Arabidopsis thaliana covering a comprehensive set of distinct tissues. By combining fluorescence-activated nucleus sorting and laser-capture microdissection with next-generation RNA sequencing, we characterized the transcriptomes of xylem vessels, fibers, the proximal and distal cambium, phloem, phloem cap, pith, starch sheath, and epidermis cells. Our analyses classified more than 15,000 genes as being differentially expressed among different stem tissues and revealed known and novel tissue-specific cellular signatures. By determining overrepresented transcription factor binding regions in the promoters of differentially expressed genes, we identified candidate tissue-specific transcriptional regulators. Our datasets predict the expression profiles of an exceptional number of genes and allow hypotheses to be generated about the spatial organization of physiological processes. Moreover, we demonstrate that information about gene expression in a broad range of mature plant tissues can be established at high spatial resolution by nuclear mRNA profiling. Tissue-specific gene expression values can be accessed online at https://arabidopsis-stem.cos.uni-heidelberg.de/.Genome-wide gene expression maps with a high spatial resolution have substantially accelerated plant molecular science. However, the number of characterized tissues and growth stages is still small due to the limited accessibility of most tissues for protoplast isolation. Here, we provide gene expression profiles of the mature inflorescence stem of  A genome-wide high-resolution gene expression map of the Arabidopsis inflorescence stem is provided, shedding light on cell type properties and the spatial organization of physiological processes. Arabidopsis thaliana (Arabidopsis) , high-resolution gene expression profiles have been established for developmental hotspots such as the root and shoot tips of idopsis) . Althougidopsis)  or hampeidopsis) . The spaPlant stems play fundamental roles in determining shoot architecture and act as transport routes that connect photosynthetically active source organs with the remaining plant body . After tArabidopsis stems, like those in most dicotyledonous species, undergo a major anatomical transition during the initiation of radial growth and extensive wood formation . In stemPopulus sp.) have provided high-resolution transcriptional profiles using serial cryo-sectioning  promoter to label fibers (VASCULAR-RELATED NAC DOMAIN PROTEIN7 (VND7) promoter for differentiating vessel elements (PHLOEM INTERCALATED WITH XYLEM (PXY)/TDIF RECEPTOR (TDR) promoter for differentiating xylem cells and proximal cambium cells (SMAX1-LIKE5 (SMXL5) promoter for differentiating phloem cells and distal cambium cells (ALTERED PHLOEM DEVELOPMENT (APL) promoter for differentiated phloem cells (SCARECROW (SCR) promoter for starch sheath cells (LIPID TRANSFER PROTEIN1 (LTP1) promoter for epidermis cells (pro:H4-GFPGENE). Microscopic inspection of cross-sections from the second bottom-most elongated internode showed that only nuclei in the expected tissues were labeled by GFP , and the phloem (APLpro) by manually chopping the second bottom-most elongated internodes of inflorescence stems, followed by FANS. From each line, we harvested GFP-positive and -negative nuclei (H4-GFP mRNA (detected through the GFP sequence) and the mRNA of the NST3, SMXL5, or APL genes whose promoters were used to express H4-GFP in the respective lines were significantly higher in extracts from GFP-positive nuclei compared with extracts from GFP-negative nuclei in all cases . These findings indicate that cell type-specific transcriptomes were thoroughly accessible following our experimental pipeline. To determine the analytical potential of the obtained datasets, we carried out differential expression analysis and found that the transcriptional activity from 23, 40, and 115 genes was significantly higher in GFP-positive nuclei of the NST3pro, SMXL5pro, and APLpro reporter lines, respectively, compared with GFP-negative nuclei [P <0.05 in the Wald test]. The relatively low number of tissue-specific genes identified in the one-to-one comparisons was mostly due to the noisy character of datasets from GFP-negative nuclei, which also resulted in a lack of significance for the higher activities of the NST3 and APL genes in the respective tissues after using BH adjustment  and phloem-related  datasets showed high correlation coefficients among each other but belonged to two different major branches within the correlation plot  and NAC DOMAIN-CONTAINING PROTEIN86 (NAC086), which are known to be expressed in phloem cells (PIN-FORMED3 (PIN3)  (XYLEM CYSTEINE PROTEASE1 (XCP1) (proVND7-positive nuclei (WUSCHEL RELATED HOMEOBOX4 (WOX4), whose expression domain is congruent with the PXY expression domain (PXYpro-positive nuclei (MORE LATERAL GROWTH1 (MOL1), PHLOEM EARLY DOF1 (PEAR1), and PEAR2, which are expressed in SMXL5pro-positive cells (proSMXL5-positive nuclei (LTP1 reads were not over-represented in LTP1pro-positive nuclei (FIDDLEHEAD (FDH) and ECERIFERUM6 (CER6), which are known to be expressed in the epidermis   were alse nuclei . Likewiser cells , showed e nuclei , and reaof VND7)  and its 1 (XCP1)  showed te nuclei . The actn domain , also pee nuclei . In addie nuclei . Althouge nuclei , the reapidermis , were moe nuclei . Finallye nuclei  had highe nuclei , furthern = 2 replicates). We then extracted RNA from the tissues, amplified mRNA, and analyzed it by RNA-seq  and CYP83B1 genes, which function in the indole and aliphatic glucosinolate biosynthetic pathways and are expressed in the phloem cap , suggesting a lower coverage compared with our FANS-based analyses . The aveloem cap , were siloem cap . In addithe pith , were ovthe pith .P-value in LRT < 0.01). Based on their activity profiles, SDE genes were categorized into 93 clusters using hierarchical clustering (SMXL5pro) and differentiated (APLpro) phloem cells. In line with their strong ontogenetic relationship, genes very active in one of the two tissues were often very active in the second one (SMXL5pro) were distinct from genes active in developing xylem cells (PXYpro). These results suggest that, although these cell types partly originate from the same procambial precursors  to our FANS-derived datasets . Based oustering . The cluustering . The notcond one . In contecursors , they quP-value in Wald test < 0.01, fold change >2). As expected . GO term analysis of this group of genes identified the term cell death (GO:0008219) as over-represented . Of the 23 promoter-reporters tested, eight behaved as predicted, with predominant activities in fibers, distal cambium, phloem, starch sheath, and epidermis, respectively , which is expressed in cambium stem cells in roots , BIG GRAIN 4 (BG4), PHYTOCHROME INTERACTING FACTOR 3-LIKE 1 (PIL1), DOF 2.2, and HEAVY METAL-ASSOCIATED PROTEIN 10 (ATHMP10), and generated promoter-reporter lines to analyze their activity patterns. In all five reporter lines, we detected signals in the cambium domain, including cambium stem cells, as indicated by our FANS-derived datasets . When analyzing respective promoter-reporter lines, we found that LYS/HIS TRANSPORTER 7 (LHT7), MYB29, and MYO-INOSITOL-1-PHOSPHATE SYNTHASE ISOFORM 3 (MIPS3) reporters were active in the phloem cap region (CLAVATA3/ESR-RELATED PROTEIN 46 (CLE46) and ARABINOGALACTAN PROTEIN 26 (AGP26) reporters were active in the pith  and xylem vessels (VND7pro) both determine wood properties in angiosperms and show distinct morphologies which are important for conducting their specific functions . Using GO enrichment analysis  and cell wall biogenesis (GO:0042546) were, as expected, over-represented within classifications of VND7pro-associated genes  and xylan metabolic process (GO:0045491) as over-represented among these genes [P < 0.05 {Bonferroni-corrected}]. The combined group of SMXL5pro and, APLpro-associated genes consisted of 2,226 genes from 13 clusters, among which the terms ion transport (GO:0006811) and transmembrane transport (GO:0055085) were over-represented in GO term enrichment analyses [P < 0.05 {Bonferroni-corrected}]. When we compared these groups with the genes expressed in the xylem or phloem of roots , we fouPopulus tremula) with a high spatial resolution along the radial sequence of tissues of the cambium domain , MYELOBLASTOSIS (MYB), and REPRODUCTIVE MERISTEM (REM) families  and VND2 . We also provide lists of tissue-specific genes derived from our FANS and LCM transcriptome data whose expression was estimated to be specific for a single tissue , these lists are rather short due to a high degree of shared gene expression patterns with other tissues. To extract genes with more complex expression patterns, the provided gene clusters  depository  under ace tissue . For somclusters  would beLTP1 activity did not peak in LTP1pro-positive nuclei may be an example of discrepancies between the activity pattern of our chosen LTP1 promoter and the distribution of endogenous LTP1 mRNA. In this context, it is important to be aware that the stability of LTP1 mRNA or the possible migration of this mRNA from cell to cell might be very different compared with H4-GFP mRNA driven by the LTP1 promoter. This difference may result in discrepancies between the patterns of H4-GFP protein accumulation and LTP1 mRNA accumulation. However, our comparison with previous datasets strongly suggested that we succeeded in isolating epidermis-specific mRNA.Several observations underline the robustness of our mRNA profiling. First, the activities of six out of seven genes whose promoters were used for tissue-specific nucleus labeling were found to peak in the respective tissues. This observation suggests that the chosen promoters mostly recapitulate the expression patterns of endogenous genes. Moreover, it shows that nuclei maintain a sufficient amount of their mRNA content during the isolation process and that the contamination by cytosolic mRNA, which is released during tissue disruption, is low. The finding that Interestingly, we identified many more genes as being expressed in specific tissues by directly comparing the different GFP-positive nucleus populations than by comparing two respective