C. difficile toxin B (TcdBFBD), which recognizes and inhibits a subclass of FZDs, FZD1/2/7, and examined whether targeting this FZD subgroup may offer therapeutic benefits for treating breast cancer models in mice. Utilizing 2 basal-like and 1 luminal-like breast cancer models, we found that TcdBFBD reduces tumor-initiating cells and attenuates growth of basal-like mammary tumor organoids and xenografted tumors, without damaging Wnt-sensitive tissues such as bones in vivo. Furthermore, FZD1/2/7–positive cells are enriched in chemotherapy-resistant cells in both basal-like and luminal mammary tumors treated with cisplatin, and TcdBFBD synergizes strongly with cisplatin in inhibiting both tumor types. These data demonstrate the therapeutic value of narrow-spectrum Wnt signaling inhibitor in treating breast cancers.Wnt signaling pathways are transmitted via 10 homologous frizzled receptors (FZD1-10) in humans. Reagents broadly inhibiting Wnt signaling pathways reduce growth and metastasis of many tumors, but their therapeutic development has been hampered by the side effect. Inhibitors targeting specific Wnt-FZD pair(s) enriched in cancer cells may reduce side effect, but the therapeutic effect of narrow-spectrum Wnt-FZD inhibitors remains to be established in vivo. Here, we developed a fragment of  Wnt signaling is transmitted via ten homologous Frizzled receptors (FZD1-10) in humans, complicating the therapeutic use of inhibitors due to side-effects. This study shows that a bacterial toxin fragment can be used as a tool to specifically inhibit Wnt signaling mediated by a subset of Frizzed receptors , with potential for cancer treatment. Basal-like breast cancer largely overlaps with triple-negative breast cancer, which lacks ER and progesterone receptor (PR) expression and HER2 overexpression  (for ERα primary antibody) and Goat anti-Chick IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Invitrogen A-32933)] (for Cytokeratin 5 primary antibody) for 30 min at room temperature. After washing with PBS again, the sections were stained with DAPI.IF staining was performed on tissue sections that were fixed in 10% formalin  and embedded in paraffin. Antigen retrieval  was performed before blocking and endogenous peroxidase activity was quenched on the slides intended for IF by incubation in 0.3% HFBD (0.1 mg/ml), FZD7 , CD31 , CD45 , and TER119 . In some experiments, FACS analysis was performed using an LSR-II analyzer  and the data was further analyzed using Flowjo v10.8 Software (BD Life Sciences). First, the cells were incubated with Zombie Violet Fixable viability dye (1:200 dilution) for 20 min at room temperature in order to discriminate live and dead cells. Then, the dye dilute was washed out and the cells were proceeded with antibody staining. The following antibodies were used and purchased from Biolegend : anti-mouse CD31- BV605 , anti-mouse CD45- BV605 , anti-mouse TER-119- BV605 , anti-mouse CD24- PE , and anti-mouse CD29- PerCP-Cy5.5 . Original FCS files are publicly available: http://flowrepository.org/id/FR-FCM-Z6Q4.Flow cytometric (FACS) analysis was performed after single cells were obtained using an Accuri C6 analyzer  and analyzed with CFlow software (BD Biosciences). The following antibodies were utilized: CD24 , CD29 , FITC-TcdBFBD or TcdBmu, PBS served as control. The frequency of TICs was calculated using the ELDA website (http://bioinf.wehi.edu.au/software/elda/index.html). The tumorsphere formation frequency in 24-well plates was calculated according to the formula F = Numbers of forming tumorspheres/Number of single cells plated. For secondary and tertiary tumorsphere formation, single cell suspensions prepared from the previous generation of tumorspheres were re-plated under the same conditions as the first generation.Single cells obtained from cell lines or tumor tissues were resuspended and plated into round-bottom 96-well ultralow attachment plates (Corning) at a density of 1, 5, 10, 100, and 200 cells per well, or at 5,000 cells per well in 24-well flat-bottom ultralow attachment plates in the sphere culture medium (DMEM/F12 media supplemented with B27 (100 units/ml), insulin (10 μg/ml), EGF (20 ng/ml), and bFGF (20 ng/ml) with 150 nM TcdBp53/BRCA1-deficient tumor organoid cells  were injected subcutaneously into nude mice in serum-free DMEM/Matrigel. The growth of tumor was evaluated daily over a 3-month period. The animal ethics endpoint was tumor reaching a size of 10 mm. Tumor volume was monitored and calculated as described above. The TIC frequency was derived as described above. For the Kaplan–Meier tumor-free survival curves, mice were considered tumor free until tumors were visible or palpable. For the Kaplan–Meier survival curves, mice were considered alive until tumors reach ethics endpoint a size of 10 mm.Total RNAs from tumors were purified by either Trizol or the Allprep DNA/RNA mini kit (Qiagen). cDNA was generated with iScript  according to the manufacturer’s protocol. Quantitative RT-PCR  and PCR (for genomic DNA) were performed using FastStart SYBR Green Master . PCR primers are listed below:FZD1:forward:5′-GAGTTCTGGACCAGTAATCCGC-3′;reverse:5′-ATGAGCCCGTAAACCTTGGTG-3′; FZD2: forward: 5′-CTTCTCGCAAGAGGAGACTCG-3′; reverse: 5′-GTGGTGACCGTGAAGAAAGTG-3′; FZD3: forward: 5′- ATGGCTGTGAGCTGGATTGTC-3′; reverse: 5′-GGCACATCCTCAAGGTTATAGGT-3′; FZD4: forward: 5′-AACCTCGGCTACAACGTGAC-3′; reverse: 5′-GGCACATAAACCGAACAAAGGAA-3′; FZD5: forward: 5′-GGTGTGCCAGGAAATCACG-3′; reverse: 5′-CACAAGCGGCCAGAATTGG-3′; FZD6: forward: 5′-TCTGCCCCTCGTAAGAGGAC-3′; reverse: 5′-GGGAAGAACGTCATGTTGTAAGT-3′; FZD7: forward: 5′-GCCACACGAACCAAGAGGAC-3′; reverse: 5′-CGGGTGCGTACATAGAGCATAA-3′; FZD8: forward: 5′-GGGTTACCTGTTGGAAGTGAC-3′; reverse: 5′-GGCACCGTGATCTCTTGGC-3′; FZD9: forward: 5′-CGCACGCACTCTGTATGGAG-3′; reverse: 5′-GCCGAGACCAGAACACCTC-3′; FZD10: forward: 5′-CATGCCCAACCTGATGGGTC-3′; reverse: 5′-GCCACCTGAATTTGAACTGCTC-3′; Gapdh: forward: 5′-GGTGAAGGTCGGTGTGAACG-3′; reverse: 5′-CTCGCTCCTGGAAGATGGTG-3′; Axin2: forward: 5′-ATGAGTAGCGCCGTGTTAGTG-3′; reverse: 5′- GGGCATAGGTTTGGTGGACT -3′; Vim: forward: 5′-CGGCTGCGAGAGAAATTGC-3′; reverse: 5′-CCACTTTCCGTTCAAGGTCAAG-3′; Zeb1: forward: 5′-GCTGGCAAGACAACGTGAAAG-3′; reverse: 5′-GCCTCAGGATAAATGACGGC-3′; Rnf43: forward: 5′-CACGAGTTTCATCGAACGTGT-3′; reverse: 5′-CTGGCGAATGAGGTGGAGT-3′.FBD or TcdBmu  was added to the culture medium every day for 10 days, PBS was used as a vehicle control. The viability of organoids was assessed using the CellTiter-Glo luminescent cell viability assay  according to the manufacturer’s protocol.Organoid lines derived from breast cancer patients are identified, as previously reported . 74T wasTumor tissues were lysed by RIPA buffer  with protease inhibitor cocktail (Roche). Total proteins were loaded on SDS-PAGE and transferred to PVDF membrane. After blocking, proteins were detected with a 1:1,000 dilution of primary antibody active β-catenin  using the enhanced chemiluminescence (ECL) method (Pierce).t test was used for comparisons between 2 experimental groups, and ANOVA was used for comparisons of more than 2 groups. Unless otherwise indicated, all results were averaged from biological triplicates and values are reported as means ± SEM. P < 0.05 was considered statistically significant.All statistical analyses were performed using GraphPad Prism 6.0 . An unpaired S1 FigFZDs in different subtypes of human breast cancer based on bc-GenExMiner online tool.Expression levels of (PDF)Click here for additional data file.S2 FigFZDs in different subtypes of human breast cancers from the METABRIC cohort, based on cbioportal online tool. Subtypes: (A) Basal-like; (B) Claudin-low; (C) Her2; (D) LumA ; (E) LumB ; (F) NC (Not classified); (G) Normal-like.Expression levels of (PDF)Click here for additional data file.S3 FigFZDs in different subtypes of human breast cancers from the TCGA, PanCancer Atlas cohort, based on cbioportal online tool. Subtypes: (A) Basal-like; (B) Her2; (C) Luminal A; (D) Luminal B; (E) Normal-like; (F) NA (not available).Expression levels of (PDF)Click here for additional data file.S4 FigFzd7 and Fzd6 in mouse models for human basal-like/triple-negative breast cancer; data was based on GEO accession # GSE25488  [C3(1)-Tag model.High expression levels of  levels) . The fir(PDF)Click here for additional data file.S5 Fig(A) Schematic diagram showing the generation of a p53/BRAC1-deficient breast cancer model by intraductal injection of Ad-K8-Cre into Trp53L/L;Brca1L/L;R26Y female mice. (B) Kaplan–Meier tumor-free results showing that Trp53L/L;Brca1L/L;R26Y female mice (n = 17) injected with Ad-K8-Cre developed malignant mammary tumors over time after Ad-K8-Cre injection. (C) Representative FACS plots of FZD7+ cells from normal mammary glands, C3(1)-Tag, p53/BRCA1-deficient, and MMTV-PyMT tumors. Numerical values are in (PDF)Click here for additional data file.S6 FigMMTV-PyMT, and C3(1)-Tag models were stained with antibodies for K5 and ERɑ. Arrows indicate ER+ luminal mammary epithelial cells in the normal MG. Scale bar = 10 μm.Normal mammary gland (MG) and mammary tumors from the p53/BRCA1-deficient, (PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fighttps://www.proteinatlas.org/). Luminal progenitors (LPs) are indicated as SOX10+TP63- cells; hormone receptor (HR)+ luminal cells are indicated as ESR1+FOXA1+ cells; basal (B) cells are indicated as TP63+ cells.Heatmap shown here is based on single-cell RNA sequencing data available at the Human Protein Atlas website ((PDF)Click here for additional data file.S9 Fig(A) Left panel: phylogenetic analysis of human FZD proteins [Fzds in MDA-MB-231 cells. (B) Cell viability measured by the MTT assay showed that TcdBFBD did not exhibit cytotoxicity to the indicated human cell lines . (C) Representative images of clonogenic growth of PaTu8988s and HPAF-II cells cultured in the presence of TcdBFBD (150 nM) or LGK974 (100 nM). (D) Representative images of sphere formation assay in PaTu8988s and HPAF-II cells cultured in the presence of TcdBFBD (150 nM) or LGK974 (100 nM). (E) Quantitation of colony numbers and sphere formation from (C and D), error bars indicate mean ± SEM, n = 4. (F) Immunoblot analysis of active β-catenin expression in PaTu8988s and HPAF-II cells cultured in the presence of TcdBFBD (150 nM) or LGK974 (100 nM). Actin serves as a loading control. (G) Quantitative analysis of expression of active β-catenin expression in (F). NS, not significant;*, p < 0.05. Numerical values are in proteins ; right p(PDF)Click here for additional data file.S10 Fig(A) Quantitation of organoids numbers in culture with or without CHIR99021(5 μM). (B) Quantitation of organoid sizes at day 5 in culture. Numerical values are in (PDF)Click here for additional data file.S11 Fig(A) Experiments were carried out as described in Axin2 and Rnf43) and EMT-associated genes (Vim and Zeb1) in tumor organoids upon TcdBFBD treatment. (B) Experiments were carried out as described in Axin2 and Rnf43) and EMT-associated genes (Vim and Zeb1) in TcdBFBD-treated xenograft tumors. (C) Experiments were carried out as described in FBD or TcdBmu). Scale bar = 50 μm. Numerical values are in (PDF)Click here for additional data file.S12 FigFBD or TcdBmu . Cell viability was assessed using the CellTiter-Glo luminescent assay. Error bars were from 3 technical replicates. Numerical values are in The viability of 2 lines of human breast cancer organoids, a luminal tumor line 74T (panel A) and a basal-like tumor line 86T (panel B), were exposed to the indicated concentrations of TcdB(PDF)Click here for additional data file.S13 Fig(A) FACS analysis using FITC-TcdBFBD and anti-FZD7 antibody showed that primary p53/BRCA1-deficient tumors cells targeted by TcdBFBD are largely FZD7+. (B) Representative images showing the tumorspheres formed from p53/BRCA1-deficient tumor cells. (C) Tumorsphere cells cultured from p53/BRCA1-deficient tumors were examined by FACS analysis using an anti-FZD7 antibody. (D) Representative images showing the tumor organoid formed from p53/BRCA1-deficient tumor cells. (E) FACS analysis of FZD7+ cells in the tumor organoids described in (D) showing that most organoid cells express FZD7. Numerical values are in (PDF)Click here for additional data file.S14 Fig(A) Representative images of p53/BRCA1-deficient mouse mammary tumor organoids treated with TcdBFBD alone (150 nM), cisplatin alone (0.2 μm), or a combination of both. (B) Quantitation of organoids sizes for the indicated treatment groups in (A). (C) Quantitation of organoid numbers for the indicated groups in (A). (D) Representative pictures of C3(1)-Tag mouse mammary tumor organoids treated with TcdBFBD alone, cisplatin alone, or a combination of both. (E) Quantitation of organoids sizes for the indicated treatment groups in (D). (F) Quantitation of organoid numbers for the indicated groups in (D). Scale bar = 100 μm. Numerical values are in (PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Correction: J Exp Clin Cancer Res 40, 258 (2021) https://doi.org/10.1186/s13046-021-02042-1Fig. Following publication of the original article , an erroThe correct Fig. The correction does not affect the overall Conclusion of the article.


