Osimertinib resistance limits the treatment of epidermal growth factor receptor-(EGFR)-mutated non-small-cell lung carcinoma (NSCLC). The mechanisms of osimertinib resistance need to be elucidated to determine alternative treatment strategies. This study explores the role of M2 type tumor-associated macrophage (TAM)-derived exosomal MSTRG.292666.16 in osimertinib resistance, and its related competing endogenous RNA (ceRNA) mechanism.M2 type TAMs were induced with 200 ng/mL phorbol 12-myristate 13-acetate, 20 ng/mL IL-4 and IL-13, and M2 type macrophage markers were measured by RT-qPCR. Next, the exosomes were isolated and characterized. Tumor formation in nude mice was conducted using H1975 cells under different treatment conditions. Small RNA sequencing was performed on exosomes derived from sensitive and resistant plasma, and ceRNA networks were constructed. Fluorescence in situ hybridization was used to observe the localization of MSTRG.292666.16, and a ceRNA network (MSTRG.292666.16-miR-6836-5p-MAPK8IP3) was selected for further validation.M2 type TAMs, and M2 type TAM-derived exosomes were successfully induced and isolated. Nude mice results showed that M2 type TAM-derived exosomes and MSTRG.292666.16 overexpression significantly increased tumor volume after administration of osimertinib for 4 weeks. M2 type TAMs were found in the resistant plasma, and MSTRG.292666.16 localized in the cytoplasm of H1975 cells. In addition, the genes in the ceRNA networks were significantly enriched in eight GO terms and seven KEGG pathways, including the MAPK signaling pathway. Subsequently, the levels of MSTRG.292666.16 and MAPK8IP3 significantly increased in both resistant plasma-derived exosomes and M2 type TAM-derived exosomes, while miR-6836-5p levels were significantly reduced. Finally, MSTRG.292666.16, miR-6836-5p, and MAPK8IP3 were part of the same network.M2 type TAM-derived exosomes promoted osimertinib resistance in NSCLC by regulating the MSTRG.292666.16/miR-6386-5p/MAPK8IP3 axis.The online version contains supplementary material available at 10.1186/s12935-022-02509-x. Lung cancer is the most common cancer worldwide and has the highest mortality rate . Non-smaExosomes are nanoscale lipid inclusions that can carry RNA and proteins and are an important system for intercellular signal transduction and substance delivery . PreviouCompeting endogenous RNA (ceRNA) can regulate the expression of downstream mRNAs by competing with shared miRNAs, and an imbalance of ceRNA networks influences cell processes and functions, thus leading to the occurrence and development of diseases , 16. In Therefore, in this study, we further confirmed the osimertinib resistance of M2 type TAM-derived exosomal lncRNA MSTRG.292666.16 to NSCLC in vivo. Moreover, small RNA sequencing of exosomes was performed and combined with the previous lncRNA sequencing data. Furthermore, the ceRNA mechanism of lncRNA MSTRG.292666.16 in NSCLC osimertinib resistance was investigated, and a ceRNA network of lncRNA MSTRG.292666.16-hsa-miR-6836-5p-MAPK8IP3 was selected for further validation. These results would provide new insights into the development of novel therapeutic targets and pathways to overcome osimertinib-acquired resistance in NSCLC.The patient samples were collected based on a previous study  with som2 at 37 °C. THP-1 cells were maintained in RPMI1640 medium (Gibco) with 10% FBS, and then used to induce the formation of M2 type TAM as previously described [Human lung cancer H1975 cells and THP-1 cells were obtained from the Cell Bank of the Chinese Academy of Sciences . The H1975 cells were cultured in Dulbecco’s modified of Eagle’s medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin (Gibco), and incubated in an incubator with 5% COg for 10 min, 2000×g for 30 min, and 10,000×g for 30 min. The supernatant was filtered using 0.22 μm filters , followed by centrifugation at 120,000×g for 70 min. The sediment, including the exosomes, was resuspended in 200 μL of PBS. For the isolation from the M2 type TAMs, the cells were centrifuged at 300×g for 10 min, 10,000×g for 30 min, and 100,000×g for 70 min. The sediment was resuspended in 1 mL PBS, and then centrifuged at 10,000×g for 60 min. The exosomes were resuspended in 200 μL PBS and stored at −80 °C until use.The exosomes were isolated from the plasma and M2 type TAMs using differential centrifugation under temperature conditions at 4 °C, as previously reported . The plaThe concentrations of the isolated