Rho–associated protein kinase (ROCK) inhibitors are used for the survival of single-dissociated human induced pluripotent stem cells (hiPSCs); however, their effects on the growth behaviors of hiPSCs in suspension culture are unexplored. Therefore, we investigated the effect of ROCK inhibitor on growth behaviors of two hiPSC lines (Tic and 1383D2) with different formation of aggregate that attached between single cells in suspension culture. The apparent specific growth rate by long-term exposure to Y-27632, a ROCK inhibitor, was maintained throughout the culture. Long-term exposure to ROCK inhibitor led to an increase in cell division throughout the culture in both lines. Immunofluorescence staining confirmed that hiPSCs forming spherical aggregates showed localization of collagen type I on its periphery. In addition, phosphorylated myosin (pMLC) was localized at the periphery in culture under short-term exposure to ROCK inhibitor, whereas pMLC was not detected at whole the aggregate in culture under long-term exposure. Scanning electron microscopy indicated that long-term exposure to ROCK inhibitor blocked the structural alteration on the surface of cell aggregates. These results indicate that pMLC inhibition by long-term ROCK inhibition leads to enhanced growth abilities of hiPSCs in suspension culture by maintaining the structures of extracellular matrices. Human pluripotent stem cells (hPSCs), which include both human induced pluripo-tent stem cells (hiPSCs) and human embryonic stem cells (hESCs), are powerful tools in clinical and industrial applications because they have the ability of self-renewal and plu-ripotency ,2. ThereThe Rho–associated protein kinase (ROCK) pathway induces cellular apoptosis through myosin hyperactivation, and ROCK inhibitor promotes cell aggregation by suppressing apoptosis in suspension culture ,11,12. RThe main objective of the research is to understand the effects of ROCK inhibitor on the growth behavior of hiPSC aggregates after cell aggregation. To accomplish this objective, two different cell line (Tic and 1383D2 cell line) with a different character of aggregate formation, are used. In the source of these cell lines, Tic cell line derived human fetus lung cells of a 14-week-old male, and 1383D2 cell line derived from peripheral blood of 36-year-old male . In aggr3 cells/cm2 with 10 μM ROCK inhibitor  at 37 °C in a humidified atmosphere with 5% CO2. The culture medium was changed every 24 h.The hiPSC lines (Tic and 1383D2) were obtained from the Japanese Collection of Research Bioresources Cell Bank and the Center for iPS Cell Research and Application at the Kyoto University , respect5 cells/mL in a 30-mL stirred suspension bioreactor  in AK02N with 10 μM CultureSure Y-27632. The agitation of the bioreactor was controlled at a rotational speed of 55 rpm and the bioreactor was set at 37 °C in a humidified atmosphere [After cell detachment from plates on day 4 following the procedure reported in our previous study , single mosphere . The culmosphere . In contFor other experiments, cells were cultured as described above and treated with 10 μM CultureSure Y-27632, 10 μM Fasudil , and 10 μM Thiazovivin . In all cases, the ROCK inhibitors were prepared as a concentrated stock solution in sterilized water, with the same volume of sterilized water added to the controls.t = 24, 72, and 120 h). Aggregates were washed with phosphate-buffered saline (PBS) and dissociated into single cells using Accumax  with 10 μM ROCK inhibitor. Cells were counted using an automatic cell counter . To calculate cell number, cell aggregates were collected at different culture times (−1), and apparent specific rate of aggregate number, −1), were calculated as follows using cell density, X (cells/mL) at culture time, t (h) and aggregate number, na (aggregates): To elucidate the kinetics of growth behaviors in aggregates in suspension culture, the apparent specific growth rate, µg is the specific growth rate (h−1), kd is the specific death rate (h−1), and kbu and kcoa are the specific break-up and coalescent rates of cell aggregates (h−1).The growth processes of hiPSC aggregates in suspension culture were evaluated based on the apparent specific growth rate and apparent specific decreasing rate of aggregates, as previously described .(3)μappS (μm2) were measured after collection of cell aggregates at 24, 72, and 120 h, as previously described [S = 3 × 102 μm2 were counted. The three experiments were summed for all conditions to make the distribution of the projected area of cell aggregates.The projected area of cell aggregates, escribed . In thre2 cells/well. Cell aggregates were collected at 72 h and reseeded onto low-attachment 96-well V-bottom plates. Coalescence between aggregates was observed using a 4× objective lens, and images were obtained every 20 min for 100 min at several positions.To understand coalescence between the aggregates in the two hiPSC lines, we monitored the coalescence process between two aggregates in static suspension culture using a time-lapse microscope  equipped with a camera for video imaging. Briefly, the cells were resuspended in a medium containing 10 μM ROCK inhibitor in low-attachment 96-well V-bottom plates  at 2.0 × 10The immunofluorescence staining of frozen sections was performed as previously described ,6,7,8. FFor SEM, samples were fixed in phosphate-buffered 2% glutaraldehyde  in 0.1 M phosphate buffer (PB) and embedded with PB overnight at 4 °C. After the aggregates were fixed, they were post-fixed in 2% osmium tetroxide  for 2 h on ice. The specimens were then dehydrated in graded ethanol  and dried by t-butyl alcohol freeze-drying . The dried specimens were coated with 20-nm thick using an osmium plasma ion coater and subjected to SEM observation .t-test for data comparison between two groups. The Mann-Whitney test was used for data that did not exhibit a normal distribution. All data sets for the distribution of aggregate size were assessed for normality using the Shapiro-Wilk test. Statistical significance was set at p < 0.05. All experiments were conducted in at least thrice to represent the mean with standard deviation. Statistical analysis was performed using unpaired Student’s 2 cells/well into low-attachment 96-well V-bottom plates using a single-cell passaging method. To understand the effect of ROCK inhibitor on morphological and growth characteristics of two lines of hiPSCs in suspension culture, cells were seeded at a density of 2 × 10Similar effects were observed for aggregate morphology and growth in treatment groups with two other ROCK inhibitors, Fasudil and Thiazovivin . This suTo examine long-term exposure-dependent differences in the growth profiles of the two hiPSC lines in suspension culture, single cells were seeded in a 30-mL bioreactor. μapp) of the Tic line was maintained at a lower value than that of the 1383D2 line after short-term exposure. Studies on cell survival and the growth of the two hiPSC lines showed that the formation of small loose aggregates of the Tic line was owing to continued cell death during the exponential phase of growth with single cells extruded from growing hiPSC aggregates. High values of μapp were maintained throughout the culture during long-term exposure, and the final cell density was 5.93-fold higher than that during short-term exposure. The μapp value of 1383D2 line after short-term exposure gradually decreased throughout the culture, whereas that of cells was continuously maintained throughout the culture during long-term exposure. The final cell density of the long-term exposed culture was 4.17-fold higher than that of the short-term exposed culture. For long-term exposure, higher cell density and no change in aggregate numbers were observed compared to those of aggregates formed after short-term exposure.The apparent specific growth rate  showing the proportion of cell aggregates relative to total aggregates. The size distribution of aggregates was in the range of 0.03 × 104 to 2.55 × 105 μm2. Aggregates larger than 2.55 × 105 μm2 are represented on the right side of each histogram. In the case of Tic cell lines under short-term exposure, the mode of distribution was maintained 2.25 × 104 μm2 at 120 h. Under long-term exposure condition, the mode of distribution at 120 h was higher and wider (8.25 × 104 μm2) than that under short-term exposure. ROCK inhibitor induced a comparatively high aggregate size in the Tic line. In 1383D2 line, during short-term exposure to ROCK inhibitor, the mode of distribution increased from 24 to 120 h (8.25 × 104 μm2 at 120 h). For long-term exposure, the mode of distribution gradually increased from 2.25 × 104 μm2 at 24 h to 6.75 × 104 μm2 at 72 h. At 120 h, cell aggregates were present in the range of 1.50 × 104 to 9.00 × 104 μm2, and larger aggregates appeared to be evenly distributed in the range of 1.20 × 105 to 2.55 × 105 μm2. In addition, much larger aggregates were present than those with a size of 2.55 × 105 μm2. The size of aggregates increased, and that of many aggregates more than twice the mode of distribution at 72 h was noticed at 120 h after long-term exposure. In 1383D2 line, right-skewed distribution was formed from 24 h to 72 h, as similar to that in Tic line. For long-term exposure, two flat distributions were formed, while right-skewed distribution was maintained for short-term exposure. Those results indicated it is possible to promote coalescence between cell aggregates by long-term exposure to ROCK inhibitor.To further confirm that long-term exposure induces coalescence between aggregates, we plated two aggregates during short-and long-term exposure to ROCK inhibitor and assessed their behavior by time-lapse observation. In both hiPSC lines, a faster fusion of aggregates was observed in cultures with long-term exposure .Next, we determined whether the enhanced growth of hiPSCs elicited by the ROCK inhibitor was owing to ECM formation. To test this hypothesis, we investigated collagen type I produced by cells to correlate between aggregate formation and collagen type I production in culture with short- and long-term exposure to ROCK inhibitor. Collagen type I expression in the aggregates was evaluated by immunostaining at the culture endpoint (120 h). For short-term exposure, collagen type I was primarily localized at the periphery of hiPSC aggregates regardless of the cell line . When coSubsequently, we examined whether long-term exposure to ROCK inhibitor affects the organization of actomyosin cytoskeleton that is responsible for most force-driven processes in cells. We prepared the sections of aggregates stained with pMLC, an active domain of myosin motor proteins, for all cell lines and conditions at 120 h . For shoTo study the surface microstructures of cell aggregates of two hiPSC lines during short- and long-term exposure to ROCK inhibitor using SEM. Both hiPSC lines formed round-shaped aggregate of approximately 80 μm, with smooth surfaces covered with stacked cells at 24 h. These cells in the aggregates were 10 μm in diameter, and most of them presented a round morphology . When coAlthough several beneficial treatments with ROCK inhibitors have been reported for cell growth ,17,18,19When the aggregate morphology and size were compared throughout the culture , the 138Long-term exposure to ROCK inhibitor resulted in different growth kinetics of the two hiPSC lines in terms of cell division and coalescence between cell aggregates based on the morphology and size of the aggregates. In both hiPSC lines, aggregates by long-term exposure to ROCK inhibitor showed higher expansion fold and apparent specific growth rate compared to those of aggregates by short-term exposure. Furthermore, the apparent specific growth rate of the 1383D2 line by short-term exposure gradually decreased in the late phase, whereas that of cells by long-term exposure was continuously maintained throughout the culture. In this study, we hypothesized that the maintenance of ECM structure consequent to inhibition of actomyosin contractility by ROCK inhibitor leads to sustained growth throughout the suspension culture of hiPSCs. Long-term exposure to ROCK inhibitor induces the stable cell aggregate formation in the Tic cell line, which is easily collapsed by mechanical stress. ROCK inhibitors have been shown to regulate cell proliferation and differentiation by activating the extracellular signal-regulated kinase (ERK) pathway ,19. ROCKIn conclusion, we demonstrated that ROCK inhibition promoted cell division related to ECM structural alterations of hiPSCs in suspension culture. The effects on cell division could be explained by the maintenance of ECM structure through inhibition of pMLC within an aggregate after long-term exposure to ROCK inhibitors. Therefore, an understanding of the structural differences in the ECM between the two hiPSC lines will facilitate the understanding growth behavior of hiPSC aggregates after cell aggregation in suspension culture.


