR-1813) were seeded in a 96-well culture plate. The number of viable cells was assessed using a Cell Titer-Glo Luminescent Cell Viability assay  according to the manufacturer’s instructions as previously described [TNBC cell lines (1 × 10escribed . For asshttp://imagej.nih.gov/ij/). Western blotting was performed for CD9, CD63, and CD81 in EV derived from HCC1937 and HCC1395 parental cell lines.Traditional western blot was performed as previously described , except Automated western blotting was performed according to the manufacturer’s protocol , and quantified as previously described . ProteinFor a detailed explanation of EV isolation please refer to Additional file https://www.R-project.org/.) in a two-tailed way. The distribution and variation within each group were assessed before statistical analysis. Two groups were compared using Student’s t-test. Multiple groups were analyzed by One-way or Two-way ANOVA followed by a post-hoc Tukey’s multiple comparisons test. The correlation between variables was determined using Pearson’s correlation test. Overall survival (OS), recurrence-free survival (RFS), and distant metastasis-free survival (DMFS) were calculated from the time of taking the first specimen until death/recurrence/distant metastasis or last contact and were analyzed using the Log-rank test. For miRNA analysis, DESeq2 normalization and statistical comparisons were performed using the HTG REVEAL software version 2.0.1. Differentially expressed miRNAs were screened using Log2 fold change (FC) > 1.5 or < − 1.5, median normalized counts > 1000. MiR expression (counts per million) was logarithmically scaled (Log10) for volcano plot data visualization. A two-sided p < 0.05 was considered statistically significant: p < 0.05, **p < 0.01, ***p < 0.001, and ns = not significant. All figures were unified using Adobe Illustrator CC  or CorelDraw graphics suite 8X .Statistical analyses were performed using the GraphPad Prism 8  or R version 4.1.2 (BRCA1 mt olaparib-resistant (OlaR) TNBC cell line was initially established as described in Additional File HCC1937 In-silico analysis using the TCGA BRCA dataset showed that miR-181a levels were significantly higher in primary BC compared to adjacent normal breast tissues   with miR-181a overexpression  but increased PARPi sensitivity in BRCA1 mt TNBC cell lines significantly  was performed in the three TNBC cell lines using lentiviral transduction  compared to normal breast and adjacent normal breast tissues , IFNB, interleukin-12A (IL12A), and interleukin-12 (IL12B) compared to respective control cell lines in RT-qPCR analysis  as the vehicle for miR-181a delivery to non-resistant cell lines. EVs derived from HCC1937 TNBC parental  and OlaR (OlaR-EV) cell lines were isolated using differential ultracentrifugation (DUC) from culture supernatants. EVs were characterized using Western blotting, NTA, FL-NTA, and ACE. By western blot, the three EVs standard markers  were detected, indicating the purity and characteristics of the EV fractions isolated  in both BC and OvCa cell lines  and BRCA1 promoter methylation predicted response to rucaparib [The early detection of drug resistance as well as the understanding of the intrinsic mechanisms that induced PARPi resistance are important for both TNBC and OvCa patients. Thus, the prospects for developing effective strategies for the early detection of PARPi resistance are a growing field. Previous studies have shown that germline and somatic and OvCa , 3. Alteand OvCa –29. A reucaparib .Tumors that are sensitive to platinum-based drugs are also sensitive to PARPi treatment , 32. OurThe present study offers additional insight into miR-181a regulatory functions controlling STING mRNA and protein levels. MiR-181a targets STING and promotes metastasis as well as recurrence in advanced stage HGSOC . Our stuSeveral studies have shown that the activation of STING and the stimulation of type I IFN production are critical for anticancer immune responses. Activation of STING signaling induces the production of type I IFN, which plays critical roles in activating both innate and adaptive immune responses . A studySeveral clinical trials have been conducted to test the efficacy of PARPi plus immune checkpoint inhibitors (ICI) in