exosomes were measured using a BCA protein assay kit . Then, a NanoSight NS300 particle size analyzer  was used to analyze the particle size of the exosomes based on the methods described by Soares et al. . Accordi2Fold change (FC)|> 1 and false discovery rate (FDR) < 0.05. Then, the Miranda and RNAhybrid algorithms (http://www.microrna.org) were used to predict the target genes of the identified DEmiRNAs. Afterwards, these screened DEmiRNAs were submitted for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. P < 0.05 was considered as the criterion for significantly enriched GO terms and KEGG pathways.The exosomes isolated from the plasma prior to osimertinib treatment (sensitive group) and their corresponding drug-resistant plasma (resistant group) were delivered to Yanzai Biotechnology (Shanghai) Co. Ltd  for small RNA sequencing . The clehttp://cbio.mskcc.org/miRNA2003/miranda.html) was used to screen the lncRNA-miRNA pair. Subsequently, the RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/) and miRanda (http://www.microrna.org/microrna/home.do) databases were used to predict the target genes of the aforementioned DEmiRNAs screened from the lncRNA-miRNA co-expression pairs, and the co-expression pairs of miRNA-mRNA with the opposite expression direction were retained. After that, ceRNA networks were established using lncRNA-miRNA-mRNA co-expression pairs and visualized using Cytoscape (version 3.7.0). Because exosomal lncRNA MSTRG.292666.16 has been reported to be associated with osimertinib resistance in NSCLC [A previous study by Deng et al.  reportedin NSCLC , we chos4 cells/well and cultured to a confluence of approximately 70%. The cells were fixed with 4% paraformaldehyde for 10 min at room temperature (RT). After washing with PBS, 1 mL of pre-cooled transparent liquid (PBS containing 0.5% Triton X-100) was added to each well and incubated at 4 °C for 5 min. After removing the transparent liquid and a washing step, a prehybridization solution (200 μL) was added and blocked at 37 °C for 30 min. After removing the prehybridization solution, 2.5 μL of 20 μM lncRNA MSTRG.292666.16 FISH probe were mixed into 100 μL hybrid solution, the mixture was added to the cells and incubated in the dark at 37 °C overnight. In the dark at 42 °C, the cells were washed with hybridization solutions I, II, III, and PBS. Finally, the cells were sealed with DAPI, and a fluorescence microscope  was used to observe the images.The localization of lncRNA MSTRG.292666.16 in H1975 cells was determined using a FISH assay kit  following the manufacturer’s protocols. The H1975 cells were seeded in a 24-well plate at a density of 5 × 105 cells/well and cultured overnight. The next day, the medium was changed to serum-free medium, and the cells were transfected with 3 μg pCDH vector and 3 μg pCDH-MSTRG.292666.16 using Lipofectamine 3000  according to the manufacturer’s instructions. After transfected for 6 h, the medium was replaced with complete medium and cultured for another 48. The level of MSTRG.292666.16 in the cells with different treatments was measured by RT-qPCR to evaluate the cell transfection efficiency. The sequences of MSTRG.292666.16 are shown in Table Our previous in vitro study found that exosomal lncRNA MSTRG.292666.16 was closely associated with osimertinib resistance in NSCLC. Therefore, in this study, we mainly explored its roles in vivo. We first constructed H1975 cells overexpressing lncRNA MSTRG.292666.16, as previously described . BrieflyThe sequences of WT/MUT MSTRG.292666.16 lncRNA inserts, miR-6836-5p mimics/negative control (NC) mimics, and WT/MUT MAPK8IP3 3′-untranslated regions (3′-UTRs) were synthesized and purchased from Yanzai Biotechnology (Shanghai) Co. Ltd. The psiCHECK2 vector (Yanzai Biotechnology (Shanghai) Co. Ltd) and pGL3 vector (Yanzai biotechnology (Shanghai) Co. Ltd) were used to generate the psiCHECK-MSTRG.292666.16 reporter plasmid (psiCHECK-MSTRG.292666.16 WT/MUT) and the 3′-UTR MAPK8IP3 reporter plasmid (pGL3-MAPK8IP3 WT/MUT), respectively. 