sets of GFP-positive and GFP-negative nuclei . We specThe second observation that supports robustness of the newly obtained profiles is that the promoters of genes we predicted to have a distinct spatial pattern recapitulated, in large, these patterns. In addition to the possible reasons resulting in differences between the accumulation of endogenous transcripts and the H4-GFP protein discussed above, it is important to note that the promoter regions upstream of the respective start codons, which we chose to drive the fluorescent reporters, may miss regulatory elements that substantially influence the expression of the endogenous genes . TherefoAlthough cytosolic and nuclear mRNA populations may differ due to the tight regulation of nuclear export or differences in mRNA homeostasis , our resUnderlining the differences between nucleus-based profiling and profiling of RNA from whole cells, the LCM-derived datasets showed substantial differences from FANS-derived datasets even after normalization . Due to In summary, considering the importance of plant stems and their tissues for land plant evolution on the one hand  and biomArabidopsis thaliana Col-0 plants were used in this study. The plants were grown in soil  alone or mixed with perlite to 20%  under short-day conditions (10-h light and 14-h darkness) for 3–5 weeks and transferred to long-day conditions (16-h light and 8-h darkness) for 3 weeks to induce reproductive growth. Light was provided by red and blue LED lights , Eindhoven, The Netherlands, #9290 004 871) at 70–120 µM photosynthetically active radiation. The SMXL5pro:H4-GFP (pIL53) line and the PXYpro:H4-GFP (pPS24) lines were described previously . A vector containing the ANT promoter region , VND7pro:H4-GFP (pTOM61), APLpro:H4-GFP (pPS02), SCRpro:H4-GFP (pPS20), and LTP1pro:H4-GFP (pPS16) were cloned using the pGreen0229 vector (APL promoter and terminator regions was described previously (H4-GFP sequence with the appropriate restriction enzyme sites are listed in ER-EYFP-HDEL)  terminator region generated by In-Fusion cloning  using previously described vectors as templates , the GFP sequence (pGGC014), the HDEL sequence (pGGD008), the RBCS terminator sequence (pGGE001), the BASTA resistance sequence (pGGF001), and the destination vector (pGGZ003)   and the emplates . The proemplates . PromotepGGZ003) .2+, Mg2+-free PBS. The sections were briefly washed in tap water and placed into a glass-bottom dish . Images were captured using a Nikon A1 confocal microscope with a 25x water immersion objective lens  and gallium arsenide phosphide detectors. GFP or YFP was excited by a 488-nm laser light, and Direct Red 23 was excited by a 561-nm laser light.Free-hand cross-sections of the second bottom-most internodes of Arabidopsis inflorescence stems were generated using razor blades  and stained with 0.1% solution of Direct Red 23 (µL RiboLock RNase inhibitor (40 U/µL) . The samples were chopped manually on ice for 10 min and transferred to a low binding tube  through a filter . Nuclei were collected by centrifugation  and gently washed twice with cold resuspension buffer (isolation buffer without Triton X-100). About 300 µL of resuspension buffer supplemented with a final concentration of 10 µg/mL Hoechst 33342 and 5-µL RiboLock RNase inhibitor were added to the nuclei, which then were transferred to a tube through a filter  for sorting.The second internodes of Arabidopsis inflorescence stems were collected in a Petri dish on ice. Each 2 g stem tissue sample was combined with 2-mL cold isolation buffer -N,N,N′,N′-tetraacetic acid (EGTA), 0.05% Triton X-100, 0.5-mM Spermidine, 0.2-mM Spermine, 15-mM 2-mercaptoethanol, 0.5-mM phenylmethylsulfonylfluorid, cOmplete Protease Inhibitor Cocktail ) supplemented with 10 TM IIIu cell sorter  using a 70-µm sort nozzle. A sheath pressure of 70 psi and a drop drive frequency of 87 kHz were applied. GFP fluorescence was excited at 488 nm using a 20 mW blue laser and Hoechst fluorescence at 405 nm using a 50 mW violet laser. A 530/30 bandpass filter was used for GFP and a 450/40 bandpass filter for Hoechst detection. Autofluorescence at 405-nm excitation was detected using a 585/42 bandpass filter. Forward scatter (FSC) detector voltage was set to 55, and a 1.0 neutral density filter was used. Side scatter (SSC) detector voltage was set to 275, Hoechst detector voltage to 352, and GFP detector voltage to 379. Events were triggered on FSC using a threshold of 5,000 and Hoechst with a threshold of 400. All the events were first filtered by FSC and SSC , except for the LTP1pro-S21 sample, for which 10,800 nuclei were collected. RNA was precipitated and washed according to the manufacturer’s protocol and resuspended in 15-µL water. One mircoliter of total RNA was used for mRNA-seq library construction using the Smart-seq2 protocol  was set based on the characteristics of the Arabidopsis genome . The GTF file was imported using makeTxDbFromGRanges in the GenomicFeatures library (v1.32.3) with the drop.stop.codons option  using the mode = "Union", ignore.strand = TRUE options  for genomic range extraction. For comparison with previous datasets, three replicates of the Mock_bdl dataset [deposited in National Center for Biotechnology Information's (NCBI) GEO database  based on the log transformed values of significantly differentially expressed genes  in the FANS/RNAS-seq-derived datasets. Distances between samples were calculated using the dist function in the stats library (3.5.0) and visualized in heat maps using pheatmap. GO enrichment analysis was carried out using PANTHER (v14.1) through the TAIR10 platform (https://www.arabidopsis.org/tools/go_term_enrichment.jsp)  . The Aras genome . Please s option . The pos options . For TPMdatabase : GSE9819database . Differedatabase . For GFPfunction , using tent.jsp) . Fisher’P < 8.8e−05.To determine the over-representation of transcription factor binding regions, 568 genome-wide DAP-Seq profiles for 387 Arabidopsis TFs were downloaded from the Plant Cistrome Database . The bacNST3 (AT1G32770), VND7 (AT1G71930), PXY (AT5G61480), SMXL5 (AT5G57130), APL (AT1G79430), SCR (AT3G54220), LTP1 (AT2G38540), H4 (AT5G59690), XCP1 (AT4G35350), WOX4 (AT1G46480), MOL1 (AT5G51350), PEAR1 (AT2G37590), PEAR2 (AT5G02460), NAC086 (AT5G17260), PIN3 (AT1G70940), FDH (AT2G26250), CER6 (AT1G68530), NST1 (AT2G46770), VND6 (AT5G62380), SEOR1 (AT3G01680), VND1 (AT2G18060), VND2 , ABI5 (AT2G36270), CAMTA1 (AT5G09410), CAMTA5 (AT4G16150), CBF1 (AT4G25490), CBF2 (AT4G25470), CBF3 , CBF4 (AT5G51990), GBF3 (AT2G46270), RTV1 (AT1G49480), SMB (AT1G79580), GT2 (AT1G76890), PHV (AT1G30490), LMI1 (AT5G03790), MYB55 (AT4G01680), MYB83 (AT3G08500), ABF2 (AT1G45249), HY5 (AT5G11260), TINY (AT5G25810), AREB3 (AT3G56850), ATHB13 (AT1G69780), ERF38 (AT2G35700), ERF34 (AT2G44940), CYP83A1 (AT4G13770), CYP83B1 (AT4G31500), ANAC028 (AT1G65910), ANAC074 (AT4G28530), LHCB3 (AT5G54270), LHCB2.2 (AT2G05070), FMO (AT1G12200), WRKY28 (AT4G18170), NCED4 (AT4G19170), RBGA6 (AT4G39260), DIN10 (AT5G20250), KIN2 (AT5G15970), COR15B (AT2G42530), MYB3 (AT1G22640), SEN1 (AT4G35770), HSFA2 (AT2G26150), CYP94B1 (AT5G63450), ANT (AT4G37750), UCN (AT1G51170), BG4 (AT3G13980), PIL1 (AT2G46970), DOF2.2 (AT2G28810), ATHMP10 (AT1G56210), LHT7 (AT4G35180), MYB29 (AT5G07690), MIPS3 (AT5G10170), CLE46 (AT5G59305), AGP26 (AT2G47930).Arabidopsis Genome Initiative locus identifiers for each gene are as follows: https://www.ncbi.nlm.nih.gov/geo/. In addition, data for genes of interest can be accessed via a website-based tool that allows gene expression profiles to be extracted (https://arabidopsis-stem.cos.uni-heidelberg.de/).Raw data discussed in this publication have been deposited into NCBI’s Gene Expression Omnibus  and are  H4-GFP reporter lines used in this study. FANS for GFP-positive and GFP-negative nuclei. PCA plot and correlation heatmap of all datasets derived from GFP-positive nuclei of seven different reporter lines. Gene expression profiles for epidermis-associated genes. Gene expression profiles for each FANS/RNA-seq-derived gene cluster. Validation of gene expression patterns determined by FANS/RNA-seq. Validation of gene expression patterns determined by FANS/RNA-seq. PCA plot and correlation heatmap for FANS-derived and LCM-derived datasets. Summary of RNA-seq results. Primers used in this study. Basic statistics of RNA-seq datasets provided in this study. Differentially expressed genes comparing GFP-positive to GFP-negative nuclei from proNST3, proSMXL5, and proAPL lines, respectively. Raw read counts for each FANS/RNA-seq dataset. Raw and normalized read counts for each LCM/RNA-seq dataset. Results of likelihood ratio test (LRT) analyses of FANS/RNA-seq datasets obtained from proNST3, proVND7, proPXY, proSMXL5, proAPL, proSCR and proLTP1 lines. Normalized read counts for each FANS/RNA-seq dataset. Clustering of genes based on their expression patterns among seven tissues analyzed by FANS/RNA-seq. SDE genes comparing the phloem cap and pith with the remaining vascular bundle using the Wald test. GO term enrichment analysis for phloem cap and pith-associated genes. Average values of normalized read counts of SDE genes in each FANS/RNA-seq-derived dataset ranked according to the highest values. SDE genes comparing proNST3-positive and proVND7-positive nuclei using the Wald test. GO term enrichment analysis for genes predominantly active in proNST3-positive versus proVND7-positive nuclei, and for genes predominantly active in proVND7-positive nuclei versus proNST3-positive nuclei. List of genes used for comparison with previously published xylem or phloem-associated expression datasets. GO term enrichment analysis for genes active in proNST3, proVND7, proPXY-positive and proSMXL5-, proAPL-positive domains. Fold enrichment values of significantly over-represented transcription factor binding regions in the upstream regions of genes from FANS/RNA-seq-derived clusters and tissue-specific genes determined by LCM/RNA-seq. List of genes with tissue-specific expression based on FANS/RNA-seq- and LCM/RNA-seq-derived datasets. Sequences of synthesized promoter regions used in this study.koaa019_Supplementary_Data_UpdatedClick here for additional data file.