ovarian, breast, prostate, lung, bladder, gastric cancers, and other solid tumors . MiR-181PIAS3) [Recently, EVs have attracted extensive attention for their role in drug resistance , 38. ThePIAS3) , which tPIAS3) . This stTNBC cell lines resistant to PARPi showed enhanced levels of miR-181a  HCC1937 cell line treated with different concentrations of olaparib (B) or cisplatin (C) (Two-way ANOVA and Sidak’s multiple comparisons test). D Cell viability assays comparing parental and OlaR HCC1937 cell line (Two-way ANOVA and Sidak’s multiple comparisons test). E Representative images for GFP positive cells with miR-181a-OV in MDA-MB-436, HCC1395, and HCC1937 cell lines using fluorescence microscopy. Scale bars: 100 µm. F–H Cell viability assays comparing miR-181a-OV and empty vector (CTRL) in MDA-MB-436 (E), HCC1395 (F), and HCC1937 (G) cell lines (two-way ANOVA and Sidak’s multiple comparisons test). I Cell viability assays comparing miR-181a-OV and empty vector (CTRL) HCC1937 cell lines treated with different concentrations of cisplatin (two-way ANOVA and Sidak’s multiple comparisons test). Cell viability assays were performed in triplicates. Figure S2. STING overexpression or downregulation does not affect cell proliferation. A Representative images for STING-OV (GFP) in MDA-MB-436, HCC1395, and HCC1937 cell lines using fluorescence microscopy. Scale bars: 100 µm. B–D Cell viability assays comparing STING-OV and empty vector (CTRL) in MDA-MB-436 (B), HCC1395 (C), and HCC1937 (D) cell lines (two-way ANOVA and Sidak’s multiple comparisons test). E–G Cell viability assays comparing si-STING and si-CTRL in MDA-MB-436 (E), HCC1395 (F), and HCC1937 (G) (two-way ANOVA and Sidak’s multiple comparisons test). Figure S3. STING is downregulated in TNBC and relates to outcomes. A STING mRNA levels in normal breast  and primary BC (Primary) tissues in the TCGA and GTEx databases (Student’s t-test). BSTING mRNA levels in tissues from tumor-adjacent normal breast , Luminal (Lum), Luminal-HER2 (Lum-HER2), HER2, and TNBC in the TCGA BRCA dataset (One-way ANOVA and Tukey’s multiple comparisons test). CSTING mRNA levels in tissues from the tumor-adjacent normal breast , Normal-like (Norm-like), Luminal-A (LumA), Luminal-B (LumB), HER2-enriched, and basal-like breast cancer (BLBC) in the TCGA BRCA dataset (One-way ANOVA and Tukey’s multiple comparisons test). D–F Survival analysis of RFS (D), OS (E), and DMFS (F) for patients with TNBC in the TCGA, GEO, and EGA databases combined (Log-rank test). G–I Survival analysis of RFS (G), OS (H), and DMFS (I) for patients with BLBC in the TCGA, GEO, and EGA database combined (Log-rank test). *p<0.05, **p <0.01, ***p <0.001. Figure S4. Analysis of the mRNA levels of the downstream components of the STING pathways. A–E Quantification by RT-qPCR of STING (A), IL6 (B), IFNB (C), IL12A (D), and IL12B (E) mRNA levels in empty vector (CTRL) and STING-OV HCC1395 cell line (Student’s t-test). F–J Quantification by RT-qPCR of STING (F), IL6 (G), IFNB (H), IL12A (I), and IL12B (J) mRNA levels in empty vector (CTRL) and STING-OV MDA-MB-436 cell line (Student’s t-test). K–O Quantification by RT-qPCR of STING (K), IL6 (L), IFNB (M), IL12A (N), and IL12B (O) mRNA levels in miR-181a-OV and CTRL in HCC1395 cell line (Student’s t-test). P-T Quantification by RT-qPCR of STING (P), IL6 (Q), IFNB (R), IL12A (S), and IL12B (T) mRNA levels in miR-181a-OV and empty vector (CTRL) in MDA-MB-436 cell line (Student’s t-test). *p<0.05, **p <0.01, ***p <0.001. Figure S5. Correlation analysis between miR-181a levels and drug activities. A–D Correlation between miR-181a levels and cisplatin (A), rucaparib (B), olaparib (C), talazoparib (D) drug activity in the BC cell lines obtained from CCLE and GDSC BRCA datasets (Pearson’s correlation coefficient). F–I Correlation between miR-181a levels and cisplatin (F), rucaparib (G), olaparib (H), talazoparib (I) drug activity in the OvCa cell lines obtained from CCLE and GDSC BRCA datasets (Pearson’s correlation coefficient). Figure S6. Uncropped western blotting images. A-C Uncropped western blotting images for Figures 3D (A), H (B), and I (C) are shown. Figure S7. Uncropped western blotting images. A–D. Uncropped western blotting images for Figures 3J (A), K (B), 4A (C), and E (D) are shown. Figure S8. Uncropped western blotting images. A Uncropped western blotting images for Figure 6A (A) and G (B) are shown.Additional file 2: Table S1. Reagents and resources utilized in the study.