293 T cells  were seeded into a 96-well plate and cultured overnight. The following day, the psiCHECK2-MSTRG.292666.16 WT/MUT (0.3 μg) or psiCHECK vector (0.3 μg), and pGL3-MAPK8IP3 WT/MUT (0.3 μg) or pGL3 vector (0.3 μg) were co-transfected into 293 T cells with NC mimics (100 nM) or miR-6836-5p mimics (100 nM) using Lipofectamine 3000 (Thermo Fisher Scientific) based on the manufacturer’s recommendations. After transfection for 6 h and culture for another 48 h, the cells were harvested to determine luciferase activity using a dual luciferase reporter system .GAPDH) served as a housekeeping gene. The RT-qPCR reaction was initiated at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The levels of lncRNA MSTRG.292666.16, miR-6836-5p, MAPK8IP3, and TAM-related markers  were calculated using the 2−ΔΔCt method. The sequences of all primers are listed in Table Total RNA was isolated from the plasma and M2 type TAMs using the mirVANA miRNA Isolation kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The quality and concentration of total RNA was assessed using a microplate reader. Total RNA was reverse transcribed into cDNA using the PrimeScriptTM II 1st Strand cDNA synthesis Kit , according to the manufacturer’s protocols. For miRNA, the stem loop RT-qPCR method was used, and U6 was used as a reference gene . For lnc7 cells/mL with PBS.Before animal experiments, H1975 cells were divided into four groups: control, H1975 + M2 type TAM-derived exosomes, H1975 + pCDH, and H1975 + pCDH-lncRNA groups. The cells in the H1975 + M2 type TAM-derived exosomes group were treated with 10 μg/mL M2 type TAM-derived exosomes, and the cells in the H1975 + pCDH and H1975 + pCDH-lncRNA groups were transfected with pCDH and pCDH-lncRNA, respectively. No treatment was applied for the cells in the control group. After that, the medium was removed, and the final concentration of cells in the different groups was adjusted to 2 × 107 cells/mL), 0.1 mL H1975 cells transfected with pCDH (2 × 107 cells/mL) and 0.1 mL H1975 cells transfected with pCDH-lncRNA (2 × 107 cells/mL), respectively. The mice in the control group were injected with PBS. On the 10th day of modeling, osimertinib (2.5 mg/kg/d) was intraperitoneally injected into each mouse for four weeks. The long and short diameters of the tumors were measured weekly to calculate the tumor volume. After four weeks, the mice were sacrificed by cervical dislocation, and the tumor was removed, weighed, and photographed. All animal experiments were performed in accordance with the National Medical Advisory Committee (NMAC) guidelines using approved procedures of the Institutional Animal Care and Use Committee at Shanghai Pulmonary Hospital.Twenty SPF BALB/C female nude mice aged 4–6 weeks were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. . All mice were maintained under controlled temperature (24 ± 2 °C) and humidity (50 ± 5%) conditions, with a 12 h light/dark cycle. The rats were given ad libitum access to food and water during the experiment. After 7 days of acclimatization, the mice were randomly divided into four groups (n = 5 for each group): control, M2 TAM-derived Exos, pCDH, and pCDH-lncRNA groups. The mice in the M2 TAM-derived Exos, pCDH, and pCDH-lncRNA groups were subcutaneously injected with 0.1 mL H1975 cells pre-treated with M2 type TAM-derived exosomes  followed by Tukey’s test was used to compare the differences among more than two groups. Statistical significance was set at P < 0.05.Data are expressed as mean ± standard deviation (SD), and GraphPad Prism 5  was used for all statistical analyses. Student’s CD206, CD163, TGF-β, IL-10, and Arg-1 were significantly increased in the M2 type TAMs compared with those in THP-1 cells  were measured. It was found that the expression levels of TEM, NTA, and western blot were used to identify the exosomes from THP-1 cells and successfully induced M2 phenotype in TAMs. TEM results showed that the exosomes isolated from the TPH-1 cells and M2 type TAMs were nearly round with a diameter of approximately 100 nm  and lncRNA MSTRG.292666.16 overexpression (P < 0.0001) pre-treatment  and 7 KEGG pathways  were significantly enriched ; while in the psiCHECK2-lncRNA MUT plasmid, the luciferase activity was significantly increased ; however, after MAPK8IP3 mutation, the relative luciferase activity was significantly enhanced , “terpenoid backbone biosynthesis” , and “MAPK signaling pathway” . ICMT has been reported to facilitate the growth, migration, and doxorubicin resistance of hepatocellular carcinoma via various carcinogenic pathways [OGDH, a subunit of the OGDH complex, is involved in the TCA cycle, energy balance, and cellular signaling. Lu et al. [OGDH, which serves as a positive regulator of gastric cancer progression, enhances mitochondrial function, and activates the Wnt/β-catenin signaling pathway. TKT was upregulated in the NSCLC cells, and its knockdown strengthened the