Radiolabelling of cells has the advantage of being able to reveal cellular kinetics in vivo over time. This study aimed to optimise the synthesis of the radiotracers [89Zr]Zr-oxine (8-hydroxyquinoline) and [89Zr]Zr-DFO-NCS (p-SCN-Bn-Deferoxamine) and to perform a direct comparison of the cell labelling efficiency using these radiotracers.There is a need to better characterise cell-based therapies in preclinical models to help facilitate their translation to humans. Long-term high-resolution tracking of the cells 89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS in order to reach a radiochemical conversion (RCC) of >95 % without purification. Radio-instant thin-layer chromatography (iTLC) and radio high-performance liquid chromatography (radio-HPLC) were used to determine the RCC. Cells were labelled with [89Zr]Zr-oxine or [89Zr]Zr-DFO-NCS. The cellular retention of 89Zr and the labelling impact was determined by analysing the cellular functions, such as viability, proliferation, phagocytotic ability and phenotypic immunostaining.Several parameters, such as buffers, pH, labelling time and temperature, were investigated to optimise the synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS were synthesised with an average RCC of 98.4 % (n = 16) and 98.0 % (n = 13), respectively. Cell labelling efficiencies were 63.9 % (n = 35) and 70.2 % (n = 30), respectively. 89Zr labelling neither significantly affected the cell viability  nor the cells’ proliferation rate. The phenotype of human decidual stromal cells (hDSC) and phagocytic function of rat bone-marrow-derived macrophages (rMac) was somewhat affected by radiolabelling.The optimised synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [89Zr]Zr-oxine was superior to [89Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency.Our study demonstrates that [The online version contains supplementary material available at 10.1007/s11307-021-01622-z. Unfortu89Zr) is attractive and feasible. 89Zr has a half-life of 3.3 days and emits positrons with a mean energy of 395 keV, which enables high-resolution PET imaging for up to 3 weeks [89Zr-complexed 8-hydroxyquinoline ([89Zr]Zr-oxine) and deferoxamine ([89Zr]Zr-DFO-NCS) exhibit great promise. A clinical trial is currently ongoing to investigate the potential of [89Zr]Zr-oxine to track lymphocytes and their capability to enter the central nervous system [89Zr]Zr-oxine over other PET tracers is its similarity to the clinically established SPECT tracer [111In]In-oxine  and include cumbersome steps such as buffer exchange, chloroform extractions, evaporations and the usage of several buffers in the synthesis [The possibility to perform long-term cell tracking using zirconium-89  using the 89Y89Zr reaction. In-house production of 89Zr is described in details in electronic supplementary material (ESM).89Zr]Zr-oxalate with 50 μL 0.1 M sodium acetate buffer  (Merck Millipore) in a centrifuge tube. Thereafter, 0.1 mg dissolved oxine (in 99 % ethanol) was added to the mixture and the pH was adjusted to 9.1 using 1 M sodium carbonate (Honeywell). The sample was incubated for 60 min at 65 °C. The RCC was determined by instant thin-layer chromatography (iTLC) and was confirmed by radio-HPLC (see details in supplementary).Several parameters were investigated to improve the radiochemical conversion (RCC) from the original protocol , 24–26, 89Zr]Zr-oxalate and 50 μL 0.5 M PBS buffer (Sigma-Aldrich). The pH was adjusted to 7.0–7.4 using 1 M sodium carbonate. Incubation was performed under mild agitation (300 rpm) for 60 min at room temperature. The RCC was determined by iTLC and was confirmed by radio-HPLC as described in supplementary.To achieve a stable RCC of >95 % without the need for purification, we optimised several conditions such as synthesis buffer, reaction time, DFO-NCS concentration and temperature (see supplementary). The RCC in all tests was determined by iTLC using a mobile phase of 50 mM DTPA (Sigma-Aldrich), pH 4, (see supplementary). After all these optimisations, a final protocol was obtained by adding 4 μg DFO-NCS to a mixture of 5–25 MBq of [89Zr]Zr-oxine or [89Zr]Zr-DFO-NCS. Cell labelling efficiency (CLE) and cell viability was determined as described in supplementary.Human decidual stromal cells (hDSCs), rat bone marrow–derived macrophages (rMac) and human peripheral blood mononuclear cells (hPBMCs) were isolated, cultured and radiolabelled with Zr-oxine from the unbound 89Zr. This protocol resulted in a loss of more than 40 % 89Zr before evaporation and an additional 10–30 % was lost when dissolving the dried [89Zr]Zr-oxine pellet in DMSO  was tested and no decrease in purity 98.4 % (0.1 SD) (P = 0.4) or change in cell labelling capability 60.9 % (4.2 SD)  but showed a higher yield for higher pH > 6. Secondly, we repeated this experiment at a temperature of 65 °C in a pH range of 5–10.5. The highest RCC of 81.2 % (11.2 SD) (n = 7) was obtained at a pH of 9.0–9.2 after 30-min incubation at 65 °C. To avoid the need for purification, we increased the incubation time further and found that the RCC reached 98.4 % (1.3 SD) (n = 16) with 9.6 mM (2.4 SD) oxine after 60 min  DFO-NCS was incubated at 37 °C for 30 min in a pH 7.4 solution of 250 μL HEPES buffer, 200 μL Tris buffer, and 50–100 μL [89Zr]Zr-oxalate. This protocol yielded an RCC of 92.4 % (4.9 SD) (n = 5). We found no significant difference in RCC between HEPES and PBS as well as the presence of Tris buffer. The final procedure was therefore performed in PBS pH 7.4, without Tris buffer. By decreasing the volume and increasing the amount of DFO-NCS, an RCC of >95 % was achieved after approximately 10 min of incubation  (n = 3). To achieve a stable RCC of >95 % at room temperature, we therefore increased the incubation time to 60 min  (n = 11) for hDSCs and 62.2 % (10.6 SD) (n = 10) for rMac (Table P = 0.19) for hDSC, 4.5 % (3.5 SD)  for rMac and 7.9 % (14.8 SD)  for hPBMC  (n = 4) for hDSCs (Table All cells were successfully labelled with [61.0 % 6. SD (n = 61.0 % 6. SD (n = 89Zr]Zr-DFO-NCS showed a decrease in cell viability with increasing concentration of [89Zr]Zr-DFO-NCS. Labelling cells with a concentration of 7.5 μM (2.3 SD) [89Zr]Zr-DFO-NCS resulted in CLEs of 71.3 % (7.2 SD) (n = 12) for hDSCs, 68.9 % (12.7 SD) (n = 9) for rMac and 70.2 % (13.5 SD) (n = 9) for hPBMC, respectively. No significant decrease in cell viability was observed under this condition (Table Viability tests on hDSC labelled with [f 7.5 μM .3 SD [8989Zr]Zr-DFO-NCS at room temperature and pH 7.4 was shorter than that of [89Zr]Zr-oxine (<1.5 hrs vs 7 days for [89Zr]Zr-oxine). The CLE of [89Zr]Zr-DFO-NCS decreased from 69 % after a waiting period of 1 h between radiosynthesis and cell labelling to 31 % for a 1.5-hwaiting period, and to less than 10 % when the waiting period exceeded 2.5 h  for hDSC, 25.5 % (1.6 SD) for rMac and 44.0 % (30.3 SD) for hPBMC. This corresponds to a radioactive efflux of 29–38 % for [89Zr]Zr-oxine and of 31–60 % for [89Zr]Zr-DFO-NCS (Table 89Zr showed that less than 1 % (n = 3) of radioactivity was taken up by the cells.The retention of [es Table . The celThe radiolabelling did not have a significant effect on the proliferation rate compared to unlabelled cells Table .89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS. No major phenotypic changes were detected in hDSCs after labelling. Since cells were previously isolated and characterised as hDSCs, the phenotype post-labelling was determined using a slightly reduced antibody panel. A positive expression of CD29, CD44, CD73, CD90 and CD105 and a negative expression of CD14, CD34 and CD45 would confirm that radiolabelling did not alter the cellular phenotype. However, radiolabelled hDSCs showed a significant increase in expression of CD29, CD44, CD73 and CD105 compared to unlabelled control cells   and cells labelled with [89Zr]Zr-DFO-NCS decreased by 13.3 % (4.7 SD)   using a QMA column [89Zr]Zr-oxalate and oxine in a sodium acetate solution. Here, [89Zr]Zr-oxine was synthesised in alkaline conditions (pH 9.1) at elevated temperature (65 °C) for 60-min incubation. The advantage of our method is the simple two-step, 80-min total procedure protocol, requiring no further purification.Our optimisation of the Zr-oxine and [89Zr]Zr-DFO-NCS are easily produced with high RCCs (> 95 %). However, the short shelf-life of [89Zr]Zr-DFO-NCS (<1.5 h), most likely due to the hydrolysis of the NCS group, greatly limits the off-site storage and research capabilities. This is not the case for [89Zr]Zr-oxine, where RCC and cell labelling capacity remains unchanged up to 7 days.In terms of radiosynthesis, [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS, showing CLEs varying between 13 and 55 % Zr-oxine and [89Zr]Zr-DFO-NCS provided a similar CLE range from 60 to 70 %. These results are comparable with previous publications using the SPECT radiotracer [111In]In-oxine but with significantly higher stability in vitro [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS after 7 days in culture was 56–60 % and 26–60 %, respectively. Previous studies on [111In]In-oxine reported a cellular efflux of 70–75 % within 3–4 days Zr-DFO-NCS relies on the abundance of free amines on the cell surface which can vary significantly between different cell types that can consequently affect the CLE of [89Zr]Zr-DFO-NCS. Additionally, the binding efficiency of [89Zr]Zr-DFO-NCS can be affected by hydrolysis and the possible presence of amines in the cell medium or residual cell fragments during cell labelling. The competition of exposed amines could therefore result in a significantly decreased CLE.Cell labelling of hDSCs, rMac and hPBMC with Zr-DFO-NCS and [89Zr]Zr-oxine did not show any statistically significant decrease in viability in any of the cell lines. Labelling rMac with either [89Zr]Zr-DFO-NCS or [89Zr]Zr-oxine resulted in a slightly decreased phagocytotic function. Radiolabelled hDSC cells showed a significant increase in CD29, CD44, CD73 and CD105 expression for both radiotracers compared to unlabelled control cells. However, the antigen expression still corresponds to the hDSC phenotype [89Zr]Zr-oxine show no significant alteration in cell functionality, phenotype or apoptotic activity at 20 kBq/106 cells [89Zr]Zr-oxine was investigated, shows no effect on the phenotype up to 1515 kBq/106 cells [et al. demonstrated that significant DNA damage is evident in white blood cells labelled with [89Zr]Zr-oxine at 32.9 kBq/106 cells [et al. established that the majority of DNA damage is repaired at the 7-day time point [111In]In-oxine [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS, instead of unlabelled oxine or DFO-NCS by themselves. Further studies are needed to assess if the stress is temporary and reparable or permanent, in which case the level of radioactivity per cell, where possible, could be decreased to minimise the DNA damage.Labelling with Zr-oxine and [89Zr]Zr-DFO-NCS. Both [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS yielded similar results in terms of radiochemical conversion and cell labelling efficiency. Although the synthesis conditions for [89Zr]Zr-DFO-NCS were more ambient , [89Zr]Zr-oxine appeared superior to [89Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency after prolonged waiting times between radiosynthesis and cell labelling. Further studies are ongoing to evaluate the two radiotracers’ applicability and feasibility for long-term cell tracking in vivo.We optimised the synthesis procedure of [ESM 1(PDF 105 kb)