C. elegans neurons causes progressive, age-associated loss of motor coordination, selective neurodegeneration, and shortened lifespan. Loss of function (LOF) mutations in the conserved genesut-2protects against progressive motor uncoordination and neurodegeneration in models of tauopathy. To determine whethersut-2LOF also protects against shortened lifespan of tau transgenicC. elegans, we conducted lifespan assays comparing four different alleles ofsut-2.We found thatsut-2LOFrobustly suppresses the shortened lifespan of tau transgenic animals. We also demonstrate that tau transgenicC. elegans exhibit hyperactive pharyngeal pumping, which is restored bysut-2LOF.Expression of human tau in C. elegans have been generated that express human 1N4R tau with the V337M FTLD-tau causative mutation (tau(V337M)) in all neurons (tau Tg). These animals exhibit uncoordinated movement, age-dependent neurodegeneration, and shortened lifespansut-2that suppress these phenotypessut-2coding sequence,sut-2(bk3011)andsut-2(bk3012),were found to similarly suppress tau movement dysfunction and neurodegenerationPathological inclusions of the protein tau in neurons and glia characterize several human neurodegenerative diseases including frontotemporal lobar degeneration (FTLD-tau), progressive supranuclear palsy (PSP), chronic traumatic encephalopathy (CTE), and Alzheimer’s diseasesut-2LOF mutants (sut-2(bk3011)andsut-2(bk3012)) have lifespan similar toN2. To test whethersut-2(bk3011)andsut-2(bk3012)mutations are able to rescue the shortened lifespan of tau transgenic worms similarly to previously characterized alleles ofsut-2,sut-2(bk87)andsut-2(bk741), we measured the lifespans of tau transgenic animals crossed to these foursut-2mutant alleles. In fact, we find thatsut-2(bk3011)andsut-2(bk3012)robustly suppress the shortened lifespan of tau Tg animals similar tosut-2(bk87)andsut-2(bk741).TheC. elegans exhibit altered pharyngeal pumping, we assayed pharyngeal muscles and neurons electrophysiology from individual animals using a microfluidic chip-based recording device. This device detects, records, and evaluates pharyngeal muscle and neuron action potentials that accompany each pump cycle. We found tau Tg animals had significantly increased frequency and reduced duration of pharyngeal pumping. We then tested whethersut-2(bk3011)andsut-2(bk3012)can modify tau TgC. elegans pumping defects. We found thatsut-2(bk3011)andsut-2(bk3012)partially suppress the increased frequency but do not suppress the decreased duration of pumping in tau Tg animals .sut-2(bk3011)andsut-2(bk3012)do not have altered pumping rates relative toN2.To test whether tau Tg expressingsut-2via a whole gene deletion ameliorates the toxic consequence of tauopathy in tau transgenicC. elegans.sut-2and its mammalian homolog MSUT2 may be compelling targets to treat tauopathies including Alzheimer’s disease.Taken together, these data show that complete elimination ofTable 1Table legendN2) = significance relative toN2(wild-type control), pvalue (tau Tg) = significance relative to tau Tg animals. N.S. = not significant. Significance was evaluated using survival curve comparisons with Mantel-cox log-rank analysis.N = number tested, Mean = average population lifespan in days, p-value (C. elegans lifespan assaysoC following a short (4-6 hour) egglay to L4 stage on NGM plates seeded withE. coli OP50, and then transferred onto seeded NGM plates with added 5-fluoro-2’-deoxyuridine  to inhibit growth of progeny. Worms were scored every 1-2 days for movement following tapping of the plate or gentle touching with a platinum wire. Failure to respond to touch was scored as dead. Statistical analysis was performed using GraphPad Prism software.Lifespan assays were modified from those described inC. elegans pumping assaysC. elegans pumping electrophysiology was evaluated using a ScreenChip System (NemaMetrix/ InVivo Biosystems) as described inC. elegans were pre-incubated in M9 buffer containing 10 mM 5HT (Sigma) for 20 minutes, to stimulate pharyngeal pumpingC. elegans were then individually loaded into the microfluidic recording device and pharyngeal muscle and neuron action potentials were recorded for two minutes using NemAquire software (NemaMetrix/ InVivo Biosystems).Action potential statistics and readouts, including frequency and duration, were computed using NemAnalysis software (NemaMetrix/ InVivo Biosystems).C. elegans pharyngeal pumping rate assay was adapted from (O'Brien 2022). The pump rates were determined by manually counting the number of grinder movements observed in individual animals over 20 seconds. Each animal was recorded 3 times and the average of the 3 recordings was used calculate pumps per minute. Data was managed in Microsoft Excel and statistical analysis was performed using GraphPad Prism Software.Strains UsedN2BristolbkIs10 IIICK10sut-2(bk3011)CK3011sut-2(bk3012)CK3012sut-2(bk3011);bkIs10 IIICK1341sut-2(bk3012);bkIs10 IIICK1342sut-2(bk87);bkIs10 IIICK185sut-2(bk741);bkIs10 IIICK187


MYCN-amplified neuroblastoma often presents as a highly aggressive metastatic disease with a poor prognosis. Activating transcription factor 5 (ATF5) is implicated in neural cell differentiation and cancer cell survival. Here, we show that ATF5 is highly expressed in patients with stage 4 high-risk neuroblastoma, with increased expression correlating with a poorer prognosis. We demonstrated that ATF5 promotes the metastasis of neuroblastoma cell lines in vivo. Functionally, ATF5 depletion significantly reduced xenograft tumor growth and metastasis of neuroblastoma cells to the bone marrow and liver. Mechanistically, ATF5 endows tumor cells with resistance to anoikis, thereby increasing their survival in systemic circulation and facilitating metastasis. We identified the proapoptotic BCL-2 modifying factor (BMF) as a critical player in ATF5-regulated neuroblastoma anoikis. ATF5 suppresses BMF under suspension conditions at the transcriptional level, promoting anoikis resistance, whereas BMF knockdown significantly prevents ATF5 depletion–induced anoikis. Therapeutically, we showed that a cell-penetrating dominant-negative ATF5 peptide, CP-d/n-ATF5, inhibits neuroblastoma metastasis to the bone marrow and liver by inducing anoikis sensitivity in circulating tumor cells. Our study identified ATF5 as a metastasis promoter and CP-d/n-ATF5 as a potential antimetastatic therapeutic agent for neuroblastoma.This study shows that resistance to anoikis in neuroblastoma is mediated by ATF5 and offers a rationale for targeting ATF5 to treat metastatic neuroblastoma. MYCN , an ATF/CREB family member, is a basic leucine zipper (bZIP) transcription factor. ATF5 is highly expressed in neural progenitor/neural stem cells, where it promotes cell proliferation and inhibits neurogenesis and gliogenesis . ATF5 alin vitro and in vivo, thereby facilitating metastasis. Mechanistically, ATF5 induces anoikis resistance by suppressing the proapoptotic BCL-2 modifying factor (BMF). We also demonstrated that targeting ATF5 using a cell-penetrating dominant-negative ATF5 peptide inhibitor (CP-d/n-ATF5) increased the anoikis sensitivity of tumor cells and inhibited metastasis.We report that ATF5 increases tumor cell survival in circulation by inducing anoikis resistance Mycoplasma testing was performed at the initiation of the project. Cell lines were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific).Human neuroblastoma cell lines were purchased from the ATCC: BE(2)-C , SK-N-DZ , and SH-SY5Y . Cell lines were authenticated by short tandem repeat profiling (ATCC). CP-d/n-ATF5 and penetratin peptides were purchased from CS Bio and were formulated in water as described previously . The fulLentivirus-mediated stable integration of the doxycycline (Dox)-inducible pTRIPZ vector (Dharmacon) encoding shATF5-1 or shATF5-2 was performed as described previously . shATF5 Cell viability was measured using the CCK-8 assay kit  solution for 10 minutes, washed, TACS-Sapphire substrate was added for 30 minutes, and the reaction was stopped with 0.2 mol/L HCl. Cell death was measured absorbance at 450 nm on a microplate reader (Molecular Devices).Suspension conditions were created by coating 96-well plates with poly-HEMA  containing 1 mmol/L phenylmethylsulfonyl fluoride (MilliporeSigma) and protease inhibitor cocktail .Immunoblotting was performed as described previously . For antBE(2)-C and SK-N-DZ cells, expressing shATF5-1 or shATF5-2, were seeded on poly-HEMA–coated 6-well plates in complete media for 24 hours. Dox (1 µg/mL) was added, and 72 hours later, total RNA was purified using an RNeasy Mini Kit (Qiagen) according to the supplier's instruction manual. cDNA was generated by performing RT-PCR using SuperScript III First Strand RT-PCR kit (Invitrogen) according to the manufacturer's protocol. The cDNAs generated were subjected to PCR with human ATF5, BMF, or ACTB cDNA-specific primers.luciferase -C tumors were randomized into two groups. One group was given drinking water containing Dox (2 mg/mL) to induce ATF5 knockdown (d-Luciferin (PerkinElmer), anesthetized with isoflurane, and imaged with a Xenogen IVIS200 (9 (photons/second).The Columbia University Institutional Animal Care and Use Committee approved all animal procedures. Mice experiments were performed in 6–8 weeks old female NCr nude mice (Taconic). BE(2)-C and SK-N-DZ cells were stably transfected with firefly ciferase . Mice wenockdown , and thenockdown . For bio IVIS200 . Mice we9 photons/second). For SK-N-DZ tumors, peptide treatment began 14 days after cell implantation. Mice were euthanized 40 days (day 40) after cell implantation.For CP-d/n-ATF5 treatment, kidney xenograft tumors, mice were treated with 50 mg/kg of the peptide, or vehicle, by intraperitoneal injection once per day for the first 3 days and then twice per week . For BE or without Dox 3 days before injection to the time of euthanasia. Bioluminescence imaging was obtained 20 minutes after injection to monitor the distribution of tumor cells. Twelve hours