Asn, tRNAGln, tRNAGlu, tRNAPro, tRNAMet, tRNALeu(UUR) and tRNASer(UCN). TRUB1 knockout (KO) cell lines generated by CRISPR/Cas9 technology exhibited the loss of Ψ55 modification in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro but did not affect other 18 mitochondrial tRNAs. An in vitro assay revealed that recombinant TRUB1 protein can catalyze the efficient formation of Ψ55 in tRNAAsn and tRNAGln, but not in tRNAMet and tRNAArg. Notably, the overexpression of TRUB1 cDNA reversed the deficient Ψ55 modifications in these tRNAs in KOTRUB1 HeLa cells. TRUB1 deficiency affected the base-pairing (18A/G-Ψ55), conformation and stability but not aminoacylation capacity of these tRNAs. Furthermore, TRUB1 deficiency impacted mitochondrial translation and biogenesis of oxidative phosphorylation system. Our findings demonstrated that human TRUB1 is a highly conserved mitochondrial pseudouridine synthase responsible for the Ψ55 modification in the mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro.Pseudouridine (Ψ) at position 55 in tRNAs plays an important role in their structure and function. This modification is catalyzed by TruB/Pus4/Cbf5 family of pseudouridine synthases in bacteria and yeast. However, the mechanism of TRUB family underlying the formation of Ψ55 in the mammalian tRNAs is largely unknown. In this report, the CMC/reverse transcription assays demonstrated the presence of Ψ55 in the human mitochondrial tRNA In mammand TRMU . The m1GTRIT1) 4,,20.Glu caused by deafness and diabetes-associated m.14692A > G mutation resulted in the destabilization of base pairing (18A-Ψ55), improper folding, and instability of tRNAGlu  and tRNASer(UCN) (TRUB1 gene. The TRUB1 knock-out (KO) (TRUB1KO) cell lines exhibited the complete loss of Ψ55 modification in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro. An in vitro enzymatic activity of TRUB1 was assayed with unmodified tRNAAsn, tRNAGln, tRNAMet and tRNAArg generated by in vitro transcription as substrates using recombinant TRUB1. To verify the defects of TRUB1 knock-out in the HeLa cells, we transferred a plasmid carrying the full-length TRUB1 cDNA into the TRUB1KO cell lines. These TRUB1KO cell lines were then evaluated for the effects of TRUB1 deficiency on the conformation, stability, and aminoacylation capacity of mitochondrial tRNAs. These cell lines were then assessed for the effects of the TRUB1 deficiency on mitochondrial translation and the biogenesis of oxidative phosphorylation (OXPHOS) system.Of core modifications, the pseudouridine (Ψ) at position 55 of TΨC arm plays an important role in the structure and function of tRNAs including proper folding and stability, and translation . In part tRNAGlu . In bact tRNAGlu . In huma tRNAGlu . Cbf5/DK tRNAGlu . TRUB2 i tRNAGlu . TRUB1 h tRNAGlu ,33. HoweSer(UCN) . To gainTRUB1KO Hela cell lines were constructed using the CRISPR-Cas9 system 458 plasmid (Addgene) containing the sgRNAs (http://crispr.mit.edu) to minimize potential off-target effects. The sequences of sgRNAs that ultimately produced successful deletion clones were 5′-CACCGCACGGCGAACACGCCGCTCA-3′ and 5′-AAACTGAGCGGCGTGTTCGCCGTGC-3′. HeLa cells were transfected with Lenti CRISPR 458 plasmid (Addgene) containing the sgRNAs using jetPRIME , according to the manufacturer's instructions. After 24 hours, the cell cultures in the same media were treated with 1μg/ml of puromycin for three days. The cells were then collected and plated in the DMEM, supplemented with 10% FBS. Subsequently, cells were cloned by limiting dilution and individual clones were isolated.The HeLa cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies) , supplemented with 5% FBS. e sgRNAs . The sgRTRUB1 mutations in each clone was performed by PCR amplification of 400 bp fragment spanning partial promoter region and exon 1 (RefSeq NC_000010.11) and followed by Sanger sequence analysis. The forward and reverse primers for this genotyping analysis were 5′-GCTTCTGAGGCGGCGGTGGTGTCTTC-3′ and 5′-AATGGTCAGTCTCCCTTTCCCCTCCTTTTG-3′, respectively.The genotyping for the KOTRUB1 cells, the full-length coding region of TRUB1 cDNA was obtained by RT-PCR amplification using the high fidelity Pfu DNA polymerase (Promega) and total RNA isolated from HeLa cells as template, with primers with NheI site: 5′-TTGCTAGCCGCCACCATGGCCGCTTCTGAGGCGG-3′ (nt.60–78) and EcoRI site: 5′-TTGAATTCTTGTCATTCTGCATCTGCACACAGGA-3′(nt.1152–1177) (GenBank accession no. NM_139169.5). The PCR products were cloned by using the TA Cloning Kit (TaKaRa), analyzed by Sanger sequencing and then subcloned into a pCDH-puro-cMyc Vector (Addgene plasmid 46970). The pCDH-puro-cMyc Vector containing TRUB1 cDNA were co-transfected with the packaging vectors pMD2.G (Addgene) and psPAX2 (Addgene) into 293T cell line by Lipofectamine 3000 (Invitrogen) to produce virus. Two days following transfection, viral supernatants were collected and used to infect the TRUB1KO cells . Twenty micrograms of RNAs were incubated with 160 mM 1-cyclohexyl-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT) for 20 min at 37°C to allow for carbodiimide (CMC) modification of Ψ residues . The reaWestern blot analysis was performed as detailed elsewhere . Twenty Asn, tRNAGln, tRNAMet and tRNAArg were generated by in vitro transcription by T7 RNA polymerase (Promega) using synthetic DNA oligonucleotides as templates. Forward oligos for all tRNAs contained T7 RNA polymerase promoter sequences. The transcripts were purified by 10% polyacrylamide gel electrophoresis. The sequences of oligonucleotides were 5′-GCTAATACGACTCACTATATAGATTGAAGCCAGTTGATTAGGGTGCTTAGCTGTTAACTAAGTGTTTGTGGGTTTAAGTCCCATTGGTCTAGCCA-3′ and 5′-TGGCTAGACCAATGGGACTTAAACCCACAAACACTTAGTTAACAGCTAAGCACCCTAATCAACTGGCTTCAATCTATATAGTGAGTCGTATTAGC-3′ for tRNAAsn, 5′-GCTAATACGACTCACTATATAGGATGGGGTGTGATAGGTGGCACGGAGAATTTTGGATTCTCAGGGATGGGTTCGATTCTCATAGTCCTAGCCA-3′ and 5′-TGGCTAGGACTATGAGAATCGAACCCATCCCTGAGAATCCAAAATTCTCCGTGCCACCTATCACACCCCATCCTATATAGTGAGTCGTATTAGC-3′ for tRNAGln, 5′-GCTAATACGACTCACTATAAGTAAGGTCAGCTAAATAAGCTATCGGGCCCATACCCCGAAAATGTTGGTTATACCCTTCCCGTACTACCA -3′ and 5′-TGGTAGTACGGGAAGGGTATAACCAACATTTTCGGGGTATGGGCCCGATAGCTTATTTAGCTGACCTTACTTATAGTGAGTCGTATTAGC -3′ for tRNAMet, 5′-GCTAATACGACTCACTATATGGTATATAGTTTAAACAAAACGAATGATTTCGACTCATTAAATTATGATAATCATATTTACCAACCA -3′ and 5′-TGGTTGGTAAATATGATTATCATAATTTAATGAGTCGAAATCATTCGTTTTGTTTAAACTATATACCATATAGTGAGTCGTATTAGC -3′ for tRNAArg.tRNA transcripts as substrates for enzymatic reactions were produced as described previously ,40. The TRUB1 gene (GenBank accession number: NM_139169) were amplified through RT-PCR by using primers (5′-CGGAATTCATGGCCGCTTCTGAGG-3′ and 5′-GCGTCGACTCAGTGGTGGTGGTGG-3′) containing an EcoRI restriction site added to the 5′ end and a SalI restriction site added to the 3′ end. The PCR products were digested and ligated into a pMAL-c2E-derived vector (pMAL-c2E-TEV-His) between the EcoRI and SalI sites, which produces a maltose binding protein (MBP)-fused protein containing a TEV cleavage site between MBP and fused partner and a His-tag fused at the C terminus of recombinant protein (Escherichia coli DH5α and then incubated at 37°C for 4h in Luria-Bertani medium containing ampicillin (100 μg/ml) after the addition of 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). The recombinant TRUB1 protein was separated via SDS-PAGE stained with Coomassie Brilliant Blue and purified through High Affinity Ni-Charged Resin FF in accordance with the manufacturer's manual (Genscript). The TEV Protease (Beyotime) was used to digest the fusion protein TRUB1-MBP to release the MBP tag.The open reading frames of  protein . The pMA4Cl, 5 mM Mg(OAc)2, 5 mM DTT, 400 units/mL RNasin) with 500 nM of in vitro transcribed tRNAs as substrates  from intact cells from various cell lines, as detailed elsewhere , tRNALeu(UUR), tRNASer(AGY), tRNALeu(CUN), tRNAMet and 5S rRNA were as detailed previously , under acid conditions as detailed elsewhere . Differences were considered significant at a Asn, tRNAGln, tRNAGlu, tRNAPro, tRNAMet, tRNALeu(UUR) and tRNASer(UCN). This approach involved CMC adduct formation with U, G and pseudouridine followed by mild alkali to remove the adduct from U and G, but not from the N3-[N-cyclohexyl-N'-β-(4-methyl-morpholinium) ethylcarbodiimide]-Ψ (N3-CMC-Ψ)  and tRNASer(UCN) but not in other 15 mitochondrial tRNAs.To examine the presence of Ψ55 in human mitochondrial tRNAs, we subjected total enriched RNAs from HeLa cells to the CMC/reverse transcription assays with DIG-labeled oligonucleotide probes specific for 22 mitochondrial tRNAs including tRNA3-CMC-Ψ) . This yiTRUB1 encodes a 349 amino acid protein with a putative mitochondrial target sequence at the N-terminus, predicted by Mitoprot program  cell lines carrying composite deletions , and truncated protein with 147 resides, while the 20 bp deletion yielded a frameshift from codon 63, the introduction of a premature stop at codon 152 (p.Pro152*), and truncated protein with 151 amino acids. The TRUB1KO2 cell line carried two alleles, which were generated by introducing a 2 bp deletion or a 1 bp insertion in the exon 1 of TRUB1, respectively. The 2 bp deletion yielded a frameshift from codon 58, the introduction of a premature stop at codon 158 (p.Pro158*), and truncated protein with 157 amino acids, while the 1 bp deletion resulted in a frameshift from codon 68, the introduction of a premature stop at codon 159 (p.Pro159*), and truncated protein with 158 amino acids. These alleles were confirmed by Sanger sequencing , and Western blot analysis  and tRNASer(UCN), we subjected total enriched small RNAs from TRUB1KO and wild type (WT) cell lines to the CMC/reverse transcription with DIG-labeled oligonucleotide probes specific for mitochondrial tRNAAsn, tRNAGln, tRNAGlu, tRNAPro, tRNAMet, tRNALeu(UUR) and tRNASer(UCN), as well as cytoplasmic tRNAThr(ACU), tRNAMet(AUG), tRNATyr(UAC) and tRNAHis(CAC), which