The populations of MDSC were increased in peripheral blood of lupus patients. The mRNA levels of immunosuppressive molecules were profoundly decreased in MDSCs from lupus patients and mice. Co-culture with splenocytes showed that PD-L1 expressing MDSCs from control mice expand both Treg cells and regulatory B cells more potently. Infusion of PD-L1 expressing MDSCs reduced autoantibody levels and degree of proteinuria and improved renal pathology of two animal models of lupus. Moreover, PD-L1 expressing MDSCs therapy can suppress double negative (CD4-CD8-CD3+) T cells, the major pathogenic immune cells and follicular helper T cells in MRL/lpr mice, and podocyte damage. Our results indicate PD-L1 expressing MDSCs have more potent immunoregualtory activity and ameliorate autoimmunity more profoundly. These findings suggest PD-L1 expressing MDSCs be a promising therapeutic strategy targeting systemic autoimmune diseases.Multiple studies have explored the potential role of programmed death-ligand 1 (PD-L1) as a mediator of Myeloid-derived suppressor cells (MDSCs) effects in various cancers. However, the role PD-L1 expression in MDSCs on autoimmune disease is still largely unknown.This study was undertaken to whether MDSC expressing PD-L1 have more potent immunoregulatory activity and control autoimmunity more effectively in two murine models of lupus (MRL/ Systemic lupus erythematosus  is an autoimmune disease characterized by the production of diverse autoantibodies and immune complex deposit in target tissues. It affects multiple organs and has significant morbidity and mortality. The etiology of SLE is multifactorial and includes contribution from the environment and genetic factors. The exact pathogenesis of lupus remains still poorly understood, although there has been much progress in researching the pathogenesis of lupus through the use of genetic variant identification, mouse models, gene expression studies, and epigenetic analyses, Nevertheless, it is evident that various immunologic abnormalities contribute to the pathogenesis of lupus , 2.Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of early myeloid progenitors, immature granulocytes, macrophages and dendritic cells. MDSCs are often generated and expanded under pathologic conditions such as tumor environment or chronic inflammation. They can play an important role because of their potent suppressive roles in the immune response. These cells express immunosuppressive factors such as arginase-1, inducible nitric oxide synthase (iNOS) or reactive oxygen species (ROS), which can induce the inactivation of various immune cells, especially T cells .Based on the well-known immunomodulatory properties of MDSCs, it can be speculated that cell therapy with MDSCs might be a promising therapeutic strategy for treating lupus. In our previous research, we demonstrated for the first time that murine MDSCs induce the expansion of regulatory B (Breg) cells and ameliorate autoimmunity in the murine lupus model . Howeverin vitro. Additionally, we examined whether in vivo treatment with PD-L1 expressing MDSCs suppress autoimmune phenotype more profoundly in two animal models of lupus (Roquin san/san mice and MRL/lpr mice).Therefore, we hypothesized that MDSCs from lupus might be dysfunctional and specific population of MDSCs could have more powerful immunoregulatory activity. In this study, we measured the population of MDSCs in lupus patients and investigated the levels of various immunosuppressive mediators in MDSCs originated from human lupus patients and lupus mice. We also examined the impact of programmed death-ligand 1 (PD-L1) expressing MDSCs on T cell proliferation and various effector T/B cell subsets lpr/J (MRL/lpr) and MRL/MpJ (+/+) mice were purchased from SLC Inc. (Japan) (n = 10 mice per group per experiment). Female Sanroque (Roquinsan/san) mice between 15–20 weeks of age were purchased from the NIH (n = 5 mice per group per experiment) and subsequently bred under specific pathogen-free conditions in an animal facility with controlled humidity (55 ± 5%), light (12 h/12 h light/dark), and temperature (22 ± 1°C). The air in the facility passed through a HEPA filter system designed to exclude bacteria and viruses. Animals were fed mouse chow and tap water ad libitum. The protocols used in our study were approved by the Animal Care and Use Committee of the Catholic University of Korea (CUMC-2017-0038-02).Female MRL/MpJ‐Fassan/san mice model, each mouse received a weekly intravenous (IV) injection of saline (n=5) or 5x105 PD-L1+MDSC (n=5) or 5x105 PD-L1-MDSC (n=5) at 14 and 15 weeks. One week after the last treatment, all mice of each groups (n=5) were sacrificed at 16 weeks. At that time, the serum, kidney and spleen were collected for analyses. In MRL/lpr model, each mice received a weekly IV injection of saline (n=10) or 1x106 PD-L1+MDSC (n=10) or 1x106 PD-L1-MDSC (n=10) from 10 to 16 weeks of age. One week after the last treatment, all mice of each groups (n=10) were sacrificed at 17 weeks. At that time, the serum, kidney and spleen were collected for analyses.All mice were randomized divided into three groups as follows: control , PD-L1+ MDSC treatment (PD-L1(+) MDSC), and PD-L1- MDSC treatment (PD-L1(-) MDSC). In Roquin6 cells/mL in complete medium supplemented with recombinant GM-CSF  for 3 days. The cultured cells were harvested and stained with anti-CD11c FITC, anti-CD11b PerCP5.5, anti-Gr-1 APC, and anti-PD-L1 PE Ab (BD Biosciences) following FcR blocking (BD Biosciences). CD11c-CD11b+Gr-1+PD-L1+ and CD11c-CD11b+ Gr-1+PD-L1-MDSC subsets were sorted using a Beckman Coulter MoFlo XDP. The purity of the sorted PD-L1+MDSC and PD-L1MDSC was > 95%.We isolated Bone marrow cells (BMCs) from the tibias and femurs of the mice by flushing the bone marrow cavity with Minimum Essential Medium . BMCs were cultured in 6-well plate at 1 × 10− HLA-DR− CD11b+CD33+ population. Informed consent was obtained from all participants according to the principles of the Declaration of Helsinki. This trial was approved by the Institutional Review Board of Seoul St. Mary’s Hospital of the Catholic University of Korea .Peripheral blood mononuclear cells (PBMCs) from SLE patients were stained with anti-Lineage, anti-HLA-DR, anti-CD11b, anti-CD33 anti-CD14, anti-CD15, anti-Arginase-1, anti-PD-L1 and anti-IL-10 Ab (Thermo fisher Scientific). Human total MDSCs analyzed as Linlpr mice by using the CD4+ T cell microbeads. Isolated PD-L1+ or PD-L1- MDSCs were cocultured with Cell Trace-violet -labelled CD4+ T cells . CD4+T cells were stimulated by 1 μg/ml of anti-CD3/CD28  and 30U/ml of recombinant IL-2 . After 5 days, proliferation of T cells was analyzed using FACS LSRFortessa cytometer .PD-L1+ or PD-L1- MDSCs were isolated from MRL/MpJ mice. CD4+T cells were isolated from spleen of MRL/PD-L1+ or PD-L1- MDSCs, CD4+T cells, splenocytes were isolated from normal C57BL/6 mice. Isolated PD-L1+ or PD-L1- MDSCs were cocultured with CD4+ T cells . Isolated PD-L1+ or PD-L1- MDSCs were cocultured with CD4+ T cells  under T cell activation condition (CD3/CD28 stimulation). For B10 analysis, PD-L1+ or PD-L1- MDSCs were cocultured with splenocyte  under LPS (100ng/ml) stimulation.Mononuclear cells were stained with various combinations of the following fluorescence-conjugated antibodies: CD19, CD1d, CD5, CD21, CD23, CD4, CD8, CD25, CD95, GL-7, CD138, PD-1, CXCR5 and B220 . These cells were also intracellularly stained with the following antibodies: IFN-r, IL-4, IL-2, IL-10, IL-17 and Foxp3 . Before intracellular staining, the cells were restimulated for 4 hr with 25 ng/mL PMA (Sigma-Aldrich) and 250 ng/mL ionomycin (Sigma-Aldrich) in the presence of GolgiStop (BD Biosciences). Intracellular staining was performed using an intracellular staining kit (eBioscience) according to the manufacturer’s protocol. Flow cytometry was performed using a FACSCalibur and FACS LSRFortessa cytometer (BD Biosciences).The concentrations of IFN-γ, IL-10 and IL-17 in culture supernatants were measured by using a sandwich enzyme-linked immunosorbent assay .Serum levels of IgG2a antibodies were measured using a commercially available ELISA kit (Bethyl Laboratories). The IgG anti-dsDNA antibody was measured by ELISA using serum samples diluted at 1:500 and alkaline phosphatase (AP)-conjugated goat-anti mouse IgG, secondary antibody at a 1:1,000 dilution.Urine albumin and creatinine concentrations were measured using a mouse albumin ELISA assay  and a creatinine assay (R&D systems), according to the manufacturer’s directions.We embedded formalin-fixed tissues in paraffin and sectioned them into 5 μm sections. We stained them with hematoxylin (Sigma-Aldrich) and eosin  for histological examination (H&E). Kidney tissues were stained with periodic acid–Schiff . Renal pathology including glomerular pathology, tubular injury, and perivascular cell accumulation was evaluated as described previously  by 2 invMessenger RNA (mRNA) was extracted using the TRI Reagent  according to the manufacturer’s instructions. Complementary DNA was synthesized using a SuperScript Reverse Transcription system . A LightCycler 2.0 instrument  was used for PCR amplification. All reactions were performed using the LightCycler FastStart DNA Master SYBR Green I mix (Takara Bio Inc.), following the manufacturer’s instructions. Primer sequences are described in the Mouse and human kidney tissue cryosections (7 μm thick) were fixed in 4% (v/v) paraformaldehyde and stained using fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, PerCP-Cy5.5-, or allophycocyanin -conjugated monoclonal antibodies to mouse nephrin (PROGEN), WT-1 (Abcam), C3, IgG and human CD11b (Abcam), CD33 (Abcam), DAPI (Invitrogen). After incubation overnight at 4 °C, stained sections were visualized by confocal microscopy .t-tests or Mann-Whitney U-test. Comparisons between multiple groups were analyzed by one‐way analysis of variance. P values less than 0.05 (2-tailed) were considered significant.Statistical analyses were performed using GraphPad Prism version 8.0. The experimental data are presented as mean ± SEM. Comparisons of numerical data between two groups were performed by Student’s in vitro. In an attempt to test whether PD-L1 positive MDSCs could be more immunosuppressive, MDSCs were generated from bone marrow cells from C57BL/6 mice as described in Materials and Method section. We first analyzed the mRNA levels of crucial immunoregulatory mediators like IL-10, Arginase-1, iNOS, and TGF-β using real-time PCR. As shown in in vitro treatment with PD-L1 positive MDSCs more profoundly decreased Th17 cells and increased Treg cells compared to PD-L1 negative MDSC (in vitro treatment with PD-L1 positive MDSCs more profoundly increased Breg cells compared to PD-L1 negative MDSC (We hypothesized that PD-L1 positive MDSCs could have potent immunoregulatory activity lpr mice) and control mice (MRL/MpJ mice). The mRNA levels of arginase-1, IDO, PD-L1 and IL-10 were profoundly decreased in MDSCs from MRL/lpr mice compared to MDSCs form control mice (lpr mice (in vitro immunoregulatory activity of MDSCs from control or lupus mice and the results showed that MDSCs from lupus mice inhibit CD4+ T cell proliferation weakly (data not shown). These findings suggest that MDSCs from lupus mice would be dysfunctional like one previous report . As expected, PD-L1 positive MDSCs from the control mice more markedly inhibit CD4+ T cell proliferation. To our surprise, both PD-L1 positive MDSCs from lupus mice and PD-L1 negative MSDSs from control mice weakly inhibit CD4+ T cell proliferation  were cultured with PD-L1 positive MDSCs, only PD-L1 positive MDSCs form control mice remarkably suppressed plasma cell (CD19-CD138+) population and expanded IL-10 producing B 10 cell (CD19+CD5+CD1d+) , resulting in excessive formation of Tfh cells and germinal centers (GC)  , 13. We lpr mice), MRL/lpr mice at the age of 8 weeks were intravenous injected with 1 × 106 PD-L1 positive or negative MDSCs obtained from MRL/MpJ mice or 1 × 106 PD-L1 positive MRL/lpr mice an equal volume of saline, which was administered once weekly for six weeks. One week after the last injection, the mice were sacrificed. As shown in lpr mice treated with PD-L1 positive or negative MDSCs or saline. The results showed that only in vivo infusion of PD-L1 positive MDSCs from MRL/MpJ mice remarkably decreased glomerular pathology score and tubular injury score (lpr mice). As shown in In order to investigate the