after intracardiac injection, mice were euthanized and blood was collected. A cohort of mice was also euthanized 31 days (day 31) after cell injection.A total of 10For peptide inhibitor studies, CP-d/n-ATF5, at a dose of 50 mg/kg, was intraperitoneally injected into mice 1 hour after intracardiac injection of tumor cells, and mice were euthanized 12 hours later or 38 days (day 38) after cell injection.g for 30 minutes. The supernatant was mixed with LARII reagent (Promega), and bioluminescence was measured using a luminometer , and incubated with an anti-GD2-FITC antibody for 1 hour at 4°C. The cells were washed twice and incubated with FITC microbeads (Miltenyi Biotec) for 30 minutes at 4°C. The cells were washed, resuspended, and applied to an MS column placed in a MACS separator (Miltenyi Biotec). The labeled cells were flushed out and counted with 1X trypan blue, and equal numbers of cells were subjected to the TUNEL assay to determine cell death or mRNA expression of ATF5, BMF, and ACTB by RT-PCR.At the time of euthanasia, blood was collected, diluted with PBS + 2% FBS, and layered on top of Lymphoprep (STEMCELL Technologies). After centrifugation at 800 × ex vivo liver imaging, mice were injected with d-luciferin, euthanized, and livers were dissected, imaged, and bioluminescence measured (For measured . Bone mameasured .t test. Bioluminescence data were transformed to Y = log(Y).Statistical analyses were performed using Prism software (GraphPad). Survival was determined using the log-rank test (Mantel–Cox). Data were analyzed using an unpaired https://ocg.cancer.gov/programs/target/data-matrix). All other data in support of the findings can be obtained from the Corresponding Author upon reasonable request.The data from the Sequencing Quality Control Consortium (SEQC) cohort used in this study can be obtained from Gene Expression Omnibus (GEO): GSE4971 . The tumATF5 expression (MYCN-amplified (MNA) tumors than in either high-risk MYCN-non-amplified (HR_NA) tumors  or stage 1 low-risk (LR) tumors . HR_NA tumors also had higher ATF5 expression than LR tumors . To determine whether ATF5 expression correlated with patient survival, we examined the SEQC cohort because it is unbiased with respect to clinical factors. Analysis of the SEQC cohort revealed that ATF5 expression (P  =  1.57E-14).Examining neuroblastoma patient datasets from the SEQC cohort  targeting ATF5. ATF5 knockdown by Dox addition reduced cell viability by an average of 40% in BE(2)-C and 50% in SK-N-DZ at 72 hours, compared with controls . ATF5 knin vivo. BE(2)-C-shATF5-2 cells were implanted intrarenally in nude mice. Mice were randomized after 10 days, and half were given drinking water containing Dox to induce ATF5 knockdown. Survival time was defined as the day when the primary tumor luciferase flux reached 4 ×  109 photons/second. ATF5 knockdown inhibited tumor growth (P  =  0.0083). Decreased expression of ATF5 in + Dox tumors was verified by immunoblotting  was also examined. ATF5 knockdown markedly decreased the liver metastatic burden, with a 22-fold lower total liver flux than controls  and F. AMYCN-non-amplified cell lines SH-SY5Y -C cells utilizing an extracellular matrix basement membrane assay . We then SH-SY5Y , and CHL SH-SY5Y . Overexp SH-SY5Y , SH-SY5Y SH-SY5Y , and CHL SH-SY5Y . In addi SH-SY5Y , SH-SY5Y SH-SY5Y , and CHL SH-SY5Y .in vivo. Twelve hours after intracardiac injection of ATF5-overexpressing BE(2)-C cells, mice exhibited a marked elevation in whole-body flux (P < 0.05).We also examined the effect of ATF5 overexpression on tumor cell survival ody flux  and F coody flux . IsolatiWe next investigated the effects of ATF5 knockdown on resistance to anoikis. Anoikis was induced by culturing the cells in suspension in poly-HEMA–coated plates. A time-course study with two different Dox-inducible shRNAs showed that ATF5 knockdown in BE(2)-C cells caused a decrease in anchorage-independent viability by 22% at 12 hours and 59% at 72 hours . An accoluciferase-expressing BE(2)-C-shATF5 cells were injected intracardially into nude mice fed with or without Dox-containing water. A significant decrease in whole-body flux was noted at 24 hours in +Dox-treated mice compared with that in −Dox control mice -C-shATF5 cells and followed whole-body bioluminescence flux over time . In the BMF mRNA in detached cells (in vivo. BE(2)-C-shATF5 cells with TF5 knocked down and isolated from mouse blood 12 hours after intracardiac injection exhibited induction of BMF mRNA -C and SK-N-DZ cells after 72 hours  and B. Ied cells , suggestBMF mRNA . These fBecause BMF has been previously linked to anoikis , we inveWe next addressed whether endogenous BMF mediates ATF5 depletion–induced anoikis. Adherent cells expressing inducible shATF5-1 or shATF5-2 were treated with either siBMF-1 or siBMF-2. After 24 hours, the cells were transferred to poly-HEMA plates (± Dox), and TUNEL assays were performed after 72 hours. ATF5 knockdown alone increased BE(2)-C apoptosis by an average of 125% compared with the controls, while BMF knockdown significantly reduced apoptosis . MoreoveBMF has been reported to trigger anoikis by binding and inhibiting BCL-2 or BCL-XL . We inveBMF has previously been shown to be a target of the transcription factor FOXO3 . ATF5 knIn vitro, under adherent conditions, CP-d/n-ATF5 dose-dependently inhibited the growth of a panel of neuroblastoma cell lines, both MYCN-amplified and MYCN-non-amplified -C and SK-N-DZ cells under suspension conditions  and undein vivo. Mice with established BE(2)-C and SK-N-DZ tumors were treated with CP-d/n-ATF5 or vehicle. Mice bearing BE(2)-C tumors treated with CP-d/n-ATF5 demonstrated decreased tumor growth as seen by bioluminescence at day 14 -C cells and treated with CP-d/n-ATF5 showed a marked reduction in viability and increased anoikis (∼3-fold) of CTC compared with vehicle-treated animals at 12 hours after treatment . In a sein vitro and in vivo, influencing their metastasizing ability. ATF5 has been linked to several adult cancers owing to its prosurvival function -C anoikis was observed as early as 12 hours after ATF5 depletion La cells .ATF5 belongs to the bZIP family of transcription factors and has been implicated in the regulation of differentiation, protection from various cellular stresses, and tumor cell survival . ATF5 knAnoikis is regulated by a delicate balance between proapoptotic and antiapoptotic protein levels . IncreasMYCN-amplified cell lines. CP-d/n-ATF5 also showed in vivo efficacy by inhibiting tumor growth, reducing CTC survival, and preventing metastatic progression. A single dose (50 mg/kg) of CP-d/n-ATF5 induced anoikis in circulating BE(2)-C and SK-N-DZ cells within 12 hours, indicating rapid CP-d/n-ATF5 activity. CP-d/n-ATF5 activity was specific, as the penetratin-only peptide showed no anoikis activity. Our findings suggest that BMF is a crucial mediator of CP-d/n-ATF5–induced anoikis. CP-d/n-ATF5 induced BMF in suspension culture but not in adherent cells. Loss of BMF prevented CP-d/n-ATF5–induced anoikis and decreased BMF was observed in the CTC of mice receiving CP-d/n-ATF5 treatments. CP-d/n-ATF5 treatment also caused the depletion of endogenous ATF5. Therefore, we speculate that CP-d/n-ATF5-induces the depletion of ATF5, leading to increased BMF, resulting in anoikis of neuroblastoma cells in suspension culture and the blood circulation of mice. Our studies identified CP-d/n-ATF5 as an effective therapeutic agent with antimetastatic efficacy and supported the clinical testing of such peptides in patients with neuroblastoma. Recent studies have identified the transcription factors CEBPB and CEBPD as CP-dn-ATF5 targets -C cells in vitroClick here for additional data file.Supplementary Figure 5Supplementary Figure 5 shows that overexpression of ATF5 promotes anoikis resistance of CHLA-255 in vitroClick here for additional data file.Supplementary Figure 6Supplementary Figure 6 shows a decrease in ATF5 after Dox addition under non-adherent suspension conditions.Click here for additional data file.Supplementary Figure 7Supplementary Figure 7 shows that ATF5 knockdown decreases SK-N-DZ CTC survivalClick here for additional data file.Supplementary Figure 8Supplementary Figure S8 shows that BMF overexpression reduces the anchorage-independent viability of BE(2)-C and SK-N-DZ cells.Click here for additional data file.Supplementary Figure 9Supplementary Figure 9 shows that BMF knockdown rescues ATF5 loss-induced reduction of anchorage-independent cell viabilityClick here for additional data file.Supplementary Figure 10Supplementary Figure 10 shows that BMF binds to BCL-XL when ATF5 is depletedClick here for additional data file.Supplementary Figure 11Supplementary Figure 11 shows that FOXO3 silencing rescues cell viability following ATF5 depletionClick here for additional data file.Supplementary Figure 12Supplementary Figure 12 shows that CP-d/n-ATF5 inhibits the growth of MYCN-amplified and MYCN-non-amplified cell lines in a dose-dependent mannerClick here for additional data file.Supplementary Figure 13Supplementary Figure 13 shows that CP-d/n-ATF5 reduces the anchorage-independent viability of neuroblastoma cell linesClick here for additional data file.Supplementary Figure 14Supplementary Figure 14 shows that CP-d/n-ATF5 induces anoikis of neuroblastoma cell linesClick here for additional data file.Supplementary Figure 15Supplementary Figure 15 shows that CP-d/n-ATF5 treatment does not induce BMF under adherent conditionsClick here for additional data file.Supplementary Figure 16Supplementary Figure 16 shows that CP-d/n-ATF5 inhibits growth of BE(2)-C tumors and increases apoptosisClick here for additional data file.Supplementary Figure 17Supplementary Figure 17 shows that CP-dn-ATF5 reduces tumor growth and metastasis of SK-N-DZ in vivoClick here for additional data file.Supplementary Figure 18Supplementary Figure 18 shows that CP-d/n-ATF5 treatment decreased viability and induced anoikis of SK-N-DZ CTCsClick here for additional data file.