Artemisinin is a natural compound extracted from Artemisia species belonging to the Asteraceae family. Currently, artemisinin and its derivatives are considered among the most significant small-molecule antimalarial drugs. Artemisinin and its derivatives have also been shown to possess selective anticancer properties, however, there are several limitations and gaps in knowledge that retard their repurposing as effective anticancer agents. Hybridization resulting from a covalent combination of artemisinin with one or more active pharmacophores has emerged as a promising approach to overcome several issues. The variety of hybridization partners allows improvement in artemisinin activity by tuning the ability of conjugated artemisinin to interact with various molecule targets involved in multiple biological pathways. This review highlights the current scenario of artemisinin-derived hybrids with potential anticancer activity. The synthetic approaches to achieve the corresponding hybrids and the structure–activity relationships are discussed to facilitate further rational design of more effective candidates. Cancer is considered to be one of the most life-threatening diseases throughout the world nowadays. World Health Organization estimated 10 million people worldwide to have died from cancer in 2020 ,4,5. To Molecular hybridization is a modern strategy used in drug discovery for rational drug design . Based oTypically, hybrid molecules containing two or more pharmacophoric units can be classified depending on the manner in which they are covalently linked. In the case of directly linked hybrids  and its derivatives were developed into drugs and became the gold standard for malaria treatment. More recent studies showed that artemisinins also exhibit interesting anticancer activities in vitro and in vivo against a wide range of cancer types ..44]. Simter bond A. Moreovnditions B. Compoue itself .12–19 and 22 were tested for their in vitro cyctotoxicity against HL-60 leukemia cells. All hybrids showed enhanced cytotoxicity with respect to unconjugated dihydroartemisinin (2) with a dihydroartemisinin (2)/hybrid ratio ranging between 3 and 10.5 . Th. Th44 waclitaxel .50) and drug concentration resulting in total growth inhibition (TGI). The anticancer agent etoposide was used as a reference standard drug. Both quinoline–bis(dihydroartemisinin) hybrids 49 and 50 showed anticancer activity against all cancer cell lines tested with GI50 values in the range of 0.03–0.08 μM and TGI values in the range of 2.21–18.50 μM. Both hybrids were found to be much more potent than the reference drug etoposide  (Quinoline–bis(dihydroartemisinin) hybrids 49 and 50  were prectively) . Pharmac3) and the appropriate indoloquinoline derivative in the presence of EDCI and 1-hydroxybenzotriazole was reported . A. A86a–c er cells .85b,c in order to obtain hybrids 88b,c characterized by stable ether linkers of different lengths  were found to be 5- and 7-fold more potent than dihydroartemisinin (2) (IC50 = 2 μM), respectively, and showed comparable cytotoxicity to that of reference drug doxorubicin (IC50 = 0.3 μM)  .93a,b were found to be significantly more effective in inducing apoptotic subG0 population and in triggering mitochondrial membrane potential loss than dihydroartemisinin (2).A mechanism insight in HL-60 cells was also reported. Both hybrids 95a–d, prepared by condensation of dihydroartemisinin (2) and a series of chalcone derivatives 94a–d, is depicted in 95a–c were found to be highly cytoselective towards cancer cell lines with respect to normal cells. Hybrid 95a was the most potent of the series against all cancer cell lines tested, showing comparable or higher cytotoxic activity with respect to reference drug parthenolide , A549 and A-460 (lung), MDA-MB-231 (breast), HeLa .Xie et al. reported ,74 two ses 96a–l . Hybrids99a–l were found to be from 2.6- to 40-fold more active than the parent dihydroartemisinin (2) in all cancer cell lines tested, proving the effectiveness of the conjugation with a chalcone