therapeutic impact of PD-L1 positive MDSCs on other lupus-prone mice .Podocytes are the essential component of glomerular filtration apparatus and critical for the maintenance of renal function . Podocytlpr mice compared to PD-L1 negative MDSCs or saline. In addition to DNT cells, Tfh (CD4+CXCR5+PD-1+) cells, which is other proinflammatory T cell subsets, were decreased by treatment with PD-L1 positive MDSCs. In contrast, follicular regulatory T  cells were increased by administration of PD-L1 positive MDSCs. We also analyzed the populations of effector B cells. The results showed that PD-L1 positive MDSCs profoundly deceased GC B cells (CD19+GL7+), but increased Breg (CD19+CD1d+CD5+IL-10+) cells (Double-negative (DN) T cells are defined by the absence of CD4 and CD8 and the ability to produce proinflammatory cytokines like IFN-γ and IL-17 which has been linked to lupus pathogenesis both in human and mice . As showWe finally examined the population of MDSCs in peripheral blood of patients with lupus and age, sex matched healthy controls. The MDSCs subset in the peripheral blood of SLE patients was analyzed by flow cytometry. Human total MDSCs (Lin-HLA-DR-CD11b+CD33+ cells), M-MDSC (HLA-DR-CD11b+CD14+ cells) and PMN-MDSC (CD11b+CD14+D15+ cells) were identified as defined previously . As showMyeloid-derived suppressor cells (MDSCs) are a heterogenous collection of myeloid cells composed of myeloid precursors, immature granulocytes, mononuclear macrophages, and dendritic cells. MDSCs are broadly characterized by CD11b+GR-1+ cells in mice and HLA-DR-CD11b+CD33+ cells in human and they typically divided into two subpopulations: polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs). Increasing evidence suggests that MDSCs with immuosuppressive effect have therapeutic effects in various animal models of systemic autoimmune diseases including multiple sclerosis , 19, rhelpr mice) also showed defective in vitro immunoregulatory activity which was determined by T cell proliferation assay (data not shown). Therefore, it could be of utmost importance to find out specific population of MDSCs with more potent immunoregulatory activity.In an attempt to clarify the reason for such discordant results about the role of MDSCs in lupus, we hypothesized that MDSCs originated from lupus could be dysfunction like one previous report showing that the function PMN-MDSCs originated from (NZB X NZW) F1 lupus prone mice was impaired which was determined by T cell proliferation assay . In our in vitro treatment with PD-L1 positive MDSCs more profoundly decreased Th17 cells and increased Treg cells and Breg cells compared to PD-L1 negative MDSC (in vitro.Programmed death-ligand1 (PD-L1), which is a ligand for the inhibitory checkpoint molecule PD-1, is a 40kDa type 1 transmembrane protein that has been speculated to play a major role in suppressing the adaptive arm of immune system during particular events such as pregnancy, tissue allografts and autoimmune diseases. Noman et al. reported that blockade of PD-L1 under hypoxia enhanced MDSC-mediated T cell activation . Therefolpr mice) . The results showed that PD-L1 positive MDSCs from control mice have more powerful immunoregulatory activity which was determined by T cell proliferation assay  . We nextsan/san mice and MRL/lpr mice)  and histopathologic findings of kidneys compared to PD-L1 negative MDSCs in two animal models of lupus (Roquin pr mice) 4B–D.in vivo infusion of MDSCs generated from control mice (MRL/MpJ mice) attenuate podocyte injury as well as immune complex deposit in kidneys of lupus prone mice (MRL/lpr mice)  that promotes survival of self-reactive lymphocytes, and a strain background (MRL/MpJ) predisposed to autoimmunity. MRL/lpr mice are characterized by massive lymphadenopathy associated with aberrant T cells, arthritis, and immune complex glomerulonephritis. Among aberrant T cells of MRL/lpr mice, double-negative (DN) T cells, defined by the absence of CD4 and CD8, are the major pathogenic immune cell subset and have the ability to produce proinflammatory cytokines like IFN-γ and IL-17 which has been linked to lupus pathogenesis both human and mice  grant funded by the Korea government (MSIT) (No. NRF-2018R1A2A2A05018848). This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (grant number HI20C1496).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CREB1, ATF1, and CREM) gene fusions are defining markers in diverse mesenchymal neoplasms . However, neoplasms harboring EWSR1-CREM/FUS-CREM fusions are rare and poorly characterized. We describe two cases  illustrating their misleading immunophenotypes. Histologically, both showed eosinophilic and focally clear epithelioid cells arranged into sheets, nests, and trabeculae. Immunohistochemistry showed ALK, EMA, and AE1/AE3 immunoreactivity suggesting ALK-rearranged renal cell carcinoma (Case 1) and coexpression of keratin, EMA, synaptophysin, and chromogranin-A, suggesting neuroendocrine neoplasm (Case 2). Targeted RNA sequencing revealed EWSR1-CREM (Case 1) and FUS-CREM (Case 2) fusions. These cases add to the spectrum of CREM fusion-positive intra-abdominal epithelioid neoplasms. Their unusual immunophenotype and unexpected sites represent major pitfalls, underline a wide differential diagnosis, and emphasize the value of molecular testing in correctly diagnosing them.CREB family ( EWSR1 on 22q) and one of the CREB family genes  have been increasingly recognized in soft tissue and visceral neoplasms with significant phenotypic and demographic diversity [EWSR1-ATF1 fusions), malignant gastrointestinal neuroectodermal tumor (mainly EWSR1-CREB1 fusions), angiomatoid fibrous histiocytoma (mainly EWSR1-CREB1 fusions), and clear cell carcinomas of the head and neck (EWSR1-ATF1 fusions) [CREB fusion family, CREM-rearranged neoplasms are rare. They include subsets of CCS, angiomatoid fibrous histiocytomas, and clear cell carcinoma of the head and neck [CREM-rearranged intra-abdominal malignant epithelioid neoplasms  displaying confusing immunophenotypes: expression of ALK and keratin (Case 1) and keratin and neuroendocrine markers (Case 2). In both, diagnosis was only made after NGS testing uncovered the underlying fusions.Gene fusions involving the Ewing Sarcoma Breakpoint Region 1 gene EWSR1 on 2q and onCases were retrieved from our consultation files. Case 2 was identified among 25 mesenchymal/non-epithelial neoplasms in a cohort of 346 neuroendocrine mimics . Immunohistochemistry (IHC) was performed on 3-µm sections using a fully automated system  and the following antibodies: keratin , vimentin , PAX8 , CK7 , CK5 , TTF-1 , ERG , CD31 , CD10 , p63 , desmin , smooth muscle actin , S100 protein , CD34 , CD30 , MUC4 , TLE1 , STAT6 , SMARCB1/INI1 , synaptophysin , chromogranin-A , SDHB , TFE3 , and MUC1  according to the manufacturer instructions. FFPE tissue from Case 2 was studied by electron microscopy  with digital image device  according to routine methods.ALK/EWSR1 (Case 1) and FUS/TFE3 (Case 2) FISH analyses were performed using ZytoLight® SPEC Dual Color Break Apart Probes  designed to detect translocations involving these genes, respectively. Case 2 was tested for SDHB mutations using the methods described previously [RNA-based NGS was performed as described previously . ALK/EWSeviously .A 55-year-old male underwent radical nephrectomy for a renal mass. His medical history was negative for other malignancies. He is currently diagnosed with metastatic disease in the pelvis 23 months from initial diagnosis.A 32-year-old female without other primary malignancy presented with a mesenteric mass that was resected together with the adjacent small bowel segment. She presented 8 months later with peritoneal, pleural, and lymph node metastases. Currently , she is alive with extensive progressive disease under palliative chemotherapy.The nephrectomy specimen contained a well-demarcated 7.5 cm renal medullary mass, distinct from the renal pelvis without extrarenal extension. Histological examination showed medium-sized epithelioid cells disposed into communicating nests and trabeculae within a prominent fibrous stroma, entrapping glomeruli and tubules Fig. . The nucThe tumor measured 5.5 cm and was intra-mesenteric distinct from the gut wall. Histological examination showed medium-sized epithelioid cells forming well vascularized sheets with variably eosinophilic, clear Fig. , rhabdoiEWSR1-CREM fusion  showed a FUS-CREM fusion  revealed no SDHB mutation .With a provisional diagnosis of ALK + renal cell carcinoma (RCC), ALK FISH failed to show rearrangement or copy number gains in Case 1. Targeted RNA sequencing revealed a ion Fig. , which wion Fig.  which waEWSR1/FUS-CREM fusions were recognized recently in a group of unclassified epithelioid mesenchymal neoplasms showing predilection for intra-abdominal organs, not fitting any known EWSR1-CREB-rearranged entity [EWSR1/FUS-CREM-positive intra-abdominal epithelioid neoplasms have been reported [EWSR1-CREM and five FUS-CREM fusions [With increasing use of targeted RNA NGS in routine surgical pathology practice, the numbers and variants of gene fusions detected in neoplasms have been increasing, and several new molecularly defined entities are emerging. d entity . We herereported , 5, 6. A fusions , 5, 6. V fusions , 7. Thes fusions , 5, 6.The morphology of these tumors seems distinctive. They all displayed monotonous epithelioid morphology with variable cytoplasmic clearing and sparse fibrous stroma. Most cases expressed keratin and EMA , 5, 6. AEWSR1-CREM-positive SEF was not illustrated [EWSR1 fusion by FISH are otherwise considered diagnostic of SEF in the context of epithelioid mesenchymal neoplasms [EWSR1-CREB3L1 and rarely by FUS-CREB3L1 fusions [Although the MUC4-positive gastric tumor was diagnosed as sclerosing epithelioid fibrosarcoma (SEF), the depicted histology suggests similarity to the current cases with variable clear cell epithelioid pattern, lacking characteristic sclerosis of SEF . Anotherustrated . Includieoplasms . HoweverEWSR1-rearranged renal sarcomas and represents the second EWSR1-CREM fusion-positive renal case [ALK-rearranged RCC [EWSR1/FUS-TFCP2 and EWSR1 but not MEIS1-NCOA2 and other fusions [ALK copy number gains, none of angiomatoid fibrous histiocytoma showed this feature [Our renal case expands the spectrum of nal case . Moreovenged RCC . Indeed,nged RCC  and rhabnged RCC . The mol fusions , 12). Wh feature . ALK mig feature , 12.TFE3-rearranged neoplasm. This is particularly important in renal cases as rare Xp11-translocation RCC may harbor EWSR1-TFE3 fusions and be indistinguishable from EWSR1/FUS-CREM neoplasms by FISH alone [EWSR1/FUS-CREM fusion driven mesenchymal neoplasms.Regarding Case 2, it is noteworthy that it labeled not only for synaptophysin but also for chromogranin-A, a combination that we encountered only rarely in mesenchymal neoplasms with neuroendocrine features (data not shown). Since chromogranin-A normally resides in the membrane of neurosecretory granules, we studied the tumor tissue by electron microscopy to search for neurosecretory granules but could not identify any. We conclude that chromogranin-A protein associates with other cell organelles than secretory granules in this neoplasm. Loss of SDHB in this case remained unexplained and represents a further confusing factor with neuroendocrine neoplasms. Finally, aberrant or equivocal TFE3 expression noted in Case 2 and in a previous case  may suggSH alone . All theEWSR1-CREM and FUS-CREM fusions. Besides their epithelioid morphology, these tumors displayed unusual misleading immunophenotypes. Recognition of these findings should help to avoid misinterpretation as SEF, ALK-rearranged RCC, and neuroendocrine carcinoma. The cases highlight the value of precise genotyping in these challenging lesions to ovoid misdiagnoses.In summary, we report two intra-abdominal malignant epithelioid mesenchymal neoplasms carrying the recently described