Correction: Mol Cancer 17, 50 (2018)https://doi.org/10.1186/s12943-018-0802-4In our publication in Molecular Cancer entitled ‘Development of a 4-aminopyrazolopyrimidine-based dual IGF1R/Src inhibitor as a novel anticancer agent with minimal toxicity ’ [018; doi:We sincerely apologize for any inconveniences these mistakes may have caused.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a contagious respiratory virus that is the cause of the coronavirus disease 2019 (COVID-19) pandemic which has posed a serious threat to public health. COVID-19 is characterized by a wide spectrum of clinical manifestations, ranging from asymptomatic infection to mild cold-like symptoms, severe pneumonia or even death. Inflammasomes are supramolecular signaling platforms that assemble in response to danger or microbial signals. Upon activation, inflammasomes mediate innate immune defense by favoring the release of proinflammatory cytokines and triggering pyroptotic cell death. Nevertheless, abnormalities in inflammasome functioning can result in a variety of human diseases such as autoimmune disorders and cancer. A growing body of evidence has showed that SARS-CoV-2 infection can induce inflammasome assembly. Dysregulated inflammasome activation and consequent cytokine burst have been associated with COVID-19 severity, alluding to the implication of inflammasomes in COVID-19 pathophysiology. Accordingly, an improved understanding of inflammasome-mediated inflammatory cascades in COVID-19 is essential to uncover the immunological mechanisms of COVID-19 pathology and identify effective therapeutic approaches for this devastating disease. In this review, we summarize the most recent findings on the interplay between SARS-CoV-2 and inflammasomes and the contribution of activated inflammasomes to COVID-19 progression. We dissect the mechanisms involving the inflammasome machinery in COVID-19 immunopathogenesis. In addition, we provide an overview of inflammasome-targeted therapies or antagonists that have potential clinical utility in COVID-19 treatment. Inf2+) efflux  efflux . After t3Inflammasomes play a double-edged role in viral infection. Appropriate activation of inflammasomes instigates innate antiviral responses, but the dysregulation of the inflammasome signaling due to defective host immunity or comorbidities can lead to the cytokine storm and hyperinflammation. NLRP3 has been detected in various cell types, including innate immune cells, lung epithelial cells and cardiac cells . It is t3.1Reportedly, NLRP3 inflammasome was activated in peripheral blood mononuclear cells (PBMCs) from COVID-19 patients . The lev3.2+CD16dim granulocytes exhibited lower NLRP3 activation potential in severe forms of COVID-19 than mild cases. The number of nonclassical monocytes was remarkably decreased in severe to critical patients compared with healthy controls, while immature neutrophils exhibited the opposite trend. In recovered patients, caspase-1 activation in CD66b+CD16dim granulocytes was restored and the percentage of immature neutrophils was comparable to the controls. Altogether, the NLRP3 inflammasome signatures in myeloid cells may represent a predictive biomarker for COVID-19 development and patient outcome and provide important guidance for therapeutic decisions involving immunoregulatory agents.Myeloid cells are a vital cellular compartment of innate immunity and consist of monocytes, macrophages and neutrophils . MyeloidhighCD16- monocytes derived from severe COVID-19 patients showed increased NLRP3 inflammasome activation potential, as shown by the formation of ASC speck/caspase-1 complexes and increased IL-1β secretion , resulting in pyroptosis and the extravasation of proinflammatory mediators . CD14higecretion . Monocytecretion . Accordiecretion . MCC950,ecretion . A recenecretion . Severe 3.3Notably, circulatory exosomes from severe COVID-19 patients were able to increase the expression of NLRP3, caspase-1 and IL-1β in endothelial cells . COVID-14Accumulating evidence has indicated the complex interaction between SARS-CoV-2 and inflammasomes. At the initial stage of infection, SARS-CoV-2 and its derived products stimulate inflammasome formation, inducing inflammatory responses to fight against viral infection. SARS-CoV-2 employs diverse strategies to hinder inflammasome activation, ensuring a beneficial environment for its replication. After that, SARS-CoV-2 inflames the inflammatory reaction to propel viral pathogenicity. Highly sophisticated mechanisms involving various viral components contribute to regulation of inflammasome functionality during SARS-CoV-2 infection Figure 34.1The inflammasome signaling performs important functions in pathogen defense. It is conceived that SARS-CoV-2 has evolved multiple mechanisms to counteract inflammasome-dependent cascades, thereby allowing for viral survival and pathogenesis. SARS-CoV-2 N protein was released into the cytosol immediately after viral entry into the host cell . N prote4.24.2.12+) flux is an important signaling event for NLRP3 inflammasome activation (2+ ion channels in ER Golgi apparatus intermediate compartment (ERGIC)/Golgi membranes and impelled reactive oxygen species (ROS)-dependent activation of NLRP3 inflammasome at the later stages of viral infection, which might contribute to advanced or complicated diseases /RIPK3 signaling pathway. The TLR8/myeloid differentiation factor 88 (MyD88) pathway was necessary for SARS-CoV-2 GU-rich RNA-stimulated IL-1β maturation. Consistently, endosomal acidification blocker chloroquine prevented GU-rich RNA-induced cytokine secretion by inhibiting endosomal TLR signaling (+ transporting accessory protein 1 (ATP6AP1), thus resulting in autophagic flux stagnation to activate NLRP3 inflammasome. Pharmacological restoration of autophagic flux dampened NSP6-mediated NLRP3 inflammasome activation. The dysfunction of autophagic flux has been associated with the priming of AIM2 inflammasome , CXCL10, interferon-γ (IFN-γ), IL-1β, IL-6, IL-18 and TNF-α . AvailabThe inflammasome machineries are needed for the synthesis of inflammatory cytokines . Particu5.2It was reported that the NLRP3 inflammasome was over-activated in elderly patients with COVID-19, which could be attributable to defects in mitochondrial functioning, enhanced production of mitochondrial ROS (mtROS) and massive release of mitochondrial DNA (mtDNA) . This ca+IL-1β+ monocytes and macrophages , thus exaggerating SARS-CoV-2-induced cytokine storm . IL-1β crophages , 110. Inrophages . Intenserophages . IL-1β-irophages . This evrophages , 146. Berophages , 148. Im5.3SARS-CoV-2 infection causes inflammation-directed coagulopathy, which may account for venous/arterial thrombosis and unfavorable outcomes in COVID-19 patients Figure 42+ influx. This activated the phospholipid scramblase transmembrane protein 16F (TMEM16F), which increased TF activity through phosphatidylserine externalization  that subsequently entered the circulation and drove coagulation through activation of prothrombin . As expelization .Extracellular traps (NETs), extracellular DNA fibers decorated with antimicrobial proteins, are extruded from activated neutrophils . NETs ar5.4SARS-CoV-2 RNA or proteins could be detected in many typical cells of central nervous system (CNS) , 124. Im5.5The NLRP3 inflammasome was constitutively activated in COVID-19 patients with underlying liver diseases such as cirrhosis, acute live failure and non-alcoholic fatty liver disease (NAFLD) . SARS-CoSARS-CoV-2 infection has been connected with various cardiovascular diseases such as acute coronary syndrome, myocardial injury, arrhythmia and myocarditis . InflammTYK2) gene could enhance the risk of becoming critically ill with COVID-19, while individuals who carried a variant in the dipeptidyl peptidase 9 (DPP9) gene were at elevated risk of developing critical disease (TLR3 and TLR7) might be connected with severe forms of COVID-19 in European populations -mediated inflammasome signaling pathway in PBMCs isolated from COVID-19 patients, hence ameliorating excessive inflammatory response and the cytokine storm. Accordingly, the nanotherapeutics may be an effective therapeutic approach for COVID-19 treatment. However, further clinical studies are necessary to comprehensively evaluate the safety and therapeutic efficiency of 25-HC@DDAB. Quercetin was able to restrict the production of NLRP3 inflammasome components such as NLRP3, ASC and caspase-1 and coordinate various inflammasome regulators   . Thus, qd TXNIP) . Mechanid TXNIP) . It alsoin vivo  derived from black rice germ and bran (BR extract) could reduce SARS-CoV-2 S1-induced secretion of IL-1β, IL-6 and IL-18 in macrophages by targeting the NF-κB/NLRP3 inflammasome pathway /MAPK/activator protein-1 (AP-1) pathway (Perilla frutescens (PFEA) and luteolin were found to quench SARS-CoV-2 S1-induced NLRP3 inflammasome activation in lung cells via downregulation of the Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3) signaling, as shown by decreased expression of NLRP3, ASC and caspase-1 (SARS-CoV-2 S protein caused NLRP3 inflammasome activation and the extravasation of proinflammatory cytokines (IL-1β and IL-6) by upregulating NLRP3 and caspase-1 . Ultrami pathway . C3G and pathway . Hespereaspase-1 . PFEA anaspase-1 . The inf6.2Colchicum autumnale, can inhibit NLRP3 inflammasome activation  . SpecifiMW and PL conceived and supervised the project. MW wrote the manuscript and drew the figures. FY and WC searched and analyzed the literatures. YZ and LZ revised the manuscript. All authors contributed to the article and approved the submitted version.


The goal of this study was to design, synthesise, and evaluate the suitability of crown-TATE as a new peptide-conjugate for radiolabelling of [155Tb]Tb3+ and [161Tb]Tb3+, and to assess the imaging and pharmacokinetic properties of each radiotracer in tumour-bearing mice. [155Tb]Tb-crown-TATE and [161Tb]Tb-crown-TATE were prepared efficiently under mild conditions, and exhibited excellent stability in human serum (>99.5% RCP over 7 days). Longitudinal SPECT/CT images were acquired for 155Tb- and 161Tb- labelled crown-TATE in male NRG mice bearing AR42J tumours. The radiotracers, [155Tb]Tb-crown-TATE and [161Tb]Tb-crown-TATE, showed high tumour targeting  and minimal retention in non-target organs at 2.5 h post-administration. Biodistribution studies confirmed the SPECT/CT results, showing high tumour uptake  and favourable tumour-to-background ratios. Blocking studies further confirmed SSTR2-specific tumour accumulation. Overall, these findings suggest that crown-TATE has great potential for element-matched molecular imaging and radionuclide therapy using 155Tb and 161Tb.Terbium radioisotopes ( Food and Drug Administration (FDA) in 2018 following the NETTER-1 Phase III clinical trial. This treatment is suitable for early stage (G1-G2) progressive, well-differentiated gastroenteropancreatic (GEP) NETs [177Lu]Lu-DOTA-TATE achieves a median progression free survival of 28.4 months Ac-DOTA-TATE) Ac3+ under mild conditions ; a clear advantage over the conventional gold-standard chelator DOTA which requires high temperatures and/or long reaction times to achieve quantitative radiolabelling Tb3+ and [161Tb]Tb3+, and assess the kinetic inertness of the resulting complexes using human serum stability studies. Preclinical assessments of both [155Tb]Tb-crown-TATE and [161Tb]Tb-crown-TATE were undertaken using longitudinal quantitative SPECT/CT in tumour-bearing mice to establish the imaging potential for each of these radiotracers. Full biodistribution studies were performed on both blocked and unblocked groups to determine the in vivo performance of this new bioconjugate for targeting NETs. Altogether, this study demonstrates the capability of crown-TATE to chelate 155Tb and 161Tb effectively and stably, and its feasibility to selectively target AR42J NETs in mice. Overall, this research confirms the in vivo viability of these radiotracers as imaging agents for use in conjunction with 225Ac TAT or as standalone 155Tb/161Tb theranostics.To further explore the scope of this promising chelator, a new peptide-chelate bioconjugate targeting NETs, referred to as crown-TATE herein, was designed and synthesised. In this research, we investigate for the first time the suitability of crown-TATE for radiolabelling of both [3]-octreotate) utilises a disulphide-bridged cyclic octapeptide which has been refined through numerous structure activity relationship studies for optimal binding towards SSTR2 —was synthesised on Wang resin using PyBop/Oxyma Pure coupling methodology, 2 and Cys7 using thallium (III) trifluoroacetate, which simultaneously cleaves the S-acetamidomethyl (Acm) side chain protecting groups and generates the disulphide-bridged cyclic peptide Tb3+ in a form that was directly suitable for radiolabelling [(Canada) . The ISAabelling . 161Tb was produced via the neutron bombardment of enriched [160Gd][Gd2O3 (98.2%) targets using the high thermal neutron flux (3 × 1014 neutrons/cm2/s) BR2 reactor at SCK-CEN (Belgium) [Belgium)  and subsBelgium) . 