contain Ψ55 modification  and tRNASer(UCN), as well as cytosolic tRNAThr(ACU), tRNAMet(AUG), tRNATyr(UAC) and tRNAHis(CAC) derived from both TRUB1KO and WT cell lines  transcripts together with tRNAMet (U55) and tRNAArg (A55) transcripts as negative controls were incubated with recombinant TRUB1 protein to examine the formation of a single Ψ residue at position 55 in these tRNAs. As shown in Figure Asn and tRNAGln transcripts (U55) in the presence of recombinant TRUB1 protein. However, the Ψ55 modification was not detected in the tRNAMet (U55) and tRNAArg transcripts (A55) in the presence of recombinant TRUB1 protein. These results verified the essential role of TRUB1 in the formation of Ψ55 in tRNAAsn and tRNAGln, as well as tRNAGlu and tRNAPro.To further examine whether TRUB1 catalyzes the isomerization of U55 in mitochondrial tRNAs, we produced human recombinant TRUB1 protein using a prokaryotic expression system and mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro. To test if the TRUB1 deficiency-induced loss of Ψ55 in tRNAAsn, tRNAGln, tRNAGlu and tRNAPro affects the conformation of these tRNAs, total RNAs from TRUB1KO cell lines or those with overexpression ofTRUB1 cDNA and WT cell lines were electrophoresed through a 10% polyacrylamide gel (native condition) in Tris-borate-EDTA buffer and then electroblotted onto a positively charged nylon membrane for hybridization analysis with DIG-labeled oligodeoxynucleotide probes for tRNAAsn, tRNAGln, tRNAGlu, tRNAPro, tRNAMet, tRNALeu(UUR), tRNASer(UCN) and tRNALys, respectively. As shown in Figure Gln, tRNAGlu in TRUB1KO cells migrated slower than those of wild type cells, while electrophoretic patterns in tRNAAsn, tRNAPro in TRUB1KO cells migrated faster than those of wild type cells. In contrast, there were no difference of electrophoretic patterns in tRNAMet, tRNALeu(UUR), tRNASer(UCN) and tRNALys between TRUB1KO and WT cell lines. These data suggested that the loss of Ψ55 in tRNAAsn, tRNAGln, tRNAGlu and tRNAPro changed the conformation of these tRNAs.It was anticipated that the destabilization of base-pairing (18A/G-Ψ55) perturbed the structure and function of tRNAGln, tRNAGlu, tRNAPro, tRNAMet and tRNALys from TRUB1KO and WT cell lines to digestion with the nuclease S1. The resultantly digested-products from TRUB1KO and WT cell lines were then followed by Northern blot analysis using tRNA probes that hybridized only to 3′ half tRNAs. As illustrated in Figure Gln, tRNAGlu and tRNAPro from TRUB1KO cell lines were more sensitive to S1-mediated digestion than those from WT cell lines and exhibited remarkable differences in S1-mediated digestion patterns of tRNAs from WT cell lines. Conversely, there was no significant difference between the sensitivity of tRNAMet and tRNALys from TRUB1KO and WT cell lines to digestion with the nuclease S1. These data validated that the inactivation of TRUB1 changed the conformation of mitochondrial tRNAs.We further evaluated whether the loss of TRUB1 perturbed the structures of tRNAs by analyzing the sensitivity of tRNATRUB1KO cell lines or those with overexpression of TRUB1 cDNA and HeLa cell lines to Northern blot with DIG-labeled oligodeoxynucleotide probes for tRNAAsn, tRNAGln, tRNAGlu, tRNAPro, tRNASer(UCN) as representatives of the whole L-strand transcription unit and tRNALeu(UUR), tRNASer(AGY), tRNALeu(CUN), tRNAMet derived from the H-strand transcription unit, as well as a nucleus-encoded mitochondrial 5S RNA (under denaturing condition)  ,45. As sTRUB1 deletion on the aminoacylation of tRNAs, we assessed the aminoacylation properties of tRNAAsn, tRNAGln, tRNAGlu, tRNAPro, tRNAMet, tRNALeu(UUR), tRNASer(UCN) and tRNAThr by the use of electrophoresis in an acidic polyacrylamide/urea gel system to separate uncharged tRNA species from the corresponding charged tRNA,electroblotting and hybridizing with tRNA probes described above  in the acidic urea PAGE system as described previously  as well as tRNAMet, tRNALeu(UUR) and tRNAThr between KOTRUB1 cells and WT HeLa cells. These data indicated the TRUB1 deficiency may not affect the aminoacylation properties of mitochondrial tRNAs.To understand the effect of ed above ,47. To feviously ,47, therKOTRUB1 and WT HeLa cells using TOM20 or GAPDH as loading control. These subunits included 8 mtDNA-encoding proteins , 10 nucleus-encoding polypeptides: NDUFA1, NDUFS1 and NDUFS1 [subunits of NADH:ubiquinone oxidoreductase (complex I)], SDHB [subunits of succinate ubiquinone oxidoreductase (complex II)], UQCRC2 and UQCRFS1 [subunits of ubiquinol-cytochrome c reductase (complex III)], COXIV and COX17 [subunits of cytochrome c oxidase (complex IV)], ATP5B and ATP5F1 [subunits of H+-ATPase (complex V)] ] .KOTRUB1 cell lines, as compared with these in the WT cell line. As shown in Figure P < 0.001) in TRUB1KO1 cells, and 79%, 70%, 78%, 102%, 87%, 49%, 94% and 102%, with an average of 83% (P < 0.001) in TRUB1KO2 cell line, relative to the mean values measured in the WT cell line. However, the overexpression of TRUB1 elevated the levels of these subunits.As shown in Figure TRUB1KO cell lines, as compared with WT cell line. As shown in supplemental Figure 5, the levels of NDUFA1, NDUFS1, NDUFA10, SDHB, UQCRC2, UQCRFS1, COXIV, COX17, ATP5B and ATP5F1 were 120%, 116%, 104%, 117%, 95%, 110%, 126%, 141%, 125% and 124%, with an average of 118% (P < 0.001) in TRUB1KO1 cell line, and 121%, 123%, 126%, 112%, 99%, 112%,116%, 133%, 120% and 117%, with an average of 118% (P < 0.001) in TRUB1KO2 cell line, relative to the mean values measured in the WT cell line. However, the levels of these proteins were reduced by the overexpression of TRUB1 cDNA. These indicated the impact of TRUB1 on mitochondrial translation.As shown in Figure TRUB1KO cell lines exhibited aberrant assembly of complex I, IV and V. In particular, the levels of complex I, II, III, IV and V were 55%, 105%, 113%, 45% and 72% in TRUB1KO1 cell line, and 53%, 114%, 99%, 23% and 65% in TRUB1KO2 cell line, relative to the mean values measured in the WT cell line, respectively  and tRNASer(UCN) but not in other 15 mitochondrial tRNAs. In E. coli, tRNA pseudouridine 55 synthase (TRUB) catalyzes the pseudouridine formation at U55 by recognizing the T-arm with a 17-base stem–loop structure at position 49–65  and tRNASer(UCN). In this investigation, two TRUB1KO cell lines with different alleles generated by with CRISPR/Cas9 system exhibited the complete loss of Ψ55 modification in tRNAAsn, tRNAGln, tRNAGlu and tRNAPro. On contrast, the TRUB1 deficiency did not affect the formation of the Ψ55 modification in the mitochondrial tRNAMet, tRNALeu(UUR) and tRNASer(UCN), as well as cytosolic tRNAThr(ACU), tRNAMet(AUG), tRNATyr(UAC) and tRNAHis(CAC). Furthermore, an in vitro enzymatic assay revealed that recombinant human TRUB1 protein indeed catalyzed the efficient formation of Ψ at position 55 in tRNAAsn and tRNAGln, but not in tRNAMet and tRNAArg. Notably, the overexpression of TRUB1 cDNA reversed the deficient formation of Ψ55 in tRNAAsn, tRNAGln, tRNAGlu and tRNAPro in KOTRUB1 cell lines. These data demonstrated that TRUB1 is responsible for the formation of Ψ55 in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro. However, the biosynthesis of Ψ55 in human mitochondrial tRNAMet, tRNALeu(UUR) and tRNASer(UCN) may be catalyzed by other pseudouridine synthase(s). In fact, the certain structural requirements may be critical for tRNA pseudouridine synthase activity  and tRNASer(UCN) are modified to m1A58 (Pseudouridine located at position 55 (Ψ55) in the TΨC arm of tRNA is a nearly universally conserved RNA modification found in all three domains of life ,3. In thon 49–65 ,54–56. Ion 49–65 . In humaon 49–65 . Notablyon 49–65 ,33. In tAsn, tRNAGln, tRNAGlu and tRNAPro, and thereby impacted their structure and function. In fact, the instability of mutant mitochondrial tRNAGlu was evidenced by electrophoretic mobility changes and sensitivity to S1-mediated digestion of tRNAGlu with the loss of Ψ55 caused by m.14692A > G (55U > C) mutation associated with diabetes and deafness  of tRNAAsn, tRNAGln, tRNAGlu and tRNAPro and the resultant aberrant structure tertiary structure, caused by the ablation of TRUB1. Furthermore, the tRNAGln, tRNAGlu and tRNAPro from TRUB1KO cell lines were more sensitive to S1-mediated digestion than those from WT cell line. However, there was no effects of TRUB1 ablation on the steady state levels of tRNAAsn, tRNAGln, tRNAGlu and tRNAPro under denatured conditions, in contrast with the decrease in the steady-state level of tRNAGlu in mutant cell lines carrying the tRNAGlu 14692A > G mutation  mutation exhibited reduced levels of mitochondrial proteins (an average decrease of ∼29%)  in the levels of CYTB, relative mild reductions (20–30%) in the levels of ND1, ND3, ND4 and ND5, but very mild decreases (3–5%) in the levels of ATP8 and CO2. In contrast to what was previously shown in cells carrying the tRNALys 8344A > G or tRNASer(UCN) 7445A > G mutation  and nuclear-encoded subunits. These mtDNA-encoded subunits appear to act as seeds for building new complexes, which requires nuclear-encoded subunit import and assembly with the assistance of assembly factors . In our of ∼29%) . In the mutation ,63, polyll lines . This coll lines ,64. The utations ,20,23,65Asn, tRNAGln, tRNAGlu, tRNAPro, tRNAMet, tRNALeu(UUR) and tRNASer(UCN). Our findings demonstrated that the TRUB1 is responsible for the formation of Ψ55 in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro and impacts on mitochondrial tRNA metabolism, translation and the biogenesis of oxidative phosphorylation system.In summary, our data highlighted the presence of Ψ55 in mitochondrial tRNAThe authors declare that [the/all other] data supporting the findings of this study are available within the article [and its supplementary information files].gkac698_Supplemental_FileClick here for additional data file.