moiety. Moreover, the presence of a chalcone moiety also improved the cytoselectivity towards HT-29 and HeLa cell lines with respect to MDA-MB-231, A549 and H460 cell lines. By comparing hybrids 99i–k it can be evidenced that the bioisosteric replacement of phenyl (99i) with a thiophene (99k) and furan (99j) rings significantly decreased the activity against the five tumor cell lines tested  whereas the extension of the length of the alkyl linker between the dihydroartemisinin and triazole moiety increased the activity (hybrid activity 108 < 109) (All hybrids were tested against selected cancer cell lines HCT116 and HT-29 (colon), MDA-MB-231 (breast) and normal MRC-5 fibroblast cells. By comparing hybrids 8 < 109) C.d, suggesting that the different electronic effect of substituents at the C-3 and C-4 skeleton of the coumarin moiety could play a positive role in the cytotoxicity of hybrids. In particular, hybrid 103d was the most potent with IC50 = 0.05 μM, comparable to that of reference drug doxorubicin. Moreover, hybrid 103d showed the best cytoselectivity towards HT-29 cancer cells with respect to normal MRC-5 cells (MRC-5/HT-29 = 1014)  C. Furtheigration .111b–e as well as of hybrids 114a–e and 115a–e conjugated through ether linkers is depicted in 111b–e, 3-chloro-4-methyl-coumarin-derived hybrid 111d showed the best activity towards the HT-29 colon cancer cell line, being 45-fold more potent than unconjugated dihydroartemisinin (2)  (The synthesis of dihydroartemisin-derived fused hybrids inin (2) C. Among inin (2) C 78]..111b–e aTamoxifen belongs to a class of anticancer drugs that are selective estrogen receptor modulators extensively used in hormone-dependent breast cancer therapy over recent decades ,122. Chr3) and deoxoartemisinin derivative 26 were reacted with two metabolites of tamoxifen, 2-hydroxytamoxifen 116 (afimoxifene) and (Z)-N-desmethyl-tamoxifen 119, to give the corresponding hybrids 117, 118 and 120, 121, respectively  and artesunate (3) against both PC-3 and MCF-7. Hybrid 121 was found to be the most potent of the series with EC50 = 2.74 and 3.91 μM in PC-3 and MCF-7 cells, respectively. Moreover, hybrid 121 showed marked improved activity compared to dihydroartemisinin (2), being 93- and 5-fold more potent in PC-3 and MCF-7, respectively.Frohlich et al. recently reported  the syntectively . Hybrids linkers . All hybSteroids are natural organic bioactive compounds present in animals as well as in plants and fungi. Steroids are considered important scaffolds in medicinal chemistry due to their molecular structure as well as to their ability to interact with various biological targets in different pathways .122 and estrone derivative 125 were conjugated with both artesunate (3) and deoxoartemisinin derivative 26 to give the corresponding hybrids with amide linkers 123, 124  and estrogen derivatives 129 and 131a,b followed by the introduction of the sulfamate moiety and deprotection. All hybrids are characterized by ester linkers. Hybrids 132 were obtained as pure anomers α (132a) and β (132b) . Al. Al133  in comparison to parent dihydroartemisinin (2) and acridine 136a (2). Compound 137b is the only hybrid with significant activity towards the HT29-AK colon cancer cell line with 1.6-fold enhanced activity with respect to unconjugated dihydroartemisinin (2). This result may be related to the length of the linker that seems to play a role in the cytoselectivity and activity in the series of hybrids 136a–c. Hybrid 136b was further investigated in HL-60 leukemia cells. Flow cytometry analysis demonstrated that this hybrid can promote apoptotic cell death and bind covalently to intraparasitic cellular targets in the presence of iron (II) . M. M144a–e 549/DDP B [85].143a,c, 145a and 146c were selected for further pharmacokinetic studies in a CD-1 mouse model by single intravenous administration (dose = 30 mg/kg). The results are reported in Hybrids 2) with isatin derivatives substituted at C-3 with different groups such as methoxy, fluorine and methyl. The synthesis of the most potent hybrids of the series is reported in 149a [149b [149a and 149b were in turn modified at the C-3 position of the isatin moiety with, among others, hydroxime and thiosemicarbazide moieties, leading to hybrids 150a