155/161Tb]Tb3+ were evaluated under mild conditions  and showed quantitative radiolabelling with both [155Tb]Tb3+ and [161Tb]Tb3+, achieving molar activities of 19.4 MBq/nmol and 11.4 MBq/nmol, respectively. Quality control studies were undertaken using radio-HPLC which showed a single sharp peak for the [155Tb]Tb-crown-TATE product (TR = 8.7 min) and no free [155Tb]Tb3+ activity (TR = 1.0 min) in the final product ), The radiolabelling properties of crown-TATE with [161Tb]Tb-crown-TATE was evaluated using a human serum stability challenge assay, Human serum contains numerous endogenous proteins  which can compete with the chelate for the binding of different metal ions. Prior to undertaking any preclinical studies, the kinetic inertness of [155Tb]Tb-crown-TATE and [161Tb]Tb-crown-TATE for imaging pancreatic exocrine tumours. The 155Tb/161Tb-labelled radiotracers were each administered to male NRG mice bearing AR42J tumour xenografts (left-shoulder) and a series of dynamic quantitative SPECT/CT scans were acquired from 0 to 2.5 h post-administration, mean) were extracted for regions of interest (ROIs) in tumour, kidneys, and bladder. The quantitative imaging studies enabled direct measurement of the activity concentrations (%ID/g) in ROIs in the SPECT/CT scans over time which can be compared to the ex vivo biodistribution results obtained after euthanising the mice following acquisition of the final SPECT/CT scan (2.5 h p.i.). Full biodistribution results for the imaging studies are provided in the Preclinical SPECT/CT studies were undertaken to evaluate the potential of [155Tb]Tb-crown-TATE in AR42J tumours (left-shoulder) beginning at 10 min post-administration (SUVmean = 5.19 g/mL) which increases over time reaching a maximum at 2.5 h p.i. (SUVmean = 8.95 g/mL), 161Tb]Tb-crown-TATE were highly comparable to those of the 155Tb-labelled radiotracer, exhibiting a similar pharmacokinetic profile and high tumour uptake (30.0 %ID/g at 2.5 h p.i.), which was further confirmed in the biodistribution studies (28.6 %ID/g).The dynamic SPECT/CT studies showed rapid uptake of Tb-crown-TATE  or [161Tb]Tb-crown-TATE . Additional blocking control studies were undertaken to confirm SSTR2 specific uptake by co-administration of excess unlabelled DOTA-TOC  with each radiotracer. Full biodistribution results for each radiotracer are provided in the Full biodistribution studies were performed for the 155Tb]Tb-crown-TATE (38.7 ± 8.0 %ID/g) and [161Tb]Tb-crown-TATE (38.5 ± 3.5 %ID/g), and elimination primarily through the renal pathway. Blocking control studies confirmed tumour uptake of each radiotracer was SSTR2 specific . Additionally, statistically significant decreases in radiotracer uptake by non-target organs expressing SSTR2  are observed in the blocking studies, further confirming SSTR2 specific uptake. Although [155Tb]Tb-crown-TATE showed an 86% reduction in tumour uptake with co-administration of the blocking agent, and a comparable decrease of 81% was demonstrated for [161Tb]Tb-crown-TATE, the blocking groups did not demonstrate complete knock-down of tumour uptake for [155Tb]Tb-crown-TATE (5.51 %ID/g) or [161Tb]Tb-crown-TATE (7.50 %ID/g); this result was anticipated since the blocking agent was co-administered with each radiotracer injection, rather than using a pre-blocking approach. This is comparable to previous reports which demonstrated that co-injection of unlabelled TATE decreased tumour uptake of [64Cu]Cu-DOTATATE by 83% in mice at 4 h post-injection Tb-crown-TATE and 5.0:1 for [161Tb]Tb-crown-TATE). Bone uptake for [155Tb]Tb-crown-TATE and [161Tb]Tb-crown-TATE was low  implying good radiotracer stability at this timepoint. The radiotracers show the typical pharmacokinetic profile exhibited by TATE-based bioconjugates, with moderate uptake in organs expressing SSTR2 , which is comparable to the biodistribution of [177Lu]Lu-DOTA-TATE —was generated using PyBop/Oxyma Pure peptide coupling methodology on Wang resin preloaded with Fmoc-Thr(tBu)-OH. Prior to coupling the first amino acid, the preloaded Wang resin was swelled in DMF (5 mL), CH2Cl2 (5 mL), and then DMF (5 mL) again. Fmoc-deprotection of the N-terminus was achieved using 20% piperidine in DMF (2 × 10 mL) and confirmed by positive Kaiser test. Successive amino acids were coupled to the N-terminus using a mixture consisting of the amino acid (4 eq.), PyBop (4 eq.), Oxyma Pure (4 eq.), and DIPEA (15 eq.) dissolved in DMF (5 mL). Each amino acid was preactivated for 5 min at RT before addition to the peptidyl resin. Coupling reactions were mechanically shaken for 2–3 h at RT under a N2 atmosphere, and reaction completion was confirmed using a negative Kaiser test. The linear sequence was cyclised between Cys2 and Cys7 on-resin by addition of thallium(III) trifluoroacetate (1.2 eq.) in DMF/anisole (19:1) to the peptidyl resin at 0 °C and shaking for 2 h. Coupling of the crown chelator was performed on-resin. Crown-tris(tBu ester) (4 eq.), PyBop (4 eq.), Oxyma Pure (4 eq.), and DIPEA (15 eq.) were dissolved in DMF (5 mL), and shaken for 5 min at RT. The solution was transferred immediately to the reaction vessel, purged with N2 gas, and the resulting mixture was shaken at RT for 21 h. Global deprotection and resin cleavage was achieved using a cleavage cocktail consisting of TFA/TIPS/H2O/thioanisole/phenol (82.5:2.5:5:5:5). After 3 h at RT, the resin was filtered, and the peptide precipitated using diethyl ether. The crude white precipitate was separated by centrifuge, washed with cold diethyl ether (3 × 10 mL), and re-dissolved in H2O (2 mL). The crude peptide solution was lyophilised and redissolved in H2O (0.1% TFA) prior to HPLC purification. Crown-TATE was purified via RP-HPLC using isocratic conditions (23% MeCN (0.1% TFA) in deionised H2O (0.1% TFA); 30 min.; TR = 7.1 min). HR-ESI-MS  found at [C69H98N14O21S2+2H]2+: 762.3385, [C69H98N14O21S2+H+K]2+: 781.3123.Crown-TATE was prepared using standardised Fmoc-based solid phase peptide synthesis. The linear peptide sequence—Tb3+ at high specific activity (~ 175 MBq/184 μL) which can be used directly without additional purification [Gd2O3 (98.2%) targets using the high thermal neutron flux (3 × 1014 neutrons/cm2/s) BR2 reactor at SCK-CEN (Belgium) [161Tb activity was purified using a semi-automated TRASIS system equipped with TrisKem solid phase extraction (SPE) resins  at TRIUMF (Canada) as described by McNeil et al. [161Tb purified using this approach.Belgium) . After cl et al. . A radio155Tb]Tb3+ and [161Tb]Tb3+ were performed to establish suitable labelling conditions and quality control methods. [155Tb]Tb3+  or [161Tb]Tb3+  were added to solutions of crown-TATE  in NH4OAc buffer ; VT = 100 μL. The final reaction pH (~6.0) was confirmed by spotting small aliquots of the reaction solution (~2–3 μL) on pH test strips. Reactions were allowed to stand at ambient temperature for 10 min, and then monitored by radio-TLC using SG-paper iTLC plates and EDTA  as mobile phase. Under these conditions free [155/161Tb]Tb3+ is bound by EDTA and migrates with the solvent front (Rf = 0.9–1.0), while [155/161Tb]Tb-crown-TATE remains at the baseline (Rf = 0.0–0.1). For all radiolabelling experiments, a separate control reaction was performed to assess the formation of any metal-hydroxo species or colloidal particles under the conditions tested. [155Tb]Tb3+  or [161Tb]Tb3+  were added to solutions of NH4OAc buffer  without any chelator present. The reaction solutions were treated under the same conditions for each radiolabelling experiment and monitored using radio-TLC under the same conditions outlined for [155/161Tb]Tb-crown-TATE. Under these conditions, colloidal terbium or metal-hydroxo species would remain bound to the baseline of the SG-paper iTLC plates (Rf = 0.0), while free [155/161Tb]Tb3+ migrates with the solvent front (Rf = 0.9–1.0). In all radiolabelling studies of crown-TATE, the control reactions showed no evidence of colloidal terbium or metal-hydroxo species when performing these studies in NH4OAc buffer below pH 6.5. Representative radio-TLC traces for [155Tb][Tb-crown-TATE], [161Tb]Tb-crown-TATE], and control reactions are provided in the Initial radiolabelling studies of crown-TATE with Tb-crown-TATE  was prepared using a similar procedure as outlined above. Aliquots of [161Tb]Tb-crown-TATE  were diluted into pooled human serum (270 μL) and incubated at 37 °C for 7 days with gentle mechanical shaking. All studies were performed in triplicate (n = 3). The radiochemical purity was determined using radio-TLC measurements under the same conditions as outlined above.For human serum stability challenge studies, a high activity stock solution of Tb-crown-TATE (60 μL) was loaded onto a Sep-Pak® C18 Plus Light Cartridge (WatersTM) (pre-conditioned with EtOH (5 × 1 mL), then saline (5 × 1 mL)). The reaction vessel was rinsed with saline (2 × 200 μL) and loaded onto the Sep-Pak cartridge to ensure quantitative transfer. The Sep-Pak® cartridge was washed with saline (5 mL) and the purified product eluted with an EtOH/saline solution . Purified [155Tb]Tb-crown-TATE (15.3 MBq) fractions were concentrated to ~50 μL under N2 gas stream and diluted with injectable saline to give suitable doses for preclinical studies. Quality control studies on the final preparation was performed using radio-TLC, radio-HPLC, and gamma spectroscopy. A similar protocol was applied for the preparation of [161Tb]Tb-crown-TATE. Final solution after purification: [161Tb]Tb-crown-TATE (14.9 MBq). All sample preparations were performed using low retention pipette tips and low-protein binding Eppendorf tubes to minimise losses of the radiolabelled bioconjugates through surface adsorption. [155Tb]Tb-crown-TATE and [161Tb]Tb-crown-TATE for imaging NETs, preclinical studies were conducted with male NRG mice bearing AR42J pancreatic exocrine tumour xenografts. For imaging studies experiments were performed using two replicates (n = 2) per radiotracer. Mice were immobilised using a Tailveiner restrainer , and administered with [155Tb]Tb-crown-TATE  or [161Tb]Tb-crown-TATE  via intravenous injection to the lateral tail vein. Mice were anaesthetised and maintained under a continuous stream of isoflurane (1.5–2.0% in oxygen) throughout the acquisition of the SPECT/CT scans, and their animal body temperature was kept using a heated animal bed. Quantitative dynamic SPECT/CT scans were acquired over the first 1 h post-injection (p.i.) of [155Tb]Tb-crown-TATE or [161Tb]Tb-crown-TATE using a VECTor/CT multimodal preclinical scanner  with an extra ultra-high sensitivity (XUHS) pinhole collimator (2 mm) /[injected dose (MBq)/animal weight (g)]. The %ID/g (injected dose/gram body tissue) in relevant organs was calculated by assuming a tissue density of 1 g/mL directly from the quantitative SPECT/CT scans using the following formula: %ID/g = [radioactivity concentration (MBq/mL) in VOIs]/[injected dose (MBq)] × 100. Maximum intensity projections (MIPs) for each time-point were generated using AMIDE software Tb-crown-TATE  or [161Tb]Tb-crown-TATE  was administered intravenously via the lateral tail using a Tailveiner restrainer. After administration, mice were allowed to roam freely in their cages. Biodistribution studies were performed at 2 h post-administration of [155Tb]Tb-crown-TATE or [161Tb]Tb-crown-TATE. Mice were euthanised by CO2 asphyxiation under 2% isoflurane anaesthesia, followed by cardiac puncture to recover activity in the blood pool. Organs of interest were completely harvested, weighed, and the activity measured using a gamma counter . To account for organs that could not be fully extracted , standardised organ weights for age- and sex-matched mice were used to correct the data Tb3+ and [161Tb]Tb3+ under mild conditions, and in vivo assessment demonstrated excellent tumour imaging contrast with minimal background organ uptake. Our investigation demonstrates the capability of crown-TATE to provide two new element-matched radiotracers which exhibit equivalent biodistribution and pharmacokinetic properties in vivo when radiolabelled with either 155Tb or 161Tb. The interchangeable metal complexation properties of the crown chelator provide a suitable platform for developing 155Tb as a diagnostic companion for 225Ac targeted alpha therapy. In addition, the favourable imaging properties of both terbium radionuclides will enable accurate staging of disease progression and assessment of patient suitability for treatment using 155Tb, as well as post-therapy scintigraphy following 161Tb administration. Further work focused on 225Ac-labelled crown-TATE is of high interest and will expand the applicability of this bioconjugate for more effective and personalised treatments of NETs. Additional therapy studies involving [161Tb]Tb-crown-TATE are the subject of ongoing research, which will allow direct comparison of the therapeutic efficacy of Meitner–Auger electron emitters versus alpha emitters.In this proof-of-principle study, crown-TATE was synthesised for the first time and evaluated as a new bioconjugate targeting SSTR2-positive NETs. The results showed that crown-TATE can be efficiently radiolabelled with both [