Radiation therapy is one of the most effective tools in cancer therapy. However, success varies individually, necessitating improved understanding of radiobiology. Three-dimensional (3D) tumor spheroids are increasingly gaining attention, being a superior in vitro cancer model compared to 2D cell cultures. This in vitro study aimed at comparing radiation responses in 2D and 3D cell culture models of different human cancer cell lines  irradiated with varying doses  of X-ray beams. Radiation response was analyzed by growth analysis, various cell viability assays  and amount of DNA damage (γH2AX Western Blot). Results showed decreasing cell proliferation with the increase of radiation doses for all cell lines in monolayers and spheroids of LNCaP and T-47D. However, significantly lower radiosensitivity was detected in spheroids, most pronounced in PC-3, evincing radiation resistance of PC-3 spheroids up to 8 Gy and significant growth inhibition only by a dose escalation of 20 Gy. Cell line comparison showed highest radiosensitivity in LNCaP, followed by T-47D and PC-3 in 2D, whereas, in 3D, T-47D showed highest sensitivity. The results substantiate the significant differences in radiobiological response to X-rays between 2D and 3D cell culture models. Radiation biology often provides the rationale for the implementation of new treatment strategies in clinical practice; hence, it is important to foster the basic understanding of radiation effects. Up until now, mostly  2D cell monolayers were used to assess therapeutic efficacy of radiation treatments in basic research. Although this technique has many advantages, being simple, cheap, and highly reproducible, it also comprises limitations when trying to compare obtained results to in vivo tumors. These limitations include unnatural cell shapes, the lack of cell differentiation and cell-to-cell communication, as well as the unnaturally equal exposure of cells to nutrients or drugs . To over® 3D and resazurin assay to complement the data. To further investigate molecular mechanisms, γH2AX was investigated by Western Blot analysis.In the early 1970s, Durand and Sutherland introduced the so-called contact effect when firstly noting the increased radioresistance of spheroids compared to monolayers after irradiation using V79 Chinese hamster lung cells . Since t2 and tested routinely for mycoplasma contamination using the MycoAlert™ Mycoplasma Detection Kit , according to the manufacturer’s instructions [Cell lines were obtained from the American Type Culture Collection (ATCC). PC-3 and LNCaP cells were cultivated in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum, 2 mM Glutamine and 1% penicillin /streptomycin . T-47D cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum and 4 mM Glutamine. All reagents were obtained from Gibco Life Technologies Ltd. . Cells were cultured at 37 °C with 5% COructions .TM  was added to the PC-3 cell suspension, followed by centrifugation at 161× g for 10 min at 4 °C. During spheroid establishment, any influence of GeltrexTM on radiosensitivity was excluded (see Spheroids were generated using the liquid overlay technique. 2000 cells/well (in 100 µL medium) were seeded into U-bottom 96-well plates, precoated with a thin layer of 1.5% low gelling agarose . LNCaP and T-47D were cultured scaffold-free, whereas 3% Geltrexuded see .X-ray irradiation  was performed at 200 kV, 20 mA with a focus size of 5.5 mm using an YXLON reference irradiator  with a 0.5 mm copper filter and a 3 mm aluminum filter. Before exposure, dose rate was measured with a cross calibrated ionization chamber . The average dose rate at the level of the respective tumor model systems was about 1.2 Gy/min . Spheroid growth was observed by both visual inspection and size measurements using an Olympus IMT-2 Inverted Microscope with an XC50 Color Camera  and the cellSens Entry  software on days 3, 5, 8, 9, 11, 13, 15, 19, 23 and 29 after seeding. Media were renewed once per week. Per experiment, one representative spheroid per cell line and radiation dose was chosen, and a mean value of three diameter measurements was calculated and plotted with Prism Version 8 .Spheroids were washed with 100 µL phosphate buffered saline (PBS), and afterwards incubated with propidium iodide  for 5 h 45 min followed by the addition of calcein acetoxymethyl ester  with further incubation for 15 min. Spheroids were then transferred to flat-bottom 96-well plates, washed with PBS and imaged under an IMT-2 Inverted fluorescence Microscope (Olympus) equipped with a 100 W Mercury Power Supply  for fluorescence detection. Diameters of total spheroid and necrotic core were measured using the Digimizer software version 5.7.2 .Cell survival and reproductive ability after irradiation were determined by performing standard clonogenic assays . 250 PC-g at room temperature. The supernatant was discarded, and the cell pellet was resuspended in medium. Single cell suspensions were mixed with Trypan blue  to determine cell viability and cell counts, using the LUNATM automated cell counter .Cells were seeded in 6-well plates: 500,000 for evaluation after 0 and 24 h, 100,000 PC-3 or 250,000 LNCaP/T-47D for evaluation after 72 h. After overnight incubation, cells were irradiated with single doses of 2, 4, 8 and 20 Gy. After the aforementioned time points, supernatants, wash fraction and harvested cells were combined and centrifuged for 4 min at 161× Cells were seeded as monolayers in 6-well plates with respect to their growth rate , incubated for 2 days and irradiated with 0, 2, 4 or 8 Gy. Cell monolayers were further cultivated under standardized conditions see  for up tIn 3D, irradiated spheroids were transferred into flat-bottom 96-well plates and incubated with resazurin (17 h) one day prior to fluorescence detection on days 7, 15 and 21, as described before. ® 3D reagent  was thawed in the fridge overnight before use. The next day, the plate and reagent were equilibrated to room temperature before the addition of 100 µL reagent per well. Afterwards, the plate was vigorously shaken for 5 min using the Synergy HTX multimode reader  and incubated at room temperature for another 25 min before measurement of luminescence.The assay was performed according to the manufacturer’s protocol . On daysg, 4 °C for 20 min. For 3D lysates, 20 spheroids were pooled and centrifuged at 135× g for 4 min at room temperature. After washing with phosphate buffered saline, 60 µL RIPA lysis buffer and a protease inhibitor were added and further treated as described for monolayers.The amount of DNA double-strand breaks in 2D and 3D cultures was assessed by determining γH2AX as surrogate. Whole cell lysates of monolayers and spheroids were prepared directly after irradiation. For monolayers, 1 million cells were seeded in petri dishes one day prior to irradiation. Lysates were obtained by mechanical scraping using 300 µL radioimmunoprecipitation assay (RIPA) lysis buffer (Gibco) together with a protease inhibitor , shaking for 30 min on ice before centrifugation at 13,523× ® TurboTM Transfer System (Bio-Rad) using a Trans-Blot Turbo 5× Transfer Buffer (Bio-Rad) and a nitrocellulose membrane . Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit . Proteins were separated on Mini-PROTEAN TGX precast Gels  with a running buffer consisting of 3 g Tris(hydroxymethyl)aminomethane (TRIS), 14,5 g Glycine and 1 g Sodium dodecyl sulfate (SDS), dissolved in distilled water. Semi-dry blotting was performed on a Trans-BlotTM ECL Western Blotting Substrate Kit; Thermo Fisher Scientific, Waltham, MA, USA) and a ChemiDoc Imaging System (Bio-Rad), according to the manufacturer.Membranes were blocked with 5% bovine serum albumin  in TRIS buffered saline with Tween  on a shaker for 1 h and at room temperature. Primary antibody incubation (anti-phospho-Histone H2A.X (Ser139), clone JBW301 , β-Tubulin antibody  was performed overnight at 4 °C. Incubation with the respective horseradish peroxidase (HRP) conjugated secondary antibodies was performed for 1 h and at room temperature before detection of chemiluminescence .t-tests. p-values < 0.05 were considered statistically significant.Data was analyzed using Prism (GraphPad). Data comparison was performed by multiple unpaired Cell viability in monolayers, evaluated by Trypan Blue Exclusion Assay, showed no significant change in viability when measured immediately (0 h) and 72 h after irradiation up to 8 Gy A,B. HoweTo further investigate this effect in monolayers, clonogenic assays for evaluation of reproductive potential were performed. All cell lines showed a decreased number of colonies with increasing radiation dose, albeit to a different extent . Values For better comparability, the coefficients alpha and beta of the survival curve were determined. Higher values correspond to higher radiosensitivity , showingPC-3 monolayers grew with a doubling time of 24 h, whereas LNCaP took 60 h and T-47D 32 h. After irradiation of the LNCaP and T-47D monolayers, further culturing was possible solely for the cells irradiated with 2 or 4 Gy. Irradiation with 8 Gy completely hampered cell proliferation in the two cell lines, as already shown with the clonogenic assay, making further culturing impossible. However, regarding PC-3, even the monolayers irradiated with 8 Gy recovered and regrew.Spheroid growth was cell line dependent and is shown in Irradiating PC-3 spheroids with 2 Gy showed no significant growth difference to control. However, 4 or 8 Gy radiation initially resulted in a decrease in growth rate, followed by a period of faster growth rate, ultimately leading to no significant size difference to control after 29 days of growth. Only the highest single fraction doses of 20 Gy resulted in a sustained growth inhibition. In contrast, LNCaP and T-47D showed significant size differences to control on day 29 after irradiation with 4 Gy or 2 Gy, respectively. Dose dependent growth inhibition was also shown for higher doses, without regrowth in the observed period after 8 Gy irradiation. Dose escalation to 20 Gy did not result in a significantly stronger growth inhibition compared to 8 Gy, neither for LNCaP nor T-47D spheroids, suggesting 8 Gy already being a highly effective treatment. Disintegration of the spheroids was shown for PC-3 and T-47D after irradiation with 20 Gy. In contrast, LNCaP retained their dense structure and only deformed slightly. Photographs of representative spheroids on day 29 are shown in To test whether irradiation directly induces necrosis, live fluorescent imaging was performed. Therefore, spheroids were stained for viable  and necrotic  cells.All spheroid models consisted of a viable outer layer and central necrosis on day 8, except T-47D whose necrotic core was first observed on day 15 of growth. This is in line with previous studies, stating that the spheroid diameter needs to be bigger than 500 µm diameter to develop a necrotic core [Viability measurements correlated well with diameter measurements of spheroids. As expected, cell viability decreases with increasing radiation doses . RadiatiTo compare the radiation response in 2D and 3D using exactly the same method, the resazurin assay was performed. Differences in metabolic activity (measured as fluorescence intensity) to control were higher in 2D than in 3D for all cell lines and all radiation doses. This difference further increased with dose and time A,B. By measuring the amount of double-strand breaks, insight into molecular mechanisms is given. As shown in In recent years, there has been increasing interest in implementing three-dimensional cell cultures as tumor model systems, being superior to 2D cell cultures when compared to in vivo models. However, there is still a lack of systematic, long term radiobiological studies and, until now, no regard to differences between 2D and 3D cell cultures in the widely used prostate cancer cell lines PC-3 and LNCaP, as well as the breast cancer cell line T-47D. In this study, a radiation response of up to 20 Gy was compared between monolayers and spheroids of these three different human cancer cell lines. 2D and 3D cell culture models were cultivated, treated with X-rays  and examined on growth behavior, amount of double-strand breaks and viability, using a range of assays. Distinct responses to radiation were shown not only by microscopic growth analysis, but also on various molecular levels.® 3D assay. Similar observations have been made for 3D cultures of HCT116 and CAL27 cells [The Trypan Blue Exclusion assay in monolayers showed no cell death upon irradiation up to 72 h, but, rather, impeded cell replicability in a dose-dependent manner. Single-cell replicability was further investigated by the commonly used clonogenic assay. Resulting data clearly supported a dose-dependent effect: a decrease in cell proliferation with increased radiation dose was observed in all cell lines in 2D. This effect was also shown for LNCaP and T-47D in 3D by means of growth analysis and was also supported by Cell Titer Glo27 cells . RegardiThe amount of DNA double-strand breaks correlates with viability data and size measurement of spheroids. Under the same experimental conditions, spheroids showed less double-strand breaks than monolayers. This is in agreement with the findings of Fazeli et al. in the prostate cancer cell line DU145 using Comet Assay . The excComparing dimensionality, the resazurin assay showed big discrepancies between 2D and 3D in all cell lines, which increase with time. The monolayers showed higher difference in metabolic activity to the control, compared to spheroids for all radiation doses, supporting earlier described lower radiosensitivity in spheroids compared to monolayers ,17,18,19Moreover, clonogenic survival and growth data provided valid indication of cell line specific treatment sensitivity. Our results evinced the highest radiosensitivity for LNCaP , followed by T-47D and PC-3 in 2D, and an order of T-47D , LNCaP and PC-3 in 3D. These results raise the knowledge on the differences in radiosensitivity not only between cell lines, but also regarding dimensionality.Our study supports previous studies, adding data of a bigger repertoire of assays for therapeutic efficacy testing of radiation treatment as well as long term (up to 29 days) radiation response data for the widely used prostate cancer cell lines LNCaP and PC-3 and the breast cancer cell line T-47D. With this, we want to inspire further investigations to gain deeper knowledge on the biological effects of radiation.However, a limitation of this study is the use of a single cell type in the 3D model. We envisioned utilizing a co-culture model comprising stromal, endothelial and immune cells. Co-culturing aims for better recapitulation of in vivo conditions, such as intercellular interactions between different cell types not represented in the current model. It also remains to be shown how fractionation affects treatment outcome, since this is the current clinical practice in cancer patients treated with external beam therapy, with the general trend of increasing doses per fraction (hypofractionation) enabled by precision radiotherapy techniques. Besides radio-oncological applications, a future step should also be the translation of the current findings into a setup of radionuclide therapy dosimetry.This study demonstrates that the radiobiological response to X-rays measured in 2D is not reflected in 3D. The spheroid model shows higher radioresistance in all cell lines, compared to the respective monolayers. This effect is immense when using PC-3 cells, which showed resistance to X-ray radiation up to 8 Gy in spheroids, whereas a dose-dependent decrease in cell proliferation was shown for monolayers. Furthermore, the order of radiosensitivity was determined, observing highest radiosensitivity in LNCaP, followed by T-47D and PC-3 in 2D, but reversed order between LNCaP and T-47D (highest radiosensitivity in T-47D) in 3D. To further foster the understanding of radiobiology in radiation oncology and radionuclide therapy, more in vitro studies need to be performed with special regard to co-culturing methods.