Long noncoding RNAs (lncRNAs) are related to colorectal cancer (CRC) development. However, the role and mechanism of lncRNA LINC01224 in CRC development are largely unknown.LINC01224, Yin Yang 1 (YY1), microRNA (miR)-485-5p, and myosins of class VI (MYO6) levels were examined using quantitative reverse transcription polymerase chain reaction and western blotting. Functional analyses were processed through CCK-8, colony formation, flow cytometry, transwell, and xenograft analyses. Dual-luciferase reporter, chromatin immunoprecipitation (ChIP), RNA immunoprecipitation, and pull-down assays were conducted to analyze the binding interaction.LINC01224 abundance was elevated in CRC tissue samples and cell lines. Elevated LINC01224 might indicate the lower 5-year overall survival in 52 CRC patients. LINC01224 was upregulated via the transcription factor YY1. LINC01224 knockdown restrained CRC cell proliferation, migration, and invasion and increased apoptosis. MiR-485-5p was sponged by LINC01224, and miR-485-5p downregulation relieved the influence of LINC01224 interference on CRC progression. MYO6 was targeted via miR-485-5p and regulated via LINC01224/miR-485-5p axis. MiR-485-5p overexpression suppressed CRC cell proliferation, migration, and invasion and facilitated apoptosis. MYO6 upregulation mitigated the role of miR-485-5p. LINC01224 knockdown decreased xenograft tumor growth.YY1-induced LINC01224 regulates CRC development via modulating miR-485-5p/MYO6 axis.The online version contains supplementary material available at 10.1186/s12957-021-02389-x. Colorectal cancer (CRC) is a commonly diagnosed cancer with a high fatality rate around the world . More thLong noncoding RNAs  with diverse cellular functions and lacking protein-coding potential are related to human cancer progression . lncRNAsMoreover, LINC01224 promotes carcinogenesis and tumor development through serving as endogenous sponge for microRNAs (miRNA), including miRNA (miR)-2467 . miRNAs Myosins of class VI (MYO6) is a unique actin-based motor protein, which has important roles in regulating multiple cellular processes . PreviouThereby, we hypothesized that LINC01224 could affect CRC progression through regulating miR-485-5p and MYO6. To confirm this, expression of LINC01224, miR-485-5p, and MYO6 was detected in CRC patients and cells, and cellular functions of LINC01224 and miR-485-5p were measured in CRC cells. Ultimately, the underlying regulatory mechanism was further confirmed via LINC01224/miR-485-5p/MYO6 competing endogenous (ceRNA) pathway and upstream regulation of the transcription factor Yin Yang 1 (YY1), a well-known transcription factor for lncRNAs [Each patient signed the written informed consent. This research was approved by the ethics committee of the first hospital of Hebei Medical University, and all procedures were processed following the guidelines of the Helsinki Declaration. Animal experiments were authorized via the Institutional Animal Care and Use Committee of the first hospital of Hebei Medical University and conducted under the National Institutes of Health.http://www.cancer.gov/tcga) [http://jaspar.genereg.net/) [http://starbase.sysu.edu.cn/index.php) [http://starbase.sysu.edu.cn/panGeneCoExp.php). The association between LINC01224 level and overall survival of CRC patients was analyzed on the Gene Expression Profiling Interactive Analysis (GEPIA) server (http://gepia.cancer-pku.cn/) [Expression of LINC01224 and MYO6 in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) was provided via The Cancer Genome Atlas (TCGA) database (ov/tcga) . The bineg.net/) . The bindex.php) . The corpku.cn/) .high (n = 26) and LINC01224low (n = 26). The 5-year overall survival of 52 CRC patients was recorded and analyzed by the Kaplan-Meier survival curve. The correlation between LINC01224 expression and the clinical-pathological features of CRC patients is shown in Table The CRC and normal control (NC) tissues were harvested from 52 CRC patients in the first hospital of Hebei Medical University from 2011 to 2014. All participants did not receive other treatment before surgery. According to the median of LINC01224 level, these 52 CRC patients were divided into two groups: LINC012242.CRC cell lines  and normal colonic mucosa FHC cells were obtained from BeNa Culture Collection  and maintained in Roswell Park Memorial Institute (RPMI)-1640 medium  plus 10% fetal bovine serum  at 37°C and 5% CO−ΔΔCt [After lysing in Trizol , the lysates were applied for total RNA extraction as previously reported . A total−ΔΔCt .Table 2YY1 and MYO6 overexpression vectors were constructed with pcDNA3.1 vector (Thermo Fisher Scientific). The siRNA targeting YY1 (si-YY1), si-NC, shRNA for LINC01224 (sh-LINC01224#1 and sh-LINC01224#2), sh-NC, miR-485-5p mimic, miR-NC mimic, miR-485-5p inhibitor (anti-miR-485-5p), and anti-NC were obtained from Genepharma , and their sequences are listed in Table Protein was obtained through RIPA . With concentration determination using BCA kit (Beyotime), 20 μg proteins were separated via electrophoresis with sodium dodecyl sulfate, and then electrophoretically transferred onto nitrocellulose membranes , which were blocked with 3% bovine serum albumin (Solarbio). Next, special proteins on the membranes were immunoblotted with corresponding primary antibodies for MYO6 , YY1 , or GAPDH , and then with universal secondary antibody  for 3 h. Eventually, an immunoblotting signal was developed by exposing to ECL reagent (Beyotime), and the blots were detected via Quantity One v4.6 (Bio-Rad) with GAPDH as control.4 cells of differently treated SW620 and LoVo cells were seeded in 96-well plates with four paralleled wells in each group. These cells were further cultured for 72 h and then added with 10% CCK-8 reagent (Beyotime) for 3 h. The optical density was determined at 450 nm on a microplate reader (Bio-Rad). Cell viability was calculated by the relative OD value with normalization to the control group (100%). For colony formation assay, 1000 cells/well of SW620 and LoVo cells were seeded in 6-well plates for 14-day cultivation. Single-cell-formed colonies were visualized and counted after crystal violet staining .1 × 105 cells of SW620 and LoVo cells were harvested for apoptosis and cell cycle analysis respectively using Annexin V-fluorescein isothiocyanate (FITC) apoptosis kit (Abcam) and propidium iodide (PI) FCM kit (Abcam). Briefly, apoptotic cells and cells distributed in cell cycle phases were examined with a flow cytometer .For cell apoptosis analysis, a group of 1 × 105 cells of SW620 and LoVo cells were re-suspended in serum-free RPMI-1640 medium for migration assay with Transwell chamber , and 5 × 105 cells were for invasion assay with Transwell chamber (BD) coated with Matrigel. In brief, these cells were seeded in the upper chamber, and a complete medium was loaded in the lower chamber as an attractant. Transwell systems were incubated in normal cell culture condition for 24 h, and cells transferred onto the lower membranes were visualized and counted after crystal violet staining (Solarbio).Transwell assay was applied to detect the migratory and invasive abilities. For migration assay, 2 × 10Cells were stained using 0.5% crystal violet. The images of migratory or invasive cells were captured under a microscope  at magnification ×100.LINC01224 promoter containing YY1 binding sites (GCCATC) or mutant type (CGGTAG) were cloned in pGL3-basic vector  to generate pGL3-WT1 (pGL3-LINC01224-WT1), pGL3-MUT1 (pGL3-LINC01224-MUT1), pGL3-WT2 (pGL3-LINC01224-WT2), or pGL3-MUT2 (pGL3-LINC01224-MUT2), which were then transfected into SW620 and LoVo cells together with YY1 overexpression vector or vector. The sequences of LINC01224 or MYO6-3′UTR containing miR-485-5p target sites or mutant sites were cloned in pGL3-basic vector to generate LINC01224-WT, LINC01224-MUT, MYO6-3′UTR-WT, or MYO6-3′UTR-MUT, which were transfected in SW620 and LoVo cells together with miR-485-5p mimic or miR-NC. Luciferase activity was detected with a dual-luciferase analysis kit (Promega).4 SW620 and LoVo cells were fixed with 1% formaldehyde (Solarbio) and then were sonicated for cutting DNA into 500-bp fragments. The DNA samples were precipitated with YY1 antibody (anti-YY1) for 6 h with anti-IgG as control, in the presence of magnetic beads. The precipitated chromatin DNA was isolated for qPCR.For the ChIP assay, a Magnetic ChIP Kit (Thermo Fisher Scientific) was used. 1 × 107 cells of SW620 and LoVo cells were collected and incubated with Protein A/G Sepharose® beads provided in the Magna RIP kit . These beads were pre-conjugated with the antibody of Ago2 (anti-Ago2) or anti-IgG for 8 h. Similarly, cell lysates containing 1 × 107 cells which were pre-transfected with biotinylated miR-485-5p mimic (Bio-miR-485-5p), Bio-NC, biotin-labeled LINC01224 probe, or oligo probe were collected and incubated with the magnetic beads supplied by Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific). Relative RNA expression levels in precipitated RNA complex on the beads were measured by qPCR.Cell lysates containing 1 × 106) were subcutaneously injected in 5-week-old male BALB/c athymic mice  (n = 6/group). The length and width of each xenograft tumor were monitored every 7 days, and tumor volume was calculated by 0.5 × length × width2 [sh-LINC01224#1 or sh-NC-transfected SW620 cells , and the significance of difference was set as P < 0.05.Data were shown as mean ± standard deviation from three repeats. The linear correlation between two variables was tested by Pearson’s correlation analysis. Association between LINC01224 level and overall survival of patients was evaluated via Kaplan-Meier plot and log-rank test. Difference was analyzed by Student’s n = 52)    of miR-485-5p and MYO6 are exhibited in Fig. To further study the role of LINC01224 in CRC progression, LINC01224-silenced SW620 cells were used to establish a xenograft tumor model in mice. After 35 days, the volume and weight of xenograft tumors were obviously lower in the sh-LINC01224#1 group comparing to the control group  and mortality  worldwide . lncRNAsAdditionally, there is a reported prognostic signature of 6-lncRNA and 12-gene for CRC , 38. LiklncRNA-mediated ceRNA regulatory network is an important mechanism in CRC . Gong etMoreover, we further identified MYO6 was targeted via miR-485-5p. Previous studies have indicated that MYO6 can facilitate CRC malignancy via boosting cell viability, cell cycle, cell motility, and glycolysis , 47–50. However, there are still some limitations in our study. Firstly, the sample size is 52 for the 5-year overall survival analysis of CRC patients. Secondly, starBase predicts other miRNAs and genes, which can be targeted by LINC01224 and miR-485-5p, and other pathways in CRC progression can be explored.In conclusion, LINC01224 knockdown represses CRC progression both in vitro and in vivo partly by regulating miR-485-5p and MYO6 via the ceRNA axis. Our study deepens our understanding on CRC pathogenesis. The study also indicates that LINC01224 high level may act as an unfavorable marker for the overall survival of these 52 CRC patients.Additional file 1: Figure S1. The enrichment of miRNAs in biotin-labelled LINC01224 probe in CRC cells.  RNA pull-down assay and qPCR determined relative miRNA expression in LINC01224 probe and oligo probe-mediated pull-down contents in LoVo and SW620 cells. ***P < 0.001.Additional file 2: Figure S2. Expression analysis of LINC01224, miR-485-5p and MYO6 in CRC patients. (A) GEPIA database showed the overall survival of COAD and READ patients (integrated from TCGA project) with High and Low LINC01224 level. (B) MYO6 level in COAD and READ was predicted via GEPIA with normalization to TCGA normal and GTEx data. Log2FC>1; p-value>0.01.  Relative miR-485-5p and MYO6 expression was determined via qPCR in CRC and normal control (NC) samples (n = 52). (E) StarBase 3.0 project analyzed the correlation among LINC01224, miR-485-5p and MYO6 levels in CRC patients (integrated from TCGA project). *P < 0.05.