By prime-boost vaccination regimens with different combinations of IN and IM inoculations of Sad23L-nCoV-S vaccine, the IN-involved vaccination stimulated higher protective mucosal or local immunity in BAL and lung, while the IM-involved immunization induced higher systemic immunity in serum and spleen. A long-term sustained mucosal and systemic NAb and T- cell immunity to SARS-CoV-2 was maintained at high level over 32 weeks by prime-boost vaccination regimens with IN and IM routes. In conclusion, priming or boosting immunization with IN inoculation of Sad23L-nCoV-S vaccine could induce effective mucosal immunity and in combination of IM route could additionally achieve systemic immunity, which provided an important reference for vaccination regimens against respiratory virus infection.The failure of COVID-19 vaccines to prevent SARS-CoV-2 infection and transmission, a possibly critical reason was the lack of protective mucosal immunity in the respiratory tract. Here, we evaluated the effects of mucosal and systemic immunity from a novel simian adenovirus-vectored COVID-19 vaccine (Sad23L-nCoV-S) in mice in comparison with Ad5-nCoV-S by intranasal (IN) drip and intramuscular (IM) injection vaccinations. As good as the well-known Ad5-nCoV-S vaccine, a single-dose IN inoculation of 1 × 10The essential goal of vaccination is to generate potent and long-term protection against diseases. Several factors including vaccine vector, delivery route, and boosting regimen influence the outcome of prime-boost immunization approaches. The immunization regimens by constructing a novel simian adenovirus-vectored COVID-19 vaccine and employing combination of intranasal and intramuscular inoculations could elicit mucosal neutralizing antibodies against five mutant strains in the respiratory tract and strong systemic immunity. Immune protection could last for more than 32 weeks. Vectored vaccine construction and immunization regimens have positively impacted respiratory disease prevention. During the past 3three years, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection caused a global pandemic of coronavirus disease-19 (COVID-19) and led to heavy losses of human life and the world economy –6, andNasal vaccines hold a superior position against infections such as SARS-CoV-2, whose invasion occurs majorly via the nasal mucosa . With thAdenoviruses as vectors are relatively easy to penetrate the respiratory mucosal barrier, achieve antigen delivery, and provoke respiratory mucosal immune responses. In addition, its ability to generate such responses at the mucosal sites of pathogen entry makes it a preferred choice for intranasal (IN) vaccination . Nasal iWe have previously developed a COVID-19 vaccine Sad23L-nCoV-S) based on a simian adenovirus type 23 vector (Sad23L) carrying the full-length S gene of SARS-CoV-2 L-nCoV-S . The comSad23L-nCoV-S vaccine carrying the full-length SARS-CoV-2 spike gene was constructed previously . Both Sa9 PFU Sad23L-nCoV-S vaccine was examined by inoculation via IN drip or IM injection in comparison with Ad5-nCoV-S vaccine in mice (50) by pVNT, respectively . From IN inoculated mice with Sad23L-nCoV-S vaccine, serum IgG S1-BAb and S2-BAb titers (104.37 and 103.54) were detected in high level, but slightly lower than those from IM injected mice (104.63 S1-BAb and 104.00 S2-BAb) (3.23) were significantly higher than those (≤102.03) from IM injected mice  and IgA S1-BAb and S2-BAb activities (102.30 and 102.06) were detected, but not from IM injected mice  against wild-type strain of pseudovirus from IN immunized mice (The immunogenicity of a single dose of 10 in mice . After 4 S2-BAb) ; serum Ited mice . From INzed mice . A singlzed mice .6 cells), while IM immunization stimulated higher specific IFN-γ-secreting T-cell response reversely in spleen but lower in lung (499 versus 281 SFCs/106 cells), respectively (+ CD8+ T-cell response (0.33%) than IM immunization in spleen , while both IN and IM immunizations induced TNF-α+ CD8+ T-cell responses varied insignificantly at low level in spleen  or flow cytometry . IN immuectively . Moreoven spleen . For comn spleen .Overall, as good as Ad5-nCoV-S vaccine, a single-dose IN or IM immunization of Sad23L-nCoV-S vaccine induced similar level of serum IgG S-BAb or NAb reactivity, while IN inoculation had stronger serum IgA, BAL S-BAb, or NAb reactivity, and higher specific T-cell response in lung but lower in spleen of immunized mice compared to IM route.4.51; 3.34; 50 >103.45) higher than those in convalescent serum from COVID-19 patients . In BAL samples from prime-boost vaccinated mice, the IN/IN, IM/IN, and IN/IM immunization regimens containing IN inoculation of vaccine showed satisfactory IgG and IgA S-BAb (S1-BAb and S2-BAb) and NAb responses, of which IN/IN appeared relatively stronger in mice, while IM/IM regimens did not raise local mucosal antibody response , suggesting the level of mucosal IgA associated with neutralization potency against this respiratory SARS-CoV-2 infection.In order to establish a proper prime-boost immunization regimens for optimizing immune efficacy with circulating and local mucosal immunity, four combinations of IM/IM, IN/IN, IM/IN, and IN/IM inoculations of Sad23L-nCoV-S vaccine were examined . All foupatients , of whicresponse . Strong 50 103.13), Beta (>IC50 102.54), Gamma (>IC50 103.16), Delta (>IC50 103.02), and Omicron (>IC50 101.81) variants, of which all were significantly higher than those from convalescent serum samples of COVID-19 patients to five pseudoviruses , excepting for no response from IM/IM inoculated mice (. Compared to a single immunization (IN or IM), the combination of IM and IN inoculations increased neutralizing antibodies , or in lung from IN/IN and IN/IM inoculations of vaccine (801 or 1005 SFCs/106 cells), respectively  cells expressing CD103 and CD69 markers on pulmonary CD8+ cells were measured by flow cytometry . The IN/IN vaccination regimens evidently presented the highest response of TRM cells in lung that was 8.45-fold higher than the control (P < 0.001), while the IM/IN and IN/IM inoculations also induced robust TRM cell responses that were 4.28-fold or 4.07-fold higher than the control (P < 0.01) and significantly higher than the IM/IM immunization regimens (P < 0.01), but insignificantly varied between each other , which generally presented a similar pattern with above ELISpot results from four vaccination regimens  up to 8 months by prime-boost vaccinations (During 8 months (32 weeks) of follow-up detection, the humoral and cellular immune responses were monitored constantly from seven separate groups of mice immunized by prime or plus boost inoculations of Sad23L-nCoV-S vaccine or vector control, of which generally prime-boost vaccination regimens induced higher and longer immune response than a single prime immunization via IM or IN inoculation of vaccine . Serum Iinations , of whicinations , and an inations , respectinations .1.35, IgA >101.12, NAb > 101.06, or T cells > 118 SFCs/106 cells), typically when an IN inoculation of vaccine was involved in prime-boost vaccination regimens  in vaccinated mice, the serum and BAL samples were measured for AdNAb to Sad23L vector in 4 weeks after prime or prime-boost immunizations . AdNAb titer to Sad23L increased to 1:960 in serum or 1:8 in BAL after priming with IM inoculation of Sad23L-nCoV-S vaccine . In contThe essential goal of vaccines is to generate potent and long-term protection against diseases. The failures to prevent SARS-CoV-2 Omicron variant infection and transmission between individuals vaccinated by most COVID-19 vaccines are considered for immune escaping of viruses , 27. HowThe relatively low level of immune response from a single dose of vaccine immunization suggested that a booster is needed for enhancing the humoral and cellular immunity. By prime-boost vaccinations, the factors including vaccine inoculation route, order, and combination are critical for deserving the optimal immune efficacy. It has been shown that administering of vaccine by different routes could have significantly different effects on the type and strength of the induced immune responses . The difThere were two limitations in this study. Firstly, the lack of live SARS-CoV-2 for measuring neutralizing antibody and challenging of vaccinated animals for vaccine efficacy by the IN involved vaccination regimens due to biosafety level-3 requirement. Secondly, the difficulty to isolate a large amount of immune cells from lung tissue of vaccinated mice for comprehensively investigating local cellular immunity. Our future studies will mechanistically investigate the kinetics of intranasal immunization through isolation of tissues in closer proximity to the nasal cavity, including mucosa-associated lymph node tissue (MALT).In conclusion, this study illustrated the superior potential for prime-boost vaccination regimens in combination of IN and IM (IN/IM or IM/IN) inoculations of Sad23L-nCoV-S vaccine for inducing strong mucosal and systemic immunity in mice, which provided an important reference for respiratory virus vaccination regimens.Sad23L, a replication defective novel adenovirus vector was constructed and produced as described previously , and Ad5BALB/c mice were obtained from the Animal Experimental Centre of Southern Medical University, Guangdong, China. All animal experiments were conducted in compliance with the guidelines for the care and use of laboratory animals and approved by the Southern Medical University (SMU) Animal Care and Use Committee, Guangzhou, China (permit numbers: SYXK [Yue] 2021–0167).n = 18/group for 5–6 weeks) were individually inoculated with a single dose of 109 PFU/100 µlL Sad23L-nCoV-S or Ad5-nCoV-S vaccine by IN drip or IM injection inoculation, respectively. A dose of 109 PFU/100 µlL Sad23L-GFP or Ad5-GFP vectorial viruses was used as sham control.Female BALB/c mice (n = 15/group for 5–6 weeks) were primed with a dose of 109 PFU Sad23L-nCoV-S vaccine, and then at 4-week interval boosted with a same dose of vaccine by IM/IM, IN/IN, IM/IN, or IN/IM immunization regimens, respectively. A dose of 109 PFU Sad23L-GFP vectorial viruses was used as sham control.Female BALB/c mice , China . All serThe eyeballs of mice were removed, and blood was collected from the eye socket. Then, mice were sacrificed and sterilized by alcohol, and the thoracic cavity was opened to expose the lung. Bronchoalveolar lavage fluid (BAL) of mice was collected by washes with PBS by tracheal intubation and centrifuged at 1,500 rpm for 5 min. The serum and BAL samples were stored at −80°C for testing.HEK293 cells infected with Sad23L-nCoV-S and Ad5-nCoV-S vaccine strains or vector controls were fixed in 4% cold paraformaldehyde, blocked, and incubated with mouse monoclonal antibody to SARS-CoV-2 S protein  in 1% BSA/PBST at 4°C overnight. Anti-mouse IgG-Dylight 594 antibody  was added to the cells for 30 min at 37°C. DAPI was used for nuclei staining. The S protein expression from cell lysates was analyzed by Western blot using an anti-SARS-CoV-2 RBD polyclonal antibody  and GAPDH as a loading control.ELISA was used to detect the binding antibodies to S protein of SARS-CoV-2 (S-BAb). The microtiter plates  were coated overnight at 4°C with 1 µg/ml of SARS-CoV-2 S1  or S2 proteins . Serum and BAL samples were threefold serially diluted, and anti-S1 and S2 binding antibodies  were tested by ELISA. Secondary antibodies of goat anti-mouse IgA-HRP  or rabbit anti-mouse IgG-HRP  at 1:4000 dilution were incubated at 37°C. The absorbance at 450 nm was recorded using a BioTek Epoch. The endpoint serum or BAL dilution was calculated by a curve fit analysis of optical density (OD) values for serially diluted sera with a signal than cut-off value (S/CO) ≥3.4 HEK293T-hACE2 cells . After 48 h incubation, the cells were lysed by Bright-Glo luciferase assay (Promega) according to the manufacturer’s instructions. NAb titer (IC50) to SARS-CoV-2 was defined as the sample dilution at which a 50% reduction (50% inhibitory concentration) in relative luciferase activity (RLU) observed relative to the average of the virus control wells.Pseudoviruses expressing SARS-CoV-2 S protein of Wild-type (Wuhan-1) strain, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), or Omicron (B.1.1.529) variants were generated, and the neutralizing antibody (NAb) to SARS-CoV-2 was measured by pseudovirus neutralization test (pVNT) . BrieflySpecific IFN-γ secreting T-cell response was measured by the enzyme-linked immunospot (ELISpot). Mouse spleen was grinded and filtered through a 70 µm cell strainer, which was centrifuged and resuspended in 5 mL ACK lysing buffer for 5 min at room temperature, diluted in 10 mL PBS and centrifuged. The single-cell suspensions were resuspended in 1 mL complete RPMI-1640 medium. The lungs were cut into small pieces and digested with collagenase IV (2 mg/mL) and DNase I (5 mg/mL) at 37°C for 60 min, and then filtered with 70 µm cell strainers. After the red blood cells were lysed, PBS was added and washed twice, and finally resuspended with 1 mL complete RPMI-1640 medium.5 cells/well) were stimulated with S peptides (5 µg/ml) in duplicate. SARS-CoV-2 S antigen-specific T lymphocyte response in mice was assessed by IFN-γ ELISpotPLUS kits (Mabtech) following the manufacturer’s instruction. S peptides were predicted (http://www.iedb.org/) and synthesized by Guangzhou IGE Biotechnology LTD. Spots were counted with a CTL Immunospot Reader (Cellular Technology Ltd). The results were expressed as spot forming cells (SFCs) per million cells.Mouse splenocytes and lung cells (5 × 105 cells/well) were stimulated with S peptides (5 µg/ml) or medium as negative control for 12 h at 37°C before a 6 h treatment with brefeldin A . Cells were collected and stained with antibodies to mouse CD45, CD4, and CD8 surface markers (BD). The cells were washed twice with PBS and fixed with IC fixation buffer, permeabilized with permeabilization buffer (BD) and stained with antibodies to mouse interferon-γ (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor α (TNF-α), and GrzB (BD). A part of the lung cells was stained with antibodies to mouse CD45, CD4, CD8, CD103, and CD69 surface markers after stimulation. Boolean gating was applied to the gated IFN-γ+, IL-2+, GrzB+ cells to determine the frequency of all cytokine+ CD4+ and CD8+ T cells. All samples were tested with a BD FACSCanto Flow Cytometer (BD) and the data were analyzed using FlowJo Software (version 10).The mouse splenocytes and lung cells  titers were defined as the maximum serum or BAL dilution that neutralized 50% of Green activity as previously described .t test and Mann-Whitney test. The correlation between S-BAb and NAb titers was analyzed using Pearson’s correlation coefficients. Statistically significant differences were indicated with asterisks . All graphs were generated with GraphPad Prism 8 software.Statistical significance between groups was analyzed using one-way analysis of variance or unpaired two-tailed


Correction: BMC Complement Med Ther 23, 206 (2023)10.1186/s12906-023-04042-4Following publication of the original article , the aut2,3The incorrect author affiliation is: Guifeng Hao3The correct author affiliation is: Guifeng HaoThe author group has been updated above and the original article has been corrected.