and 150b, respectively. The new hybrids were tested for their antiproliferative activity against breast cancer cell lines MCF-7 and MDA-MB-231 as well as their corresponding multidrug-resistant cell lines. Most of the hybrids were found to be significantly more potent than parent dihydroartemisinin (2) in all cancer cell lines tested and, in particular, hybrids 149a,b and 150a,b were found to be 4- and 5-fold more potent (50 > 100 μM).Cao et al. reported ,87 a ser in 149a  and 149be potent . Accordi2) was reacted with isatin carboxylic acid homologue derivatives 151a–d (153a–c is reported in 153a [2) towards triple-negative MDA-MB-231 and MDA-MB-231/ADR breast cell lines and 75- and 55-fold more potent than in MCF-7 and MCF-7/ADR cell lines, respectively (153b (R = OBn) [153a (153b (R = OEt) [50 = 1.27 μM (MDA-MB-231/MCF-7 = 11.8) (153c [50 = 2.85–10.18 μM). In particular, hybrid 153c was found to be 29-fold more active than dihydroartemisinin (2) in multidrug-resistant triple-negative MDA-MB-231/ADR (2) with isatin through ester bonds could be helpful to overcome drug resistance and to tackle triple-negative breast cancer.Xu et al. reported ,89 a sers 151a–d  and theiectively . AnalogoR = OBn) , with a n) [153a . On the R = OEt) , with a  = 11.8) . Hybrid -231/ADR . The ove154, Sulfasalazine 154,  is an an155 obtained by condensation reaction of dihydroartemisinin (2) and sulfasalazine 154  and sulfasalazine 154 as well as their 1:1 combination. In particular, treatment of TN22 cells with 5 μM of hybrid 155 led to a 60% reduction of cell viability whereas unconjugated dihydroartemisinin (2) and dihydroartemisinin (2) and sulfasalazine 154 1:1 combinations reduced the cell viability by only 20%. No effect on cell viability was found by treatment with sulfasalazine 154 alone. These data prove a relevant effectiveness of molecular hybridization. A deep mechanism insight demonstrated that the remarkable reduction of cell viability can be attributed to apoptosis and cell cycle arrest. Moreover, hybrid 155 showed significant inhibition of cell migration which is an important property to fight malignancies with invasive growth such as glioma.Hybrid 135a in lung carcinoma [The research on artemisinin’s anticancer activity has demonstrated that despite challenges, repurposing of artemisinin and its derivatives as anticancer drugs is possible. Indeed, artemisinins have shown anticancer activity against different cancer types by acting through various pathways. Anticancer efficacy of artemisinins has also been proved in in vivo studies with evidence of tumor growth inhibition in xenograft models. Human trials reported so far have demonstrated that some artemisinins are safe and have significant anticancer efficacy. However, artemisinins are characterized by poor pharmacokinetic properties such as low tissue distribution and short half-life that limit their applicability in clinical anticancer therapy. To avoid these limitations, different strategies have been implemented, such as the use of combination therapy and nanoformulations, that can overcome the pharmacokinetic barriers of artemisinins, as well as molecular hybridization. This last option can help not only to overcome the intrinsic limitations of artemisinins but also to improve anticancer activity by lowering the toxicity as a result of enhanced cytoselectivity and to improve the efficacy against multidrug-resistant cancers. The discovery of artemisinin’s anticancer activity is relatively recent, therefore, the research on artemisinin moiety molecular hybridization has been developed mainly over the last 10–15 years. Although many efforts have been made, as demonstrated by the numerousness of the hybrids reported in the literature, the lack of systematic studies on the molecular mechanism of action of hybrids as well as the lack of in vivo studies still limit the development of new artemisinin-based hybrid drugs. Indeed, while many hybrids among the herein reported ones showed interesting improved anticancer activity against selected cancer cell lines, few cases have been extensively studied, for instance, ursodeoxycholic bile acid hybrids 13 and 22 in hepatocellular carcinoma ,46, cinnarcinoma  and sulfarcinoma . Up to narcinoma  and quinarcinoma . More in