Lung cancer is one of the most common malignancies with the highest mortality rate and the second-highest incidence rate after breast cancer, posing a serious threat to human health. The accidental discovery of the antitumor properties of cisplatin in the early 1960s aroused a growing interest in metal-based compounds for cancer treatment. However, the clinical application of cisplatin is limited by serious side effects and drug resistance. Therefore, other transition metal complexes have been developed for the treatment of different malignant cancers. Among them, Ru(II/III)-based complexes have emerged as promising anticancer drug candidates due to their potential anticancer properties and selective cytotoxic activity. In this review, we summarized the latest developments of Ru(II/III) complexes against lung cancer, focusing mainly on the mechanisms of their biological activities, including induction of apoptosis, necroptosis, autophagy, cell cycle arrest, inhibition of cell proliferation, and invasion and metastasis of lung cancer cells. Primary bronchogenic carcinoma, also known as lung cancer, is a malignant tumor that originates from the bronchial mucosa or gland. With a high incidence and mortality rate, lung cancer poses a serious threat to human health, while its cases and deaths rise every year . AlthougLung cancer can be classified into central and peripheral lung cancer depending on the anatomical part affected, as well as into two main pathological entities: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). NSCLC can be further subdivided into three histological subtypes: lung squamous cell carcinoma, lung adenocarcinoma, and large cell carcinoma. NSCLC, which accounts for about approximately 85% of lung cancer cases, has shown an increased mortality rate in recent years. Radical surgery is the most common treatment applied to early-stage NSCLC patients, while chemotherapy is mainly used for NSCLC patients in advanced or recurrent stages . MoreoveConsidering the effectiveness of cisplatin and its derivatives, other transition-metal complexes, such as Ru-, Ir-, Rh-, Pd-, Au-, and Os-based complexes, have emerged as a new generation of promising anticancer agents due to their potential anticancer properties and selective cytotoxic activity ,11,12,13trans-RuCl4(1H-imidazole) (DMSO-S)  (NAMI-A administered in combination with gemcitabine was given to patients with advanced NSCLC [trans-RuCl4(1H-indazole)2] (KP1019) [trans-RuCl4(1H-indazole)2] (KP1339)  (Ru3) and fac- [RuCl3(PPh3)(dmpbt)] (Ru4)  benzothiazole) (III(HL) Cl2(PPh3)2] (Ru5), [RuII(L)(CO)(PPh3)2] (Ru6), ([RuIII(HL) Cl2(AsPh3)2] (Ru7), and [RuII(L)(CO)(AsPh3)2] (Ru8)} (The tryptamine-based mixed ligand Schiff base Ru(III) complexes Ru(Cl)e) (Ru2)  showed misplatin . Furtherisplatin . In the hiazole) , apoptos] (Ru8)}  were preNAMI-A, which can greatly reduce lung metastasis and the formation of solid metastasizing tumors in mice 2PtCl2}, AH197 (Ru11/AH197) was also synthesized [4(DMSO-S)(μ-pyz)Pt(DMSO-S)Cl2] (IT127), NAMI-A, and Na [trans-RuCl4(DMSO)(pyz)] (AH403) [2Pt trinuclear species showed higher inhibitory activity in the order Ru11 > IT127 > NAMI-A > AH403  . The Ru2 > AH403  78], wh, whNAMI-Ru(II) complexes are also known for their low toxicity, different modes of action, and non-cross resistance to traditional Pt-based drugs, especially cisplatin. Therefore, recent studies have focused on understanding the anticancer mechanism of Ru(II) complexes to develop more effective Ru(II)-based drug candidates.6-p-cymene complexes have been reported as potential anticancer drugs, as they show distinct anti-proliferative activity and can effectively induce apoptosis [6-p-cymene)Cl)2] (RAPTA-C)  cymene complex, [(Ru(η6-p-cymene) Cl)2 pyrimidin-4-yl) benzene)] (Ru12)  polymerase(PARP) and triggering the p53-dependent pathway  (Ru12) , showed nd light . Mitocho pathway . Moreovetastasis ,83. In amembrane .2(η6-p-cymene)22Cl2]X2 (X = Cl− (Ru13), I− (Ru14), NO3− (Ru15), BF4− (Ru16), PF6− (Ru17), and CF3SO3− (Ru18); 1,3-bib = 1,3-di(1H-imidazol-1-yl) benzene) were designed and synthesized PF6] (dppm = bis(diphenylphosphino)methane) (Ru30), with stability in DMSO monitored by 31P {1H} NMR experiments, showed high cytotoxic activity against Leishmania promastigotes  and Ru32 [Ru(dmp)2(SA)] , two Ru(II) complexes bearing O,O-chelated ligands with low toxicity to BEAS-2B, could also induce apoptosis in A549 cells via caspase family proteins and PARP activation, ROS accumulation, DNA damage, MMP reduction, and Cytochrome c release from mitochondria (ClO4)2  (Ru33) could also downregulate the expression of BCL-2, BCL-x, BAK, and BIM, while upregulating the expression of BAG-1 and BAD, thus inducing apoptosis of NSCLC A549 cells via an intrinsic ROS-mediated mitochondrial dysfunction pathway (PF6)2 (Ru34), [Ru(phen)2(dmbpy)](PF6)2 (Ru35), [Ru(bphen)2(dmbpy)](PF6)2 (Ru36), [Ru(BPS)2dmbpy]Na2 (Ru37) } (Ru36 was the only lipophilic complex and exhibited the highest cellular uptake and significant phototoxicity [4(p-cpip)]2+ (p-cpip = 2-(4-chlorophenyl)-1H-imidazo  phenanthroline, MeIm = 1-methylimidazole) (Ru38) has also been recently synthesized and characterized, exhibiting relatively high cytotoxicity against lung cancer (A549) cells, as well as high selectivity to tumor vs. normal cells compared to cisplatin 2+  (Ru39)  signaling pathway. Eventually, the accumulation of Ru39 in mitochondria could induce autophagy and compete with mitochondria-mediated apoptosis , induced autophagy in Caki-1 renal cancer cells  and rat ) (Ru39) . Ru39 capoptosis . Further pathway . Resembl pathway .2+, causing lysosomal membrane permeabilization, and release cathepsins into the cytosol   triazine with different ancillary ligands have been recently prepared and used to induce necroptosis by Xiong K et al. (Ru40–46)  60]. Al. AlRu40– mitosis . The cha mitosis . Moreove mitosis .Ru25–29  compounds can not only suppress the primary tumor but also effectively inhibit malignant tumor metastasis. A series of recently synthesized Ru(II)-containing polypyridyl ligand complexes (Ru25–29  53] and andRu25–29 (Ru47  61]) sh) shRu25–2(bpy-2-nitroIm)]Cl2  propyl]-2,2′-bipyridine)} (Ru47) could reduce the activity of MMP enzymes to inhibit tumor metastasis  and Ru49/Ru-hq2 [(η6-p-cymene) Ru  Cl]  Cl] , have ald models . Similarisplatin . TherefoThe cell cycle, which involves four main phases , contributes significantly to the regulation of cell self-renewal and differentiation, while cyclin-dependent kinases (CDKs) and cycle proteins play a key role in the process . TherefoRu12  (L = carbazolone benzhydrazone ligands) [Ru30 [Ru48–49 [The cytotoxicity of {[Ru(pipe)(dppb)(bipy)]PF pathway . The numrylation . Furtheron [Ru47 , arresteligands)  -based compounds proved to be the most effective, as they show high cytotoxicity, which induces apoptosis, necroptosis, or autophagy and cell cycle arrest, thus inhibiting cell proliferation, invasion, and metastasis . Most stIn this review, we summarized the recent developments of Ru(II) and Ru(III) complexes and discussed their biological activity and mechanism of action against lung cancer. The existing findings clearly support that Ru complexes can be used to develop effective chemotherapeutic agents for human lung cancer, while they may serve as a guide for the design of other metallodrugs with higher efficiency and better clinical application potential. Nevertheless, the anticancer mechanisms of Ru(II/III) complexes require further investigation, and their efficiency against other cancer types should also be explored in future studies.


Endogenous and heterologpusly expressed human IFITM3 restricts early and late replication steps of chikungunya and Mayaro virus infection and is downregulated at the protein level in infected cells. IFITM3 that may associate with severe influenza in humans, restricted CHIKV, MAYV, and influenza A virus infection as efficiently as wild-type IFITM3. Antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several nonstructural protein(s) of CHIKV. Finally, IFITM3-imposed reduction of specific infectivity of nascent particles provides a rationale for the necessity of a virus-encoded counteraction strategy against this restriction factor.Interferon-induced transmembrane (IFITM) proteins restrict membrane fusion and virion internalization of several enveloped viruses. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies are insufficiently understood. Here, we characterized the impact of human IFITMs on the entry and spread of chikungunya virus and Mayaro virus and provide first evidence for a CHIKV-mediated antagonism of IFITMs. IFITM1, 2, and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in loss of antiviral activity. rs12252-C, a naturally occurring variant of  Infection of humans by mosquito-transmitted chikungunya virus (CHIKV) and Mayaro virus (MAYV) causes, in most of the infected individuals, an acute febrile illness accompanied by symmetric joint pain and inflammation. In a subset of infected individuals of varying size depending on the outbreak, long-term morbidity manifesting as chronic arthritis with debilitating pain has been reported for both members of the Semliki Forest Virus complex and is causing growing medical concern and socioeconomical loss in affected countries. Although likely being endemic in East Africa since several centuries, chikungunya virus outbreaks of increasing frequency have occurred worldwide in the past 15 yr. MAYV is causing increasing attention in the neotropics. Neither approved vaccines nor antivirals are available against either alphavirus infection. Despite increasing importance of emerging arthritogenic alphaviruses for the human population, the biology of their replication cycle and their interplay with host proteins only begin to be elucidated. Specifically, the entry process of CHIKV and MAYV is greatly facilitated in the presence of target cell–expressed adhesion molecule MXRA8 , and FHLIFITM genes, only IFITM1, IFITM2, and IFITM3 have antiviral properties by restricting virus entry  proteins are broadly active against numerous enveloped RNA viruses, including HIV-1 , West Nius entry . IFITM gtopology . IFITM2 topology . The mectopology . IFITM3 topology . Experimmutation  or deletmutation , nullifimutation .The potential importance of human IFITM3 protein as antiviral factor has been addressed in clinical observation studies of influenza A. Specifically, the single-nucleotide polymorphism (SNP) rs12252-C allele might associate with increased influenza A mortality and morbidity , althougMus musculus, a non-natural host of alphaviruses from the Semliki Forest Virus complex, IFITM3 shares 65% homology in amino acid sequence with the human ortholog and inhibits multiple arthritogenic alphaviruses, including CHIKV, and encephalitogenic alphaviruses in vivo  or by deleting a large part of exon 1 of IFITM3 (Δexon1) in both alleles. In addition, we introduced a T-to-C transition at position 89 in both IFITM3 alleles to express the minor C allele of the SNP rs12252 (rs12252-C), which has been suggested to associate with severe IAV infections (st ATG) (IFITM3 (IFITM2 gene (data not shown) had been edited. Immunoblotting showed that although all cell lines and clones clearly up-regulated expression of IFITM1 and the prototypic ISG product