Acute myeloid leukemia (AML) is a hematological malignancy characterized by the impaired differentiation and uncontrolled proliferation of myeloid blasts. Tumor suppressor p53 is often downregulated in AML cells via ubiquitination-mediated degradation. While the role of E3 ligase MDM2 in p53 ubiquitination is well-accepted, little is known about the involvement of deubiquitinases (DUBs). Herein, we found that the expression of YOD1, among several DUBs, is substantially reduced in blood cells from AML patients. We identified that YOD1 deubiqutinated and stabilized p53 through interaction via N-terminus of p53 and OTU domain of YOD1. In addition, expression levels of YOD1 were suppressed by elevated miR-221/222 in AML cells through binding to the 3′ untranslated region of YOD1, as verified by reporter gene assays. Treatment of cells with miR-221/222 mimics and inhibitors yielded the expected effects on YOD1 expressions, in agreement with the negative correlation observed between the expression levels of miR-221/222 and YOD1 in AML cells. Finally, overexpression of YOD1 stabilized p53, upregulated pro-apoptotic p53 downstream genes, and increased the sensitivity of AML cells to FLT3 inhibitors remarkably. Collectively, our study identified a pathway connecting miR-221/222, YOD1, and p53 in AML. Targeting miR-221/222 and stimulating YOD1 activity may improve the therapeutic effects of FLT3 inhibitors in patients with AML. Acute myeloid leukemia (AML) is a genetically heterogeneous and clonal hematopoietic stem cell malignancy characterized by chromosomal abnormalities, recurrent gene mutations, and/or epigenetic modifications affecting chromatin structures , 2. AlthAs the most important tumor suppressor, p53 plays a key role in protecting genome integrity and preventing the occurrence of tumors . Unlike The human genome encodes nearly 100 DUBs divided into seven different families: ubiquitin-specific proteases (USPs), ovarian tumor (OTU) proteases, ubiquitin C-terminal hydrolases (UCHs), Machado–Joseph Disease (MJD) DUBs, motif interacting with ubiquitin (MIU)-containing novel DUB family (MINDY), recently discovered zinc finger-containing ubiquitin peptidase 1 (ZUP1), and JAB1/MPN/MOV34 metalloprotease DUBs (JAMMs) . DUBs re–n = 20) and bone marrow (n = 6) and compared them with those from the peripheral blood of AML patients. qPCR data revealed that YOD1 and OTUB1 decreased in AML samples, with YOD1 showing the most significant changes . FAB classifications and gene mutations for AML patients are provided in Supplementary Table U2OS, MOLM13, and MV-4–11 cells were cultured in RPMI medium  supplemented with 10% fetal bovine serum (FBS) . HCT116 and 293 T cells were cultured in DMEM medium (Gibco) supplemented with 10% FBS. Cultured cells were transfected with plasmid DNAs, miRNA mimics, miRNA inhibitors, or siRNAs using Lipofectamine 3000 reagent .Full-length human wild-type (WT) YOD1 cDNA was cloned into the pFlag-CMV2 vector . Full-length human wild-type p53 was cloned into the pCMV-Myc-N vector , whereas full-length human wild-type and various mutant forms of ubiquitin were cloned into the pCMV-HA-N vector . Point and domain-truncation mutants of YOD1 and p53 were made through site-directed mutagenesis in the original vectors. The primers used are shown in Supplementary Table A lentivirus packaging system was purchased from Hanbio . The system contained two helper plasmids (pSPAX2 and pMD2G) and a shuttle plasmid LV011-pHBLV-CMV-MCS-3flag-EF1-T2A-Zsgreen-Puro (LV011) for gene overexpression or pLKO.1-U6-EF1a-copGFP-T2A-puro (pLKO.1) for gene knockdown. The shRNA sequence was CCGGGCACTGGAATTAGCAGATGAAC-TCGAGTTCATCTGCTAATTCCAGTGCTTTTTGAATT. The lentiviral miR-221 and anti-miR-221 sequence was CCGGA AATCTACATTGTATGCC- AGGTCTCGAGACCTGGCATACAATGTAGATTTTTTTTGAATT and CCGGACCTGGCATACAATGTAGATTTCTCGAGAAATCTACATTGTATG CCAGGTTTTTTGAATT.Total RNA was extracted using the MiniBEST Universal RNA Extraction Kit , and reverse transcription with equal amounts of RNAs was performed using the PrimeScript RT-PCR Kit . qPCR was conducted using SYBR Green I  as the fluorescent dye and analyzed using Precision Melt Analysis software . The primers listed in Supplementary Table http://www.mirbase.org/). Primers were designed according to the method described by Varkonyi-Gasic and Hellens [The miR-221/222-3p sequences were based on miRBase  and staining with DAPI. Images were captured under a Zeiss LSM 510 confocal laser scanning microscope.Immunostaining was performed as described , 77. TheFor cell viability assays was measured using the cell counting Kit-8 (CCK-8) method. For apoptosis analysis, cells were stained with APC-Annexin V  and propidium iodide. To assess cell cycle arrest, cells were fixed with ethanol overnight and stained with propidium iodide in the presence of RNase A. A CytoFlex LX Flow Cytometer  was used according to the manufacturer’s instructions. FlowJo  was used for apoptosis and cell cycle analyses.Luciferase constructs were made using the pmirGLO Dual-Luciferase miRNA Target Expression Vector System . A YOD1 3′ UTR fragment was generated by RT-PCR from MV-4-11 cell total RNA. The primer sequences are provided in Supplementary Table p < 0.05 was considered significant.Optical intensity was measured using the AlphaEasy program . Data were analyzed using GraphPad Prism version 8.0.2 . All numerical values are presented as the mean ± SD. Difference between two groups was calculated using unpaired student t-test. Differences among multiple groups were calculated using two-way ANOVA. Supplementary infoOriginal Data FileCertificate of editing


C. briggsaeis an excellent genetic model for comparative and evolutionary studies involving its well-known cousinC. elegans. The vulval system in these two species has been used extensively to investigate genes and pathways involved in cell proliferation and cell differentiation. Here we report initial characterization of twoC. briggsaemultivulva (Muv) mutants,Cbr-lin(bh1)andCbr-lin(bh3).The nematode Caenorhabditisare routinely used to investigate genes, genetic processes, and conserved signaling pathways. The vulva, a reproductive organ inC. elegansandC. briggsae, is often utilized for comparative analysis due to its invariant cell lineage and well-defined cell fatesC. briggsaeshould enable further studies of evolutionary differences between the two species.Nematodes of the genusCbr-lin(bh1)andCbr-lin(bh3), were recovered in a forward genetic screen in our lab. The phenotype ofCbr-lin(bh1), but notCbr-lin(bh3),was found to be heat sensitive such that the penetrance of theCbr-lin(bh1)Muv phenotype positively correlated with temperature (Cbr-lin(bh1)heterozygous mothers, n = 309, vs. expected ~7%, i.e., one-quarter of 27% Muv; and 9.3% Muv in the progeny ofCbr-lin(bh3) heterozygous mothers, n = 313, vs. expected ~10%, i.e., one-quarter of 38% Muv). Nomarski microscopy revealed that P3.p, P4.p and P8.p VPCs were ectopically induced in mutant animals  and Cbr-zmp-1::GFP (Félix 2007), were used to examine the VPC fates. While ectopically induced VPCs inCbr-lin(bh3) animals adopted both 10and 20fates , only 20fates were observed inCbr-lin(bh1) animals .The multivulva (Muv) mutants,erature . There animals . Two ceCbr-lin(bh1)animals grow slower, taking almost 20 hours more than the wild-type AF16 to reach adulthood. TheCbr-lin(bh3)strain showed no such growth delay. Indel-based polymorphism mappingCbr-lin(bh1)to the right arm of chromosome I andCbr-lin(bh3)roughly to the center of chromosome III (Cbr-lin(bh1)with existing chromosome I Muv mutants,Cbr-spr-4(gu163)andCbr-pry-1(sy5353), suggested that it represents a distinct gene .We also observed thatome III . CompleMaterial and methodsWorm culture maintenance and genetic screenOP50bacteriaAF16worms were mutagenized by feeding them with 25 mM EMS for four hours. Individual P0s were allowed to grow on plates and F2 animals were screened for a Muv phenotype. Candidates were isolated and phenotype was confirmed in successive generations.Cbr-lin(bh1)andCbr-lin(bh3)strains were outcrossed 2x before characterizing their phenotypes. The strains used in this study are listed in the following table.All strains were grown on the Nematode Growth Medium (NGM) plates seeded withPolymorphism mappingCbr-lin(bh1)andCbr-lin(bh3)was carried out using medium indelsHK104males. Muv animals were selected in the F2 generation and were pooled for PCR analysis. See Sharanaya et al., (2015) for a mapping scheme and the protocol. The details of indel polymorphism markers and primers are mentioned in the table below. PCR products were analyzed on 1% agarose gel and the linkage was determined based on qualitative assessment of the intensity ofAF16andHK104bands.Mapping ofMuv penetrance and VPC induction analysisAnimals were grown for at least three generations at a given temperature  before scoring the phenotype. Each genotype was scored in triplicates. In each case 40 - 50 worms were examined under a Nikon Eclipse 80i Nomarski microscope.L4 stage worms were analyzed for VPC induction by mounting them on agar pads containing 1mM Sodium Azide. 50 - 60 animals, pooled from three batches, were examined for each genotype at 20℃.