action of gefitinib in NSCLC cells [ZMPSTE24 accelerates senescence associated with cancer [RPS6KA1, CACNA1B, and MAPK8IP3 were significantly enriched in the MAPK signaling pathway. Fan et al. [ICMT, OGDH, TKT, ZMPSTE24, RPS6KA1, CACNA1B, and MAPK8IP3, as well as pathways for biosynthesis of antibiotics, terpenoid backbone biosynthesis, and MAPK signaling pathway may play important roles in osimertinib resistance in NSCLC.Previous studies have shown that ceRNAs influence the progression of liver cancer, and cytoplasmic lncRNAs can function as ceRNAs regulating mRNA stabilization or translation by sponging miRNAs, and thus affecting signaling pathways , 39. In pathways . OGDH, au et al.  showed tLC cells . Loss ofh cancer . The MAPh cancer , and ourn et al.  demonstrRPS6KA1, CACNA1B and MAPK8IP3 were significantly enriched in “MAPK signaling pathway”, and lncRNA MSTRG.292666.16 was the hub node. Therefore, a ceRNA network of lncRNA MSTRG.292666.16- miR-6836-5p-MAPK8IP3 was chosen for further validation. The levels of MSTRG.292666.16 and MAPK8IP3 were significantly increased in resistant plasma-derived exosomes and M2 type TAM-derived exosomes, while miR-6836-5p levels were significantly evidently reduced, which was consistent with the expression trend of ceRNA mechanisms. The dual-luciferase reporter gene assay indicated that MSTRG.292666.16, miR-6836-5p, and MAPK8IP3 could interact with each other. A previous study reported that HOTAIR was the target of miR-331-3p, and HER2 was the target of miR-331-3p through a luciferase assay, which suggested that lncRNA HOTAIR could regulate HER2 expression by sponging miR-331-3p in gastric cancer [The MAPK signaling pathway has been reported to be activated in cancer drug resistant samples, and its abnormal activation could contribute to cell loss of differentiation and apoptosis , and couc cancer . Anotherc cancer . TherefoHowever, this study has some limitations. First, the specific ceRNA mechanism of MSTRG.292666.16/miR-6836-5p/MAPK8IP3 through M2 type TAM-derived exosomes in osimertinib-resistant NSCLC needs to be further investigated in vitro. Moreover, a larger sample size is needed to verify our conclusions, and further experiments should be conducted to investigate the effects of the identified genes and pathways on osimertinib resistance in NSCLC.ICMT, OGDH, TKT, ZMPSTE24, RPS6KA1, CACNA1B, and MAPK8IP3, as well as pathways for biosynthesis of antibiotics, terpenoid backbone biosynthesis, and MAPK signaling pathway may play important roles in osimertinib resistance in NSCLC. Additionally, M2 type TAM-derived exosomes may promote osimertinib resistance in NSCLC via the MSTRG.292666.16/miR-6836-5p/MAPK8IP3 axis. Our work illuminated a novel mechanism of osimertinib resistance in NSCLC, and implied that M2 type TAM-derived exosomal MSTRG.292666.16/ miR-6836-5p/ MAPK8IP3 serves as a novel therapeutic target and may have potential therapeutic value in treating osimertinib resistance in NSCLC.In conclusion, through sequencing, it was found that Additional file 1: Table S1. All differentially expressed miRNAs between the exosomes isolated from the sensitive and resistant plasma with the thresholds of |log2Fold change (FC)|> 1 and false discovery rate (FDR) < 0.05.


The Ad26.COV2.S vaccine expresses a stabilized spike protein from the WA1/2020 strain of SARS-CoV-2, and has recently demonstrated protective efficacy against symptomatic COVID-19 in humans in several geographical regions—including in South Africa, where 95% of sequenced viruses in cases of COVID-19 were the B.1.351 variant3. Here we show that Ad26.COV2.S elicits humoral and cellular immune responses that cross-react with the B.1.351 variant and protects against B.1.351 challenge in rhesus macaques. Ad26.COV2.S induced lower binding and neutralizing antibodies against B.1.351 as compared to WA1/2020, but elicited comparable CD8 and CD4 T cell responses against the WA1/2020, B.1.351, B.1.1.7, P.1 and CAL.20C variants. B.1.351 infection of control rhesus macaques resulted in higher levels of virus replication in bronchoalveolar lavage and nasal swabs than did WA1/2020 infection. Ad26.COV2.S provided robust protection against both WA1/2020 and B.1.351, although we observed higher levels of virus in vaccinated macaques after B.1.351 challenge. These data demonstrate that Ad26.COV2.S provided robust protection against B.1.351 challenge in rhesus macaques. Our findings have important implications for vaccine control of SARS-CoV-2 