ISG15 upon IFN stimulation, IFITM3 protein detection was abrogated in HeLa cells encoding IFITM3 KO and IFITM3 Δexon1 (st ATG), arguing against expression of a truncated protein and rather for absence of expression  . In all  -HA and IFITM3(Δ1-21)-HA, which localize to the plasma membrane (20A)-HA and IFITM3(Δ1-21)-HA (20A)-HA and IFITM3(Δ1-21)-HA failed to impair infection -HA . IFITM3(1-21)-HA . To asse1-21)-HA . IFITM3.Most of the cell culture studies on CHIKV are conducted using cell-free, purified virus. An early report published in 1970 suggested that CHIKV spreads via cell-to-cell contacts when free virions are immunologically neutralized . Along t20A; Δ1-21) IFITM3 proteins were reduced in quantity in CHIKV-infected cells. This specificity was observed despite all IFITM-HA proteins being heterologously expressed under the control of the identical CMV immediate early promoter, which is unlikely to be targeted by CHIKV evasion strategies. In CHIKV-infected HeLa cells, endogenous IFITM3 protein levels were reduced in the absence of a detectable net decrease of IFITM3 mRNA, again pointing towards a specific counteraction strategy directed against the IFITM3 protein. Interestingly, IFITM3 degradation has been reported to occur through ubiquitination of a highly conserved PPxY motif that overlaps with the aforementioned YxxФ endocytosis motif by the E3 ubiquitin ligase NEDD4 , HEK293T , Vero E6 cells , and TZM-bl cells (obtained from the NIH AIDS Reagent Program) were cultured in DMEM with 10% FBS, 100 µg/ml streptomycin, and 2 mM L-glutamine. For an agarose-overlay, pre-warmed DMEM with 2% FBS mixed with liquid SeaPlaque Agarose (Lonza) to a final concentration of 0.8% agarose was added to cells 2 h postinfection. For plaque assays, Vero E6 cells were overlayed with 2.5% Avicel (Merck) after 1 h of virus inoculation. HEK293T cells expressing vector or IFITM-HA proteins were generated via retroviral transduction and subsequent puromycin selection. Interferon stimulation was performed using indicated concentrations of Roferon .IFITM3-edited HeLa clones were generated by electroporation of pMAX-CRISPR plasmids encoding EF1α promotor-driven Cas9-2A-EGFP and U6 promoter-driven chimeric gRNAs via the Neon Transfection System (Thermo Fisher Scientific), settings were as recommended by the manufacturer : 5 × 106 cells/ml, 1,005 V, 35 ms pulse width, two pulses. 50 µg/ml of each plasmid was used, for generation of rs12252 point mutation via HDR, 5 µM ssDNA repair template (Integrated DNA Technologies) was added. After electroporation, EGFP-expressing cells were FACS-sorted and single cell clones were obtained by limiting dilution. For gRNA target sequences and HDR-template sequence see st ATG): CACCCTCTGAGCATTCCCTG, IFITM3 locus reverse2 (Δexon1): GTGCCAGTCTGGAAGGTGAA, IFITM2 locus forward: CCCTGGCCAGCTCTGCA and IFITM2 locus reverse: CCCCTGGATTTCCGCCAG.20A were generated by cloning corresponding gblocks (Integrated DNA Technologies) into pQXCIP using the NotI and AgeI restriction sites. Retroviral particles were generated by lipofection of HEK293T cells with pQCXIP-IFITM-HA constructs, and plasmids encoding MLV gag pol (226V); E1(A226V/M269V/), E2(K252Q): sl1; E1(K211N/A226V), E2(V222I): sl2; E1(A226V/M269V), E3(S18F): sl3A; E1(A226V/M269V), E2(R198Q), E3(S18F): sl3B; E1(A226V/M269V), E2(L210Q): sl4. pCHIKrep1 EGFP, encoding CHIKV non-structural proteins 1–4 was kindly provided by Gorben Pijlman . MAYV (strain TRVL15537) was passaged on Vero cells. HSV-1 stocks were prepared as previously described +)Lox-pMCMVGFP, which expresses GFP under the control of the major immediate-early promoter of murine cytomegalovirus  and processed according to the recommended protocol. The lysate was mixed with Laemmli buffer and boiled for 10 min at 95°C. Proteins were run on a 10% SDS–PAGE and immobilized on a nitrocellulose membrane  using the Trans-Blot Turbo system (Bio-Rad). Blocked membranes were incubated with the following antibodies: mouse anti-MAPK , rabbit anti-Tubulin , mouse anti-HA , mouse anti-IFITM1 , mouse anti-IFITM2 , rabbit anti-IFITM3 , rabbit anti-CHIKV antiserum , or mouse anti-HIV-1 p24 . Secondary antibodies conjugated to Alexa 680/800 fluorescent dyes were used for detection and quantification by Odyssey Infrared Imaging System (LI-COR Biosciences).Cells were fixed with 4% PFA (Carl Roth) and permeabilized in 0.1% Triton X-100 (Thermo Fisher Scientific) in PBS before immunostaining, if not stated otherwise. Cells were immunostained with the following antibodies: rabbit anti-IFITM3 , mouse anti-ISG15 , rabbit anti-MX2 , mouse anti-BST2 BV421  and mouse anti-HA . Secondary antibodies conjugated to Alexa Fluor 488 or 647  were used for detection. Flow cytometry analysis was performed using FACS Calibur, FACS Celesta, or FACS Accuri with BD CellQuest Pro 4.0.2 Software (BD Pharmingen) and FlowJo V10 Software (FlowJo).Cells were grown in µ-slide eight wells (Ibidi). Cells were fixed with 4% PFA and permeabilized with 0.5% Triton X-100 in PBS. Immunostaining was performed with primary antibodies for 1 h at room temperature for HA  or rabbit anti-IFITM3  and secondary antibodies conjugated to Alexa Fluor 488 and 647  for 1 h at room temperature. Nuclear DNA was stained with 2.5 µg/ml DAPI (Invitrogen) for 5 min at room temperature. Microscopic analysis was performed using a Nikon Ti-E microscope equipped with a Yokogawa CSU-X1 spinning-disc and an Andor DU-888 camera. ImageJ was used to prepare microscopy images and for quantification of signal intensity of the immunostaining.IFITM3 (assay ID Hs03057129_s1), IFIT1 (assay ID Hs01911452_s1), and RNASEP (#4316849). Influenza virus RNA replication was assessed by quantifying HA mRNA levels with forward primer CAGATGCAGACACAATATGT and reverse primer TAGTGGGGCTATTCCTTTTA. Relative expression was calculated with the ΔΔCT method, using RNASEP or GAPDH mRNA as reference.Cells were lysed and RNA extracted using the Promega Maxwell 16 with the LEV simplyRNA tissue. cDNA was prepared using dNTPs (Thermo Fisher Scientific), random hexamers (Jena Bioscience) and M-MuLV reverse transcriptase (NEB) with buffer. Quantification of relative cellular mRNA levels was performed with the 7500 Fast Real-Time PCR System (Applied Biosystems) or the LightCycler 480 PCR System (Roche) in technical triplicates using Taq-Man PCR technology with the following Taqman probes and primers (Thermo Fisher Scientific): human t tests. P-values < 0.05 were considered significant (*), <0.01 very significant (**), <0.001 extremely significant (***); n.s., not significant (≥0.05).If not otherwise stated, bars and symbols show the arithmetic mean of indicated amount of repetitions. Error bars indicate SD from at least three or SEM from the indicated amount of individual experiments. Statistical analysis was performed with GraphPad Prism 8.3.0 using two-tailed unpaired


We used the BinaxNOW COVID-19 Ag Card to screen 1,540 asymptomatic college students for severe acute respiratory syndrome coronavirus 2 in a low-prevalence setting. Compared with reverse transcription PCR, BinaxNOW showed 20% overall sensitivity; among participants with culturable virus, sensitivity was 60%. BinaxNOW provides point-of-care screening but misses many infections. In August 2020, the US Food and Drug Administration granted emergency use authorization to the BinaxNOW COVID-19 Ag Card , and college; the study was conducted in accordance with applicable federal law and CDC policy.Project staff directed students to self-collect 2 anterior nasal swab samples by inserting 1 swab into each nostril and then switching the swabs to obtain sample secretions from the other nostril. We tested 1 swab immediately using BinaxNOW and sent the other swab to CDC for rRT-PCR.We conducted BinaxNOW assays on-site in accordance with manufacturer instructions (https://www.thermofisher.com) with cold packs; samples were transported daily to CDC and refrigerated at 4°C. We isolated nucleic acid from the specimens using the MagNA Pure 96 Instrument  within 48 hours of collection, then analyzed the nucleic acid using the CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay (t] <40 for the SARS-CoV-2 target), SARS-CoV-2–negative, or invalid .Swab samples collected for rRT-PCR were stored using Remel R12587 viral transport media . We extracted nucleic acid from the wells exhibiting cytopathic effects and confirmed the presence of SARS-CoV-2 by rRT-PCR. We considered a specimen to be culture-positive if the first viral passage had a Ct value >2 less than the clinical sample.We cultured residual frozen SARS-CoV-2–positive samples in 100 μL viral transport media. We limited dilution in Vero CCL-81 cells and monitored 96-well plates daily for cytopathic effects . Students received rRT-PCR results within 72 hours. No specimens tested positive for influenza A or B viruses. All 8 persons who tested positive by BinaxNOW and rRT-PCR later reported symptom onset. Among the 32 students who provided samples that tested negative by BinaxNOW and positive by rRT-PCR, 10 (31.3%) later reported symptom onset (median Ct 34.9), 16 (50.0%) later reported no symptoms (median Ct 35.1), and 6 (18.8%) did not report information on symptoms (median Ct 34.9).In total, 1,540 asymptomatic students provided paired samples . Forty (t 28.9) that tested negative by BinaxNOW but was culture-positive; symptoms later developed in this person, who tested positive by a different antigen test  the next day. Symptoms did not develop in the other person who provided a sample that tested negative by BinaxNOW and positive by culture (Ct 37.3).We detected culturable virus in 5 (12.5%) samples that tested positive by rRT-PCR, including 3 (60%) that also tested positive by BinaxNOW . One pert values, which suggest lower viral load. BinaxNOW did not identify 32 persons who tested positive by rRT-PCR.BinaxNOW provides rapid, point-of-care results; students received BinaxNOW results 3 days earlier than rRT-PCR results. However, BinaxNOW had low sensitivity, especially among persons with higher Ct value than discordant samples (22.5 vs. 33.9); we observed 88.9% (8/9) sensitivity among samples with Ct values <32. However, Prince-Guerra et al.  reported 41.2% sensitivity and 98.4% specificity among 871 asymptomatic college students (CDC provided guidance on expanded screening testing of asymptomatic individuals to reduce spread of SARS-CoV-2 and for interpretation of antigen tests (Isolation of SARS-CoV-2 in cell culture demonstrates viral replication. However, because many factors affect the culture performance, lack of culturable virus does not necessarily indicate a lack of infectious virus. The presence of culturable virus in samples that test negative for SARS-CoV-2 antigens suggests that BinaxNOW does not identify some persons with infectious virus. However, the speed of BinaxNOW enabled the immediate identification of 8 SARS-CoV-2–positive persons, thereby limiting transmission that might have occurred during the additional 2 days that students waited for rRT-PCR results.Although rRT-PCR tests remain standard for SARS-CoV-2 detection, point-of-care antigen tests such as BinaxNOW could increase access to serial screening, enabling the rapid identification and isolation of infectious persons. Because presymptomatic and asymptomatic persons can transmit SARS-CoV-2 (