Characterized by an aggressive course with a poor overall survival due to treatment refractoriness, plasmablastic lymphoma (PBL) is a rare variant of diffuse large cell B cell lymphoma. Gorham's lymphangiomatosis or Gorham–Stout disease (GSD) is a rare skeletal condition of unknown etiology characterized by progressive bone loss and nonmalignant proliferation of vascular and lymphatic channels within the affected bone. Neither disease has a standard of care. We present a 23-year-old HIV-negative woman with GSD, managed medically with octreotide and sirolimus, who developed PBL. After progressing on V-EPOCH , she was treated with daratumumab, lenalidomide, and dexamethasone (DRD) therapy and achieved complete remission after two cycles with progression after eight cycles. This is a report of treatment of PBL with DRD therapy. Clinical investigations of the DRD regimen in PBL in conjunction with other agents to improve both depth and durability of response are warranted. Plasmablastic lymphoma (PBL) is highly associated with a compromised immune system, with the majority of the cases found in HIV-positive individuals. HIV-negative disease occurs with a slight male predominance , a median age of 55 years, oral mucosal tropism, and is associated with a worse overall median survival . Among tPlasmacytic differentiation, CD38+, high MYC expression secondary to translocation 8; 14), high Ki-67 activity, positive MUM-1, and absence of B cell markers such as CD20 are the key features of the histology of PBL , high Ki.While the exact mechanism of GSD development is unknown, lymphangiogenic growth factors may be drivers , 11. AltWe present a case of PBL associated with sirolimus in a patient with GSD who achieved a complete response to treatment with daratumumab, lenalidomide, and dexamethasone.The patient was a 23-year-old Hispanic female in whom GSD was diagnosed at age 5 when she was found to have an absent left clavicle. Her manifestations of GSD were lymphedema of left upper extremity, resorption of the left clavicle, scapular head and humerus, chylothoraxes requiring chest tubes, and recurrent pericardial effusions requiring a window. Treatment with sirolimus and octreotide, for nearly 3 years, controlled lymphedema and effusions.She developed pneumonia and hypoxic respiratory failure which required intubation. No oropharyngeal lesions were detected. CT scan of the chest, abdomen, and pelvis showed markedly enlarged supraclavicular, mediastinal, left axillary, and thoracic wall conglomerate, as well as enlargement of the thyroid with a significant mass effect on the trachea. Due to critical illness, biopsy was limited to a fine needle aspiration (FNA) of a left axillary lymph node and revealed sheets of cells with plasma cast and revealed sheets of cells with plasmablastic morphology. Immunohistochemical stains showed that the malignant cells were kappa restricted with MYC overexpression (>80%) and MUM-1 and CD 138 expression, Ki-67 > 90%. The cells were negative for CD20, CD30, CD3, ALK, BCL6, BCL2, and HHV8. Notably, EBV in situ hybridization was negative . PD1 andFlow cytometry from FNA revealed an abnormal population of CD45-negative events, expressing bright CD38 and partial dim CD20. No definite surface light chain expression was detected. CD19, CD5, CD10, and any T cell antigens were not coexpressed.Fluorescence in situ hybridization (FISH) was positive for MYC gene rearrangement t and negative for both BCL2 and BCL6 translocation.PET/CT revealed hypermetabolic lymphadenopathy above and below the diaphragm, extensive conglomerate mass of the chest wall, left upper extremity, mediastinum, and left breast, and hypermetabolic involvement of the thyroid gland bilaterally with narrowing of the trachea. No bone lesions were found. SUV ranged from 4 to 12.8 with mean 9.4 and median 10.1 . Given tSirolimus was discontinued. Dose-adjusted V-EPOCH was initiated and was complicated by neutropenic fever despite high-dose granulocyte colony stimulating factor support. Cycle 2 of V-EPOCH was complicated by small bowel obstruction, prolonged neutropenia, and slow functional recovery. Repeat CT of the chest, abdomen, and pelvis revealed progression of disease in the small bowel and peritoneum. Treatment with lenalidomide, dexamethasone, and daratumumab as per the DRD regimen was initiated .Repeat PET-CT after 2 cycles of DRD showed significant improvement in the size of the nodes and a metabolic complete response in all nodes (Deauville 2) . In addiAutologous stem cell transplantation (ASCT) was considered. After 8 monthly cycles of DRD, PBL progressed. No IVIG was given. Standard dose carfilzomib was added to the regimen for one cycle without improvement. ICE  regimen was initiated without improvement. She expired 13 months after diagnosis.This patient had two diseases that required active management: GSD and HIV-negative and EBV-negative immunosuppression-associated PBL. As stated above, both HIV-negative and EBV-negative statuses have been independently linked to worse overall survival , 3.Plasmablastic lymphoma is a rare disease which makes up approximately 2% of HIV-associated lymphomas. The incidence of the disease is unknown, and it has been reported in patients of all ages.Review of the database from eighteen Surveillance, Epidemiology, and End Results (SEER) registries showed median age at diagnosis was 52 years with male predominance (81.9%) and majority of patients were non-Hispanics (76.6%). Patients were slightly more frequently diagnosed with advanced stage (III-IV) (59.0%). Extranodal presentation was more common than nodal (54.8% vs. 45.2%). Oral and gastrointestinal (GI) sites were the most frequent primary extranodal locations . Oral primary location was inversely associated with the presence of B symptoms and advanced Ann Arbor stage .PBL in HIV-negative patients is more heterogeneous in sites of involvement with advanced clinical stage, B symptoms, and bone marrow involvement being less common (25%) than in HIV-positive patients.MYC gene rearrangements in addition to the association with EBV infection as important pathogenic mechanisms [The specific pathogenesis of PBL is not well understood, and the cell of origin is thought to be the plasmablast, an activated B cell that has undergone somatic hypermutation and class switching recombination and is in the process of becoming a plasma cell. Recent studies have identified the presence of chanisms .MYC dysregulation mediated by translocation or amplification may cause plasmablast development and prevent further differentiation. MYC translocations may allow PBL cells to escape apoptosis [EBV infection is associated with prevention of apoptosis in B cells by several mechanisms related to EBV antigens. poptosis .MYC, in these lymphomas [Finally, recurrent mutations in PRDM1, a gene that encodes the plasma cell transcription factor Blimp1 protein, have been described. These mutations in PRDM1 could alter the regulation of different targets, including ymphomas .GSD is an extremely rare condition with no current consensus on management, as options are limited to case reports. One of the complications of the disease is the development of a chylothorax, which has a reported mortality rate of as high as 64% with medical management and 36% with surgical treatment , 15, andPBL is a difficult condition to treat with a poor overall survival of 6–19 months and without standard treatment regimens , 19–21.Improved survival was reported in PBL patients treated with chemotherapy recently. The 3-year and 5-year overall survival (OS) rates of treated PBL patients in SEER registries were 54% and 52.8%. HIV status did not affect survival outcomes in unadjusted and adjusted analyses .In a study of 135 PBL patients reported by the Lymphoma Study Association (LYSA), 80% of patients received chemotherapy and their median OS was 32 months .V-EPOCH has been associated with high complete response (CR) rates and improved overall survival , 19. In Surface signaling molecule expression can yield opportunities for utilization of antibody or cell-based therapeutics: CD19 (CAR-T), CD38 , CD 30 , and PD1/PDL1/PDL2 (checkpoint inhibitors) .The strong CD38+ expression that could be responsive to daratumumab, as well as the potential for dual activity of lenalidomide in PBL and GSD, supported the use of the DRD regimen in this patient.To our knowledge, there are no reported cases of attaining a complete response of PBL with DRD therapy. Eight cases of use of daratumumab in the treatment of plasmablastic neoplasms have been published. In one HIV-negative patient with plasmablastic myeloma, three cycles of daratumumab and CHOP  resulted in a partial response . The secNCT01192464 and NCT02917083), with Ramos et al. showing durable responses in patients with refractory NHL [CD30 is a viable target for PBL as positive expression has been found in approximately 30–50% of PBL cases . Two castory NHL .+PBL [In a study by Laurent et al. on 82 patients with PBL, nearly all cases expressed PDL1 and PD1 in the immune infiltrate, and one-quarter of them strongly expressed PDL1 in tumor cells and in immune cells. PD1/PDL1 were more overexpressed in the microenvironment in EBV+PBL . In anot+PBL .CD19 CAR-T therapy was described in a case report about a patient with relapsed PBL who had complete response with axicabtagene ciloleucel at 4 months with an undetectable CD-19+ count 2 months after administration. Unfortunately, the patient relapsed at 5 months with loss of CAR T cell activity .Stem cell transplantation in PBL has been assessed as either consolidation in chemotherapy sensitive disease or as salvage therapy . There aBased on this patient's experience, the DRD regimen has a significant activity in treatment of both PBL and GSD. The response and drug mechanisms suggest noncross resistance. We speculate that maximizing immunocompetency as soon as possible, e.g., by minimizing intensive corticosteroids and myelosuppressive chemotherapies after initial deep remission, may be a beneficial strategy. Furthermore, given that the DRD regimen is not readily available as an emergency intervention in the inpatient setting, we anticipate that an EPOCH-based regimen will remain as the preferred upfront induction regimen. We believe that testing DRD regimen in conjunction with other therapeutics such as checkpoint inhibitors, cellular therapeutics such as CAR-T, and/or stem cell transplantation with curative intent in PBL is warranted.