Sphingosine kinases (SphK), including SphK1 and SphK2, are important enzymes promoting progression of prostate cancer. SKI-178 is a novel and highly potent SphK1/2 dual inhibitor. We here tested the potential anti-prostate cancer cell activity of SKI-178. Bioinformatics analyses and results from local tissues demonstrated that that both SphK1 and SphK2 are upregulated in human prostate cancer tissues. Ectopic overexpression of SphK1 and SphK2, by lentiviral constructs, promoted primary prostate cancer cell proliferation and migration. In primary human prostate cancer cells and immortalized cell lines, SKI-178 potently inhibited cell viability, proliferation, cell cycle progression and cell migration, causing robust cell death and apoptosis. SKI-178 impaired mitochondrial functions, causing mitochondrial depolarization, reactive oxygen species production and ATP depletion.SKI-178 potently inhibited SphK activity and induced ceramide production, without affecting SphK1/2 expression in prostate cancer cells. Further, SKI-178 inhibited Akt-mTOR activation and induced JNK activation in prostate cancer cells. Contrarily, a constitutively-active Akt1 construct or the pharmacological JNK inhibitors attenuated SKI-178-induced cytotoxicity in prostate cancer cells. In vivo, daily intraperitoneal injection of a single dose of SKI-178 potently inhibited PC-3 xenograft growth in nude mice. SphK inhibition, ceramide production, ATP depletion and lipid peroxidation as well as Akt-mTOR inactivation and JNK activation were detected in PC-3 xenograft tissues with SKI-178 administration. Together, targeting SphK1/2 by SKI-178 potently inhibited prostate cancer cell growth in vitro and in vivo. Prostate cancer is the second most common malignancy among males , 2. It iSphingosine kinases (SphK), including SphK1 and SphK2, are key enzymes phosphorylating sphingosine to sphingosine 1-phosphate (S1P) –9. IncreOverexpression of SphK1 in prostate cancer is correlated with cancer grade and poor overall survival . Lee et Recent studies have developed a novel SphK1 and SphK2 dual inhibitor SKI-178, competing for sphingosine binding site in SphK1/2 –15. SKI-Chemical and reagents. Culture medium, N-Acetyl-L-cysteine (NAC) and ATP, fetal bovine serum (FBS) and antibiotics as well as the caspase-3 inhibitor z-DEVD-fmk and the pan caspase inhibitor z-VAD-fmk were from Invitrogen . Poly-d-Lysine was from BD Biosciences . Cell counting kit -8 (CCK-8) was provided by Dojindo Co. . EdU (5-Ethynyl-2’-deoxyuridine) and TUNEL  and JC-1 ,were from Thermo-Fisher Invitrogen . Antibodies for SphK1 (#12071), SphK2 (#32346), cleaved caspase antibody sampler kit (#9929) and β-Tubulin (#2146) were purchased from Cell Signaling Technologies . SP600125 and JNK inhibitor II (JNKi-II) were obtained from Selleck .PTEN-null, PI3KCA-positive and androgen receptor-positive. Short-tandem repeat (STR) profiling, population doubling time, and cell morphology were always checked for the cells. The human prostate cancer tissues and matched adjacent normal prostate tissues of ten (10) written-informed consent castration-resistant prostate cancer (CRPC) patients were provided by Dr. Mi [The established PC-3/LNCaP prostate cancer cell lines and the non-transformed RWPE1 prostate epithelial cell line were reported previously . The priy Dr. Mi . All proCells were seeded in 96-well plates and treated with SKI-178. Afterwards, 40 μL of caspases-3-Glo substrate or the caspase-9-Glo substrate  was added into each well for 1 h. A FlexStation 3 microplate reader  was utilized to quantify Glo luminescence.Western blotting and The quantitative reverse transcription PCR (qRT-PCR) protocols were described early . Figure The GV369 lentiviral vectors encoding SphK1 shRNA and SphK2 shRNA were synthesized by Shanghai Genechem Co . The construct, together with the lentivirus Helper plasmids, was co-transfected to HEK-293T cells (Genechem). The generated lentivirus were added to prostate cancer cells (cultured in polybrene medium) for 24 h. Cells were then cultured in puromycin (5 μg/mL)-containing complete medium for another 48 h, and stable cells established. SphK1/2 expression in stable cells was tested by Western blotting and qRT-PCR assays. The scramble control shRNA lentivirus  was added to the control prostate cancer cells.The lentivirus-packed constitutively-active Akt1  was reported in our previous study . It was The GV369 lentiviral SphK1-expressing construct and the GV369 lentiviral SphK2-expressing construct were both provided by Genechem . Each construct was transfected to HEK-293 cells along with lentiviral packaging constructs (Genechem), thereby generating lentivirus. The virus was then filtered and enriched, and was added (at MOI = 15) to cultured prostate cancer cells (in polybrene medium). Stable cells were then established after selection using puromycin. SphK1/2 overexpression was verified by the Western blotting assays. Control cells were infected with vector control lentivirus.4 cells per chamber) and were allowed to migrate for 16 h. Cells on the lower surface were then fixed, stained and were photographed by an microscope.Cells in no serum basic medium were added in the upper surface of the chamber  and Ethic Committee of Soochow University. The nude mice  were obtained from the Animal Center of Soochow University. Mice were housed under standard procedures and were injected subcutaneously (s.c.) with PC-3/pCan1 cells (at six million cells/mice) in Matrigel-serum medium. Tumor volumes were measured by the modified ellipsoid formula: (π/6) × mor mass . Tissue mor mass .t-test (Excel 2007), and overall significance of multiple groups was evaluated by ANOVA followed by Tukey’s multiple comparison test of variance . All statistical tests were two-sided and the level of significance was set at P < 0.05.Statistical analyses. Experimental data were expressed as mean ± standard deviation (SD). The statistical significance of differences between the means of two treatment groups was evaluated by unpaired two sided Student’s SphK1/2 expression in human prostate cancer. A total of 553 tissues samples were collected, including 501 prostate cancer tissue samples and 52 para-cancerous normal prostate tissue samples. As shown in Fig. SphK1 transcripts in the prostate cancer tissues (“Tumor”) was significantly higher than that in the normal prostate tissues . Moreover, upregulation of SphK2 transcripts was detected in the prostate cancer tissues from the TCGA database  database was first consulted to examine ase Fig. .Fig. 1SpSphK1 and SphK2 was significantly elevated in human prostate cancer tissues (“T”) from 10 different CRPC patients  , the protein expression of both SphK1 and SphK2 was significantly elevated in the oe-SphK1+oe-SphK2 pCan1 cells Fig. . In SphKThe primary human prostate cancer cells, pCan1, were cultivated in completed medium and treated with SKI-178 at gradually increased concentrations (from 1-25 μM). Cells were then cultured for different periods, and cell viability examined by CCK-8 assays. As shown, SKI-178 potently inhibited pCan1 cell viability (CCK-8 OD) in a concentration-dependent manner Fig. . SKI-178The EdU staining assays were employed to test cell proliferation. SKI-178 efficiently decreased the EdU-positive nuclei ratio in pCan1 cells Fig. , supportThe potential effect of the SphK1/2 dual inhibitor on other prostate cancer cells was examined as well. The primary human prostate cancer cells-derived from another patient, pCan-2, as well as the established cell lines, PC-3 and LNCaP, were treated with SKI-178 (10 μM) and cultured for applied time periods. As shown SKI-178 potently decreased the cell viability (CCK-8 OD) in the prostate cancer cells Fig. . ResultsNext experiments were performed to examine the potential effect of SKI-178 in non-cancerous prostate epithelial cells. The primary human prostate epithelial cells (“pEpi”) and established RWPE-1 cells , 25 wereThe PI-FACS assay was employed to examine cell cycle distribution. In SKI-178 -treated pCan1 primary cells, the G1 phase cell percentage was significantly increased Fig.  and the Whether apoptosis was induced by SKI-178 in other prostate cancer cells was tested next. As shown, in the pCan-2 primary prostate cancer cells and established lines (PC-3 and LNCaP), the SphK1/2 dual inhibitor induced significant apoptosis activation, evidenced by significantly increased TUNEL-positive nuclei ratio Fig. . InteresResults in Fig. Whether mitochondrial disruption played an important role in SKI-178-induced prostate cancer cell death was studied next. As shown the well-known anti-oxidant NAC and supplementing ATP attenuated SKI-178 (10 μM)-induced CCK-8 viability reduction Fig.  and cellSphK1 and SphK2 in pCan1 primary cancer cells  and protein phosphatase 2 A (PP2A) –44. ConsCeramide accumulation after SphK inactivation could also provoke JNK activation, thereby promoting cancer cell apoptosis and death , 49, 50.6 cells per mouse) were subcutaneously (s.c.) injected to the flanks of the nude mice. Within 20 days, the PC-3 xenografts were established (labeled as “Day-0”) with tumro volumes close to 100 mm3. The PC-3 xenograft-bearing mice were thereafter randomly assigned into two groups, with 10 mice per group (n = 10). The SKI-178 treatment mice received intraperitoneal injection of SKI-178 (25 mg/kg body weight), daily for 18 days. The control group mice received vehicle control administration (“Veh”). In Fig. At last we tested the potential anti-cancer activity of SKI-178 in vivo. A significant number of PC-3 cells  days after cell injection, xenografts were measured. The volumes of oe-SphK1+oe-SphK2 pCan1 xenografts were significantly higher than those of vector control pCan1 xenografts (Fig. To further support the role of SphK1/2 in prostate cancer cell growth in vivo, the oe-SphK1+oe-SphK2 pCan1 cells (see Fig. fts Fig. . Moreovefts Fig. . The micfts Fig. . When anfts Fig.  and Akt-fts Fig. . These rProstate cancer is the fifth leading cancer-caused death among men , 2. It iRecent studies have shown that SphK inhibition by the pharmacological agents could potently inhibit prostate cancer cell growth. Pchejetski et al., have shown that FTY720 inhibited SphK1 to sensitize prostate cancer cells to radiotherapy . FTY720 SKI-178 is a small-molecule, non-lipid and highly-efficient SphK1/2 dual inhibitor . It has Here the TCGA database results and results from local tissues demonstrated that that both SphK1 and SphK2 are upregulated in human prostate cancer tissues. Targeting SphK1/2 by SKI-178 induced significant anti-prostate cancer activity. In primary human prostate cancer cells and established cell lines, SKI-178 potently inhibited cell viability, proliferation, cell cycle progression and cell migration. Significant cell death and apoptosis were detected in SKI-178-treated primary and established prostate cancer cells. It however failed to induce significant cytotoxicity in prostate epithelial cells. In vivo, daily intraperitoneal injection of a single dose of SKI-178 potently inhibited PC-3 xenograft growth in nude mice. Contrarily, ectopic overexpression of SphK1 and SphK2, by lentiviral constructs, further increased promoted primary prostate cancer cell growth in vitro and in vivo.We here provided evidence that SphK inhibition should be the primary cause of SKI-178-induced activity in prostate cancer cells. SKI-178 largely inhibited SphK activity and induced ceramide production, without affecting SphK1/2 expression, in prostate cancer cells. K6PC-5, the SphK1 activator, or supplement with S1P, largely ameliorated SKI-178-induced cytotoxicity in primary prostate cancer cells. Significantly, shRNA-induced silencing of SphK1/2 not only mimicked SKI-178-induced actions in prostate cancer cells. More importantly, SKI-178 was unable to induce further prostate cancer cell apoptosis. Since SKI-178 can block both SphK1 and SphK2, it was not surprising to show that SKI-178-induced SphK inhibition and prostate cancer cell death were significantly more potent than the SphK1 inhibitor PF-543 and the SphK2 inhibitor ABC294640. The SphK1/2 dual inhibitor also failed to induce significant cytotoxicity in SphK1/2-low prostate epithelial cells. Further, SKI-178 failed to induce apparent toxicity to the nude mice. These results suggested that SKI-178 could be a promising therapeutic target for prostate cancer.Studies have shown that SphK1 inactivation/silencing could result in ceramide accumulation in cancer cells, which then inactivates Akt-mTOR cascade by activating phosphatases PP2A and PP1 , 55, 56.Sustained activation of JNK cascade could cause growth inhibition and apoptosis in prostate cancer cells . Shi et SphK1/2 inhibition by SKI-178 suppresses prostate cancer cell growth in vitro and in vivo.Original Data Fileaj-checklist FORMAuthor contribution FORM


Scientific Reportshttps://doi.org/10.1038/s41598-023-44257-6, published online 09 October 2023Correction to: The original version of this Article contained an error in the Materials and methods section, under the subheading ‘Animals’.Acomys cahirinus  95-220) were used for behavioral testing and 32 adult male A. cahirinus (PND 60-512) were used for immediate early gene (IEG) studies.”“Fourteen adult male now reads:Acomys cahirinus  95-220) were used for behavioral testing and 32 adult male A. cahirinus (PND 60-512) were used for immediate early gene (IEG) studies.”“Forty adult male The original Article has been corrected.