variants of concern.The emergence of SARS-CoV-2 variants that partially evade neutralizing antibodies poses a threat to the efficacy of current COVID-19 vaccines SARS-CoV-2 challenge of rhesus macaques demonstrates that the Ad26.COV2.S vaccine induces robust protection against both the WA1/2020 isolate and the B.1.351 variant of concern. Ad26.COV2.S is a replication-incompetent human adenovirus type 26 vector8 that expresses a prefusion stabilized SARS-CoV-2 spike protein (S)10 from the Wuhan 2019 strain of SARS-CoV-2. It was previously reported that Ad26.COV2.S demonstrated protective efficacy against SARS-CoV-2 WA1/2020 challenges in hamsters and nonhuman primates13, and also showed safety and immunogenicity in humans15. A recent phase III efficacy trial has shown that Ad26.COV2.S provided 86%, 88% and 82% protection against severe COVID-19 disease by day 28 after vaccination in the USA, Brazil and South Africa, respectively3.SARS-CoV-2 variants of concern have shown increased transmissibility and pathogenicity in humansn = 6 macaques per group) as follows: groups 1 and 3 received a sham vaccine (sham control macaques), and groups 2 and 4 received a single immunization with 5 × 1010 viral particles of Ad26.COV2.S; after vaccination, groups 1 and 2 were challenged with the original SARS-CoV 2 strain WA1/2020, and groups 3 and 4 were challenged with the SARS-CoV-2 variant B.1.351.We developed a B.1.351 challenge stock by expansion of a seed stock  in Calu-3 cells (ATCC HTB-55). We immunized 24 rhesus macaques in 4 experimental groups . Live-virus neutralizing antibody assays19 showed a greater reduction of neutralizing antibody titres against B.1.351 . We also used an electrochemiluminescence assay (ECLA)20 to evaluate S- and RBD-specific binding antibody responses to WA1/2020, B.1.1.7, P.1. and B.1.351 -specific enzyme-linked immunosorbent assay (ELISA) titres in macaques that received Ad26.COV2.S vaccine were less than 25 at week 0, and were 4,050, 3,186 and 805 against the WA1/2020, B.1.1.7 and B.1.351 strains, respectively, in Ad26.COV2.S-vaccinated macaques at week 6 Fig. . These dses Fig. .+ and CD4+ T cell responses using multi-parameter intracellular cytokine staining assays at week 6  assays in peripheral blood mononuclear cells at week 4. ELISPOT responses were comparable among the WA1/2020, B.1.351, B.1.1.7, P.1 and CAL.20C strains, with no evidence of decreased responses against the variants Fig. . We alsoins Fig. . Similarnts Fig. . These d5 50% tissue culture infectious dose (TCID50) of SARS-CoV-2 WA1/202022 or B.1.351 by the intranasal and intratracheal routes. We assessed viral loads in bronchoalveolar lavage (BAL) and nasal swabs by reverse-transcription PCR (RT–PCR) specific for subgenomic mRNA (sgRNA), which is believed to measure replicating virus24. All sham control macaques were infected and showed higher median peak sgRNA of 6.16 (range of 4.93–6.80) log10(sgRNA copies per ml) in BAL for B.1.351, as compared to 4.80 (range of 4.70–5.52) log10(sgRNA copies per ml) for WA1/2020  in BAL for B.1.351, as compared with less than 1.69 (range of <1.69 to 3.23) log10(sgRNA copies per ml) in BAL for WA1/2020  in nasal swabs for B.1.351, as compared with 5.48 (range of 4.44–6.00) log10(sgRNA copies per swab) for WA1.2020  in nasal swabs for B.1.351, as compared with 2.64 (range of <1.69 to 3.89) log10(sgRNA copies per swab) in nasal swabs for WA1/2020   Fig. . By day ts) Fig. . Ad26.COOn day 10 after challenge (study week 8), sham control macaques developed both humoral and cellular immune responses, as expected  in BAL  that lead to partial evasion of natural and vaccine-elicited neutralizing antibodies7. Binding and neutralizing antibody titres were suppressed 4–5-fold against B.1.351 as compared to WA1/2020, but Fc functional antibody responses were affected less, and T cell responses were not affected at all by the SARS-CoV-2 variants. Ad26.COV2.S provided robust protection against both high-dose WA1/2020 and B.1.351 challenges. These data have important implications for the potential utility of current vaccines and inform boosting strategies against SARS-CoV-2 variants of concern.It has previously been reported that Ad26.COV2.S provided robust protection against challenge with SARS-CoV-2 WA1/2020 in both rhesus macaques and hamsters3. Robust protection was observed in all geographical regions, with similar levels of protection against severe COVID-19 disease regardless of variant, including in the USA, in