Limonium species represent a source of bioactive compounds that have been widely used in folk medicine. This study aimed to synthesize the anticancer and anti-proliferative potential of Limonium species through a systematic review. Searches were performed in the electronic databases PubMed/MEDLINE, Scopus, and Scielo and via a manual search. In vivo or in vitro studies that evaluated the anticancer or anti-proliferative effect of at least one Limonium species were included. In total, 942 studies were identified, with 33 articles read in full and 17 studies included for qualitative synthesis. Of these, 14 (82.35%) refer to in vitro assays, one (5.88%) was in vivo, and two (11.76%) were designed as in vitro and in vivo assays. Different extracts and isolated compounds from Limonium species were evaluated through cytotoxic analysis against various cancer cells lines . Limonium tetragonum was the most evaluated species. The possible cellular mechanism involved in the anticancer activity of some Limonium species included the inhibition of enzymatic activities and expression of matrix metalloproteinases (MMPs), which suggested anti-metastatic effects, anti-melanogenic activity, cell proliferation inhibition pathways, and antioxidant and immunomodulatory effects. The results reinforce the potential of Limonium species as a source for the discovery and development of new potential cytotoxic and anticancer agents. However, further studies and improvements in experimental designs are needed to better demonstrate the mechanism of action of all of these compounds. Among the clinically approved anticancer drugs, over 50% are derivatives of medicinal plants, as these have been recognized as a source of biologically active compounds with therapeutic potential, being historically used to treat, among others, different types of tumors ,10. In tLimonium Mill., which has 607 accepted species, six of which have been recorded as synonyms [Limonium species demonstrated the presence of different classes of metabolites, such as anthocyanins, flavonoids, proanthocyanidins, hydrolysable tannins, phytosterol, saponins, phenolic acids, and essential oils [Limonium includes species used in folk medicine that have been associated with a range of biological activities, such as antioxidant activities and free radical-scavenging abilities, antibacterial, antifungal, antimalarial, antileishmanial and neuroprotective effects, and promising cytotoxic activity against cancer cells [In this setting, Plumbaginaceae is a family that has 22 accepted genera including synonyms ,12. Previal oils ,14,15. Aer cells ,21,22,23Limonium species, articles that have systematically synthesized all available evidence on the potential roles of this genus in the oncology field are scarce [Limonium species by means of a broad systematic review of the literature.Although some primary studies evaluated the anticancer activity of e scarce . Thus, tOverall, 942 records were identified in the database after duplicate removal, of which 909 were excluded during screening (based on reading the title and abstract). Of the 33 articles read in full, 16 were eligible for inclusion. One additional record was found during manual searches, resulting in 17 studies for synthesis as shown in in vitro assessments, one study (5.88%) was in vivo, and two (11.76%) evaluated both in vivo and in vitro parameters. No ex vivo and in silico studies were found. Studies were mostly performed in China  and published between 2003 and 2021.The main baseline characteristics of the included studies are depicted in The main reported human cancer cell lines were acute lymphoblastic leukemia (MOLT-4), acute promyelocytic leukemia (HL-60), breast adenocarcinoma (MCF-7), breast carcinoma (T-47D), cervix adenocarcinoma (HeLa), colorectal adenocarcinoma , colorectal carcinoma (HCT116), chronic myeloid leukemia (K562), diffuse large cell lymphoma or non-Hodgkin’s B cell (Toledo), fibrosarcoma (HT-1080), gastric adenocarcinoma (BGC-823), hepatocellular carcinoma (HepG2), lung carcinoma (A549), malignant melanoma (SK-MEL-28), osteosarcoma (U2-OS), and pancreas epithelioid carcinoma (PANC-1); meanwhile, non-human cancer cell lines included Abelson murine leukemia virus-induced tumor (RAW-264.7), mouse melanoma (B16-F10), mouse sarcoma (J774), and rat glioma (C6). For human normal cell lines, studies used embryonic kidney (HEK-293), lung fibroblast (WI-38), primary peripheral blood mononuclear cells (PBMC), and skin fibroblast (WS1); for non-human normal cell lines, mouse bone marrow (S17), mouse microglia (N9), and monkey kidney (Vero) were reported.in vitro cell line was HepG2 for human hepatocellular carcinoma . Thirteen studies (76.47%) also evaluated other biological activity of Limonium species, such as immunologic effects; antioxidant, anti-diabetic, anti-inflammatory, antimicrobial, and antiviral activities; and anti-migration and anti-clonogenic effects in cells.The most evaluated in vivo or in vitro activities using only crude extracts, while the other four (23.53%) evaluated just isolated compounds of Limonium species. Both crude extracts and fractions of the plant were analyzed in one study (5.88%), while two others (11.76%) assessed only the fractions. The remaining three articles (17.64%) analyzed crude extracts, fractions, and isolated compounds of the plant. The phytochemistry of Limonium species included in the systematic review is summarized in The aerial parts were the most used, followed by the underground plant organs. Only one study (5.88%) used the whole plant, and other four (23.53%) were unclear about the part of the plant used. Most studies  analyzed the Limonium tetragonum (Thunb.) Bullock, and Bae et al. [L. tetragonum via tyrosinase and tyrosinase-related proteins. Hamadou et al. [Limonium duriusculum (Girard) Kuntze, and Cordeiro et al. [Limonium brasiliense (Boiss.) Kuntze compounds.Around one-third of studies n = 5; 29.41%) assessed the potential mechanism of action of the tested compounds. Bae et al. [.41% assee et al.  further e et al.  tested tu et al.  assessedo et al.  used floin vitro studies (18.75%) did not present data on the cell culture conditions; only one in vitro study (6.25%) calculated the selectivity index (SI). The SI can be defined as the ratio of the toxic concentration of a sample against its effective bioactive concentration. For evaluating any anti-proliferative activity of a sample, its cytotoxicity against normal and cancer cell lines must be determined in order to calculate the SI value [Other anti-neoplastic drugs were also used as positive controls . Three of the sixteen SI value .in vitro assays and from 6 to 312 h for in vivo assays. The results based on the 50% inhibitory concentration (IC50) of cell proliferation  against A. salina (both larvae and adults), and the chronic toxicity was considered higher than acute toxicity, as set by Lellau and Liebezeit [D. magna adults, L. vulgare reached relative toxicity of around 40%. The authors demonstrated that the L. vulgare extract was the second lead sample with the maximum activity for the inhibition of tumor induction based on a potato disc assay [The toxic activity of iebezeit . For D. sc assay .Limonium sinense (Girard) Kuntze (LSP) had no significant growth inhibition effect in vitro against HeLa and K562 cell lines. Although LSP could inhibit the growth of HepG2 cells, the maximal inhibition rate of LSP was no more than 30% for the concentration tested of 500 µg/mL. On the other hand, for all three different doses of LSP, in vivo tests demonstrated an important anticancer activity on Heps tumor cells. The greatest tumor inhibition rates achieved with 400 mg/kg of LSP were 38.03%. The LSP improved macrophage phagocytosis functions in immune-suppressed mice, suggesting that the anticancer activity of this compound can be related to the regulation of immune functions in mice [L. sinense  revealed that LSP21 has the most significant dose-dependent inhibitory effect on the growth of HepG2 tumor cells (inhibitory rate of 48.13%). This isolated compound induced cell body shrinkage, chromatin condensation, and a decrease in the number of tumor cells with normal morphology, which suggested that its cytotoxicity can be related to the inhibition of cell proliferation and induction of cell death [The study of Tang et al.  showed t in mice . Anotherll death .Limonium franchetii Kuntze (1) had moderate in vitro cytotoxic activity against the C6 cell line, with a proliferation inhibition rate of 77.09% (100 µg/mL). However, other isolated compounds had no significant cytotoxic activity against BGC-823 and HepG2 cell lines [Limonium densiflorum Kuntze, performed by Medini et al. [50 values of 29 µg/mL and 85 µg/mL, respectively. Furthermore, this extract was not significantly cytotoxic against the normal human WS1 cell line [A new flavonoid glycoside isolated from ll lines . The firi et al. , showed ell line .L. tetragonum extract was cytocompatible with the human HT-1080 cell lines and inhibited the enzymatic activity and mRNA expression of matrix metalloproteinase (MMP-2 and MMP-9). In another study, Bae et al. [L. tetragonum extract against HT-1080 cell lines, focusing on the inhibition of matrix metalloproteinases (MMP-2 and MMP-9) and the regulation of MMPs by intracellular inhibitors called tissue inhibitors of metalloproteinase (TIMPs). The authors demonstrated that 85% methanol and n-butanol fractions of the plant had potential antimetastatic effects and can regulate cell proliferation, differentiation, and death through their inhibitory effects on the enzymatic activity of MMPs (MMP-2 and MMP-9), regulation of MMPs and TIMP expression, and suppression of the mitogen-activated protein kinase (MAPK) pathway. However, n-hexane and 85% methanol fractions exhibit increased cytotoxicity following high concentrations. All fractions were cytocompatible at concentrations below 50 µg/mL. Similar results were found by Lee et al. [n-butanol fractions of L. tetragonum had antimelanogenic activity due to tyrosinase-inhibitory effects, the prevention of L-3,4-dihydroxyphenylalanine (L-DOPA) oxidation, and suppression of melanin production [Bae et al.  suggestee et al.  evaluatee et al.  who addioduction .Limonium algarvence Erben flowers against mammalian cell lines  and brine shrimp eggs  was evaluated by Rodrigues et al. [in vitro study resulted in rates of cellular viability higher than 80% at the concentration of 100 µg/mL, and non-toxic effects were observed at the maximal concentration of 1000 µg/mL against A. salina. According to the authors, all ethanoic extracts of L. algarvence flowers, peduncles, and leaves had no toxicity against human normal and cancer cell lines, HEK 293 and HepG2 cells, respectively. However, few extracts were able to reduce the viability of the non-human cancer cell line (RAW 264.7), with cellular viabilities ranging from 67.4% to 78.2% [The preliminary toxicity screen of an aqueous extract of s et al. . The in to 78.2% .Limonium bicolor Kuntze flowers. Both luteolin (4) and quercetin (9) were cytotoxic against the LoVo cell line, with rates of 89.10% and 79.78% for cell proliferation inhibition, respectively, at 100 µg/mL. The compounds acacetin (15) and eriodictyol (16) were cytotoxic against the U-2OS cell line at 100 µg/mL, with cell proliferation inhibition of 96.83% and 82.06%, respectively. Only acacetin was able to inhibit the proliferation of the MCF-7 cell line (97.05% at 100 µg/mL and 68.39% at 20 µg/mL). The authors suggested that the presence of 3-O-glycosylation in the isolated flavonoid of L. bicolor is not paramount for cytotoxic activity [Chen et al.  evaluateactivity .O-galatte (28), samaragenin A (29), and samaragenin B (30)) of L. brasiliense rhizome was evaluated by Cordeiro [50 = 8.23 ± 0.83; IC50 = 7.35 ± 0.36 µg/mL, respectively). The subfraction G showed an IC50 value of 7.92 ± 0.86 µg/mL against the MOLT-4 cell line, while samaragenin A resulted in an IC50 value of 29.24 ± 17.64 µg/mL for the K562 cell line. According to flow cytometry results, subfraction G required the lowest concentration for cell death mediated by apoptosis induction for K562 cell line (10 µg/mL) and the highest percentage of cell death mediated by late apoptosis (37.8%) and necrosis (24.7%) at 50 µg/mL for the MOLT-4 cell line, after 48 h of treatment. The isolated compound, samarangenin A, did not cause significant cell death (p < 0.05) [The cytotoxicity of crude extracts, fractions, subfractions, and isolated compounds  .Limonium virgatum (Willd.) Fourr. The crude extracts were more cytotoxic against the non-human tumor cell line (J774) than against the human non-tumor cell line (WI-38). However, the extracts of the species Silene succulenta Forssk. (stem and leaves) and Cirsium scabrum (Poir.) Bonnet & Barratte (leaves) showed the most significant cytotoxic activities when compared with those of L. virgatum [Sahli et al.  evaluatevirgatum .Limonium sokotranum (Vierh.) Radcl.-Sm. leaves and flowers exhibited the highest cytotoxic activity against HepG2 tumor cells, with an IC50 value of 9.97 ± 0.79 μg/mL, which was close to that of the positive control, doxorubicin (7.38 ± 0.11 μg/mL). On the other hand, IC50 values in the test against the MCF-7 cell line ranged from 8.70 ± 0.08 to 21.8 ± 1.30 μg/mL, and the lowest IC50 value was recorded with the methanol extract of L. sokotranum leaves and flowers (8.70 ± 0.08 μg/mL) [According to Al-Madhagi et al. , a petro8 μg/mL) .n-butanol extract from aerial parts of Limonium bonduellei (T.Lestib.) Kuntze against two human cancer cell lines (HT-29 and HeLa) was evaluated by Amrani et al. [n-butanol extract of L. bonduellei (250 μg/mL) showed the highest proliferation inhibition in all cell lines (92.6% in HT-29 and 98.9% in HeLa) [The anti-proliferative activity of an i et al. . The extin HeLa) .n-butanol extract and isolated compounds (apigenin (31) and apigenin7-O-β-D-(6”-methylglucuronide) (32)) of L. duriusculum against the HCT116 cell line were assessed for the first time by Hamadou et al. [50 value of 7.60 μg/mL, while the results for the apigenin IC50 were 25.74 μM. Apigenin7-O-β-D-6”-methylglucuronide did not affect cell proliferation [The anti-proliferative and pro-apoptotic activities of an u et al. . The autferation .Limonium gmelinii Kuntze roots against tumor cell lines was evaluated by Tuohongerbieke et al. [50= 25.25 μg/mL), and compounds (33) (IC50 = 19.24 ± 1.62 μM) and (50) (IC50= 12.85 ± 2.65 μM) and compounds (37), (43), (33), and (50) demonstrated moderate cytotoxicity against the MCF-7 cell line, with IC50 values ranged from 14.14 ± 1.08 to 28.85 ± 2.33 μM. Other lignanamides showed low or no cytotoxicity (IC50 >30 μM).The cytotoxic activity of the ethyl-acetate extract and isolated lignanamides of e et al. . The ethin vitro studies did not properly describe the conditions of cell culture  in the in vitro and in vivo studies were unclear about the domain of baseline characteristics, allocation concealment, and incomplete outcome data. It is unclear whether both in vitro and in vivo studies were free of selective data reporting, especially due to the lack of conflicts of interest or funding statements in some articles. Two-thirds of the articles were unclear about other sources of bias .However, despite i et al.  and duriin vivo and in vitro screening of natural products is to discover new promising agents, such as those with anticancer activity, and guide the development of new drugs [in vitro anticancer screenings of plant extracts, we can consider significant or strong cytotoxicity values of IC50 below 20 µg/mL for extracts and below 10 µM for isolated compounds. Usually, anticancer drugs from natural compounds act by inhibiting DNA synthesis (antimetabolites), damaging DNA , or inhibiting the function of the mitotic spindle based on microtubes  [One of the aims of the ew drugs . Accordiew drugs  for in vtaxanes) ,46,47. Itaxanes) ,49,50.in vitro studies were based on the colorimetric assay 3--2–5-diphenyltetrazolium bromide (MTT), a cell proliferation assay that measures the activity of mitochondrial dehydrogenase enzymes in living cells, and it is one of the most widely used assays for evaluating the preliminary anticancer activity of natural products [In vitro cell viability and cytotoxicity assays using cultured cells are widely employed for drug screening and have some advantages, such as quick assays, reduced costs, and room for automation. Currently, these assays are also used in anticancer drug development to evaluate the cytotoxicity and tumor cell growth inhibition of different compounds. However, in vitro assays are not technically advanced enough to promptly replace animal tests due to the lack of a physiological environment. This is not the case of in vivo assays that are able to measure several behavioral and physiological parameters and guide the understanding of the pharmacological activity of the tested compound on the entire organism [Most products ,52. Otheorganism ,54.Limonium species was evaluated through bioassays using the organisms A. salina and D. magna. A. salina (brine shrimp) is a highly sensitive crustacean, and it has been extensively used for toxic screening of bioactive compounds since 1956 [D. magna was first mentioned by Flücker and Flück (1949) as another organism used in toxicity testing. It is a simple, sensitive, and reproducible laboratory model for the toxicity screening of compounds [L. vulgare was toxic against A. salina and can be considered a promising candidate for new anticancer compounds [L. algarvense was non-toxic against A. salina, which could explain the lack of toxicity against the human hepatocellular carcinoma cell line [in vitro mammalian cells and in vivo brine shrimp assays.The toxicity of nce 1956 . D. magnompounds ,58,59. Tompounds . The ethompounds . The aquell line . This suL. algarvense have low cytotoxicity against the RAW 264.7 mouse cell line, and all extracts were not toxic against the human normal HEK 293 cell line and the human tumor HepG2 cell line. The authors suggest a possible correlation between the presence of several flavonoids in L. algarvense and the in vitro and in vivo hepatoprotective effect as demonstrated by other literature studies with this class of secondary metabolites [L. sinense against HepG2 tumor cells, this compound inhibited the growth of transplanted mouse tumors and demonstrated a synergistic action when used in association with the anti-neoplastic agent 5-fluorouracil. It was suggested that the anti-cancer effects could be related to the in vivo immunomodulatory activity [On the other hand, few ethanolic extracts of different organs of abolites . DespiteL. tetragonum, the most evaluated species among the included studies, was shown to be a potential source of bioactive agents with proven anti-MMP activity and anti-melanogenesis properties, including compounds that can prevent hyperpigmentation [L. tetragonum include, but are not limited to, flavonoid glycosides  and quercetin 3-O-β-galactopyranoside (14)). These compounds inhibit the activity of MMP, suppress MAPK associated with MMP upregulation, and act as anti-melanogenic compounds, demonstrating the nutraceutical potential of L. tetragonum as a source of anti-MMP compounds [L. densiflorum demonstrated promising anti-proliferative effects against human lung carcinoma (A549) and human colorectal adenocarcinoma (DLD-1), with results that were similar to those from the positive control etoposide. Furthermore, this extract was not significantly cytotoxic against a human normal cell line (WS1), which suggests the possible selectivity of the extract for cancer cells [entation ,31,34. Bompounds ,31,34. Ter cells .L. densiflorum crude extracts and isolated compounds was investigated. All extracts reduced nitric oxide (NO) production in a concentration-dependent manner, suggesting interesting anti-oxidant activities. These results can be due, in part, to the majority presence of polyphenolic compounds (flavonoids and phenolic acids) that can be related to the anticancer potential of L. densiflorum [The antioxidant, total phenolic content, and anti-inflammatory activity of siflorum . In addisiflorum ,62.L. brasiliense against neoplastic cells (HL-60) vs. normal cells (PBMCs), and the favorable anti-proliferative activity of subfractions F and G against the HL-60 cell line, which can be related to the immunomodulatory activity of crude extracts and fractions of L. brasiliense. In the literature, it is suggested that tumor growth and proliferation can also be restrained by targeting and modulating the immune response. Natural immunomodulators can stimulate humoral and cell-mediated immune responses against the tumor [Cordeiro  demonstrhe tumor ,64.L. sokotranum leaves and flowers, which displayed a profile similar to that of the positive control doxorubicin [50 value against the MCF-7 tumor cells (8.70 ± 0.08 µg/mL). Finally, the isolated compound apigenin (31) of L. duriusculum had the lowest IC50 value against the HCT116 cell line (25.74 µM). The n-butanol extract and isolated compound apigenin promote apoptosis in HCT116 cancer cells, associated with reduced signaling from MAPK, activation of the p53 response pathway, and poly(ADP-ribose) polymerase (PARP) cleavage [L. gmelinii roots showed moderate cytotoxicity against the HeLa cell line (25.25 µg/mL). The possible anti-cancer mechanisms of Limonium species suggested in the included studies are summarized in Other important cytotoxic activity, against HepG2 tumor cells, was obtained with the petroleum ether extract of orubicin . A methacleavage . The rescleavage  is the fLimonium species. Several of the compounds have already been described in Limonium spp., such as flavonoids and their glycosides derivatives [Limonium species could be investigated as a source of bioactive phytochemicals, including polyphenolic compounds that might combat oxidative stress, act in cell cycle regulation, and could possibly be used in the nutraceutical field. This can be supported by the fact that Limonium spp. are mainly composed of flavonoids, phenolic acids, and tannins  Kuntze (green tea). The authors demonstrated that L. algarvense flowers had similar or higher in vitro antioxidant and anti-inflammatory properties than green tea, based on radical-scavenging activities and the decrease in NO production, respectively [Many isolated or identified compounds, as primary and secondary metabolites, and principally phenolic compounds were described in the literature sourced. The extraction method and solvent polarity were some factors related to the phytochemical diversity observed in the crude extracts, subfractions, fractions, and isolated compounds of ivatives ,37,38. Oivatives . Thus, w tannins . These ps et al.  in a comectively .Limonium species associated with their high polyphenol content [Limonium species reported in included studies, can contribute to the anticancer effect observed in some of these species. Furthermore, as Limonium species are considered halophytes, which means that they can adapt to salinity conditions via physiological and biochemical processes, a consequent increase in the enzyme and antioxidant metabolites may occur depending on the environment [In addition, several studies demonstrated the antioxidant potential of  content ,36,65. O content . Thus, tironment ,67,68. Bironment .in vitro and in vivo studies, should be performed using appropriate and validated methods aimed at obtaining accurate and reliable results that can be reproduced by other studies. However, some of the included studies were unclear or lacked in their reporting of relevant information, such as the correct and accepted species name , the organ of the plant material used for extraction, the number of plant voucher specimens, culture conditions of cell lines, and the use of positive and negative controls. It was also found that in SYRCLE’s tool, the general quality of the articles was moderate. In this scenario, it was suggested that a checklist grounded on pharmacognostic literature of medicinal plants should be completed by researchers and authors prior to publication to standardize the conduction and reporting of studies in this field , atmosphere conditions (exact 0.0% CO2), and methods used).The assessment of cytotoxicity and other biological activities from plant species, both ield see . It was Limonium species, only a few studies were included because this field of the anti-proliferative and anticancer effects of this genus is still recent (studies published between 2003 and 2021). Despite the popular use of Limonium, few species had their biological potential evaluated. Due to the nature of the data, the differences among Limonium species, cell lines, and compounds evaluated, quantitative analyses were not possible.This conducted systematic review has some limitations. Although there is extensive literature on Limonium species have a wide worldwide distribution, with vast chemical and biological potential, and are popularly used in several countries. The results obtained from this systematic review reinforce these data and bring a new perspective in the search for useful anticancer agents from natural sources, mainly polyphenols. In this context, it is necessary to carry out more in vitro studies for a better understanding of the mechanism of action of these compounds and in this way, direct future in vivo and clinical studies, reinforcing the use of natural products in the discovery of less toxic, more selective, and effective phytochemicals for the treatment of different types of cancer.This systematic review was performed following the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement , CochranCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).A systematic search was performed based on the electronic databases PubMed/MEDLINE, Scopus, and Scielo with no time or language restrictions (updated on 24 June 2021). The full search strategy is available in the in vitro, in vivo, ex vivo, or in silico experimental studies that evaluated the anticancer or anti-proliferative activity of Limonium species . Studies evaluating other biological activities of Limonium species and with other study designs  and those published in non-Roman characters were excluded.We included in vitro assays and the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) tool were used to assess the studies’ risk of bias and methodological quality in the A standardized form was used to collect data on the studies’ general characteristics , bioassay type, methodological aspects, and main results. The adapted tool for Limonium species mostly refers to preliminary assessments of the cytotoxicity of different compounds obtained from crude extracts, fractions, subfractions, and isolates and their impact on the viability of a range of cancer cell lines. Limonium tetragonum was the most evaluated species, with promising in vitro anti-melanogenesis effects. Isolated compounds of the flavonoid class, such as apigenin of L. duriusculum, also demonstrated a favorable cytotoxic effect against colorectal cancer, as well as lignanamides of L. gmelinii against cervix and breast adenocarcinoma cell lines. However, the complete mechanism of action of all isolated compounds and their effect in in vivo models remain unclear for most species.The literature on the potential anticancer effects of Limonium spp. as a source for the discovery and development of new potential cytotoxic phytochemicals. However, better planning, experimental designs, and reporting of the results will make future studies more robust and provide better proof to demonstrate the mechanism of action of these compounds.These findings reinforce the biological potential of


In vitro drug combination screens have not been performed on NF1-associated HGG, hindering our ability to develop informed clinical trials. Here we present the first in vitro drug combination screen  on the only human NF1 patient derived HGG cell line available and on three mouse glioma cell lines derived from the NF1-P53 genetically engineered mouse model, which sporadically develop HGG. These mouse glioma cell lines were never exposed to serum, grow as spheres and express markers that are consistent with an Oligodendrocyte Precursor Cell (OPC) lineage origin. Importantly, even though the true cell of origin for HGG remains elusive, they are thought to arise from the OPC lineage. We evaluated drug sensitivities of the three murine glioma cell lines in a 3D spheroid growth assay, which more accurately reflects drug sensitivities in vivo. Excitingly, we identified six compounds targeting HDACs, BRD4, CHEK1, BMI-1, CDK1/2/5/9, and the proteasome that potently induced cell death in our NF1-associated HGG. Moreover, several of these inhibitors work synergistically with either MEK or PI3K inhibitors. This study forms the basis for further pre-clinical evaluation of promising targets, with an eventual hope to translate these to the clinic.Neurofibromatosis Type 1 (NF1) patients develop an array of benign and malignant tumors, of which Malignant Peripheral Nerve Sheath Tumors (MPNST) and High Grade Gliomas (HGG) have a dismal prognosis. About 15–20% of individuals with NF1 develop brain tumors and one third of these occur outside of the optic pathway. These non-optic pathway gliomas are more likely to progress to malignancy, especially in adults. Despite their low frequency, high grade gliomas have a disproportional effect on the morbidity of NF1 patients.  Neurofibromatosis 1 (NF1) is an autosomal dominant tumor predisposition syndrome impacting approximately one in every 3000 births , 5. AbouNF1 tumor suppressor gene due to inactivating mutations or deletions [NF1 individuals have a germline mutation in the NF1 gene that encodes neurofibromin, a GTPase activating protein that negatively regulates the RAS pathway. Mechanistically, neurofibromin accelerates the conversion from the active GTP-bound RAS to its inactive GDP-bound form. NF1-HGG have complete functional loss of the eletions , leadingeletions . Recentleletions , 10.https://cellmodelpassports.sanger.ac.uk/passports/SIDM01460). To our knowledge, this is the first drug sensitivity study performed on a HGG cell line derived from an NF1 patient.To date, limited drug sensitivity studies have been performed on NF1 associated High Grade Gliomas. This is in part due to the lack of HGG cell lines derived from NF1 patients; only a single human NF1-HGG is publicly available (TM-31). This cell line has inactivating mutations of NF1 , ATRX , CDKN2A (homozygous deletion) and TP53 (C238G)  sporadi- in cis . Signali- in cis –15. Our In our study, we sought to identify NF1-HGG sensitivities against agents targeting downstream RAS effector pathways (MAPK and AKT pathways) in combination with a broad array of approved and investigational anti-cancer drugs. Although NF1-HGG arises from aberrant RAS pathway hyperactivation, it is well established that monotherapy targeting a single pathway often renders limited therapeutic benefit especially against malignant, high-grade tumors. In recent years, combinatorial drug therapies have significantly altered the prognosis of many cancers in children and adults . ConsideThis research was determined to be none-human subject research by the Children’s Hospital Philadelphia Committees for the Protection of Human Subjects (IRB). The need for consent was waived.The NF1 patient-derived HGG cell line TM-31 (RRID:CVCL_6735) , purchasNf1 and Tp53; mutant alleles are located in cis. The 17, 5653, and 5746 glioma cell lines are grown in NeuroCult™ Mouse and Rat Basal Medium  supplemented with NeuroCult™ Mouse and Rat Proliferation Supplement , 1% Penicillin Streptomycin , 20 ng/mL human recombinant epidermal growth factor  and 10 ng/mL human recombinant fibroblast growth factor .Mouse NF1-HGG cell lines 17, 5653, and 5746 were derived from three distinct high-grade murine brain tumors sporadically developed by the NPcis genetically engineered mouse model. The germline of these mice is heterozygous for ® Nuclight Red Lentivirus  such that they will produce an EF1α promoter driven nuclear restricted red fluorescent label (nuclear RFP) and flow sorted to select for successfully transduced cells .All cells are cultured at 37°C in a humidified 5% CO2 incubator. Cell lines were transduced with IncucyteThe following antibodies were purchased from Cell signaling Technologies: pERK , total ERK , pAKT , total AKT , Ubiquitin , MYC , pCHEK1 S296 , BMI1 , H3K27Ac , total H3 , CDK9 , Vinculin , CPNase  and MAG . Beta-Actin  was purchased from Novus, pS2 RNApol II  from EMD Millipore, SOX2  from R&D systems and NG2  from Invitrogen.All drugs and compounds were purchased from Selleckchem . A complete list of compound names, sources, catalogue numbers, are listed in ® S3 Live-Cell Analysis System  and serve as a proxy for the number of cells present. Analysis parameters are listed in Two thousand cells/well of 17, 5653, or 5746 glioma cell lines are plated in 96-well round-bottomed ultra-low attachment plates  and used for spheroid drug screening. Cells are plated in 100uL of complete culture media, centrifuged  and incubated overnight to stimulate a single colony from forming at the bottom of the round well. Twenty-one drugs were screened, using three concentrations . We used the Incucyte® S3 Live-Cell Analysis software to identify and count the number of individual cells, as defined by the number of RFP+ nuclei, across the 72 hours. Analysis parameters are listed in The TM-31 cells were plated  in 96-well flat-bottom tissue culture treated plates (Corning 3596). Cells were plated in 100uL culture media and incubated overnight before adding drugs on Day 0. Twenty-one drugs were screened, using three concentrations  for each time point relative to 0 hours was calculated. Drugs were considered for a broader follow-up combinatorial evaluation if our initial treatment caused more than 50% cell death in 72h  at concentrations below the maximum serum concentration that can be safely attained during pharmacokinetic drug studies for clinical trials (Cmax). These Cmax obtained from the literature (Table 2), and values were converted from mg/ml to nM.https://synergyfinder.fimm.fi/; parameters: LL4 curve fit, ZIP method for synergy score calculation).The synergy of drug combinations was evaluated in the mouse 5746 and TM31 glioma cell lines. The 17 glioma cell line was used for the PTC596/Trametinib combination as this combination showed limited sensitivity than the 5746 line . Cell liOur initial 21 compound drug screen was performed in triplicates, the synergy study was performed in duplicates. IC50 and Lethal Dose 50 (LD50) values and 95% confidence intervals were calculated to assess drug sensitivity (GraphPad Prism 9 software) and tabulated in Nf1  and Tp53  was performed [GAACATGATTCTCTTTTGGACCAC and ACCTGTTTCAAATGTGACCCTTT and sequencing primer GGATACCATACTTGCAGAGC) and NF1 mutation  in the TM-31 cell line was confirmed by sequencing. Western blot was performed to evaluate the expression of OPC lineage markers and for the expression of TP53 and CDKN2A in TM-31. Target inhibition of the drugs that caused potent cell death in our glioma lines inhibited was evaluated by western blot as well. Glioma cell line 17 was treated for 24h with 20nM Trametinib, 1μM GDC-0941, 5nM bortezomib, 25nM Dinaciclib, 500nM JQ1, 100nM LY2606368, 500nM PTC596, or 1μM Vorinostat and target inhibition was evaluated by comparing the expression levels of pERK, pAKT, Ubiquitin, pS2 RNApol II and CDK9, MYC, pCHEK1, BMI1 and H3K27Ac respectively.To characterize our mouse glioma cell lines, a genotyping PCR for erformed . The repNf1 and Tp53, in cis on mouse chromosome 11. Tumors form when a cell loses the wildtype copy of Nf1 and TP53, usually due to whole chromosome loss. Importantly, the loss of the wildtype chromosome occurs sporadically, each tumor thus is unique. About 10% of these NPcis mice form high grade gliomas [Nf1 and TP53 as evidenced by the loss of the wildtype Nf1 and Tp53 alleles of the genotyping PCRs  and the MEK inhibitor Trametinib (GSK1120212) were selected. Both agents are currently in active or recently completed clinical trials for solid and brain malignancies. https://www.cancerrxgene.org/compounds) or in the literature [The sensitivity to 21 compounds as single agents and in combination with Trametinib or GDC-0941 was determined. terature , 24 , culture media (serum versus stem cell media), culture method (2D vs 3D assays) and/or species (human versus mouse) and will require the evaluation of other human NF1 HGG once they become available. As mentioned before, it is believed that 3D stem cell cultures represent more accurately the biology of tumors than 2D serum grown lines. Our screen provides valuable insight into potential drug targets for NF1 associated HGG. Follow-up in vivo pre-clinical studies will have to confirm if the identified drug targets and drug combinations are able to shrink tumors in an in vivo setting at well tolerated doses and enhance survival in preclinical models of NF1-HGG.Six drugs combined with MEK or PI3K and the combination of MEK and PI3K inhibitors worked synergistically in our mouse NF1-HGG stem cell line. None of the combinations worked synergistically in the human TM-31 line (12 additive and 1 antagonistic combination). Crucially however, baseline sensitivity to these compounds remains high in our human NF1 HGG line. Moreover, while synergistic effects of drug combinations point to a mechanistic interaction between both compounds, the fact that a combination therapy work additively should not exclude them from clinical trials or further investigation. It is unclear if the observed differences between our mouse and human line are due to differences in driver mutations Click here for additional data file.S1 TableOverview of the drugs used in this study including targets and catalogue numbers.(XLSX)Click here for additional data file.S2 TableDrug sensitivity data expressed in log2 fold change in the different cell lines. A value <0 denotes that less cells are present compared to day 0.(XLSX)Click here for additional data file.S3 TableIncucyte analysis settings and parameters used to analyze our 3D spheroid assays.(XLSX)Click here for additional data file.S4 TableIncucyte analysis settings and parameters used to analyze our 3D spheroid assays.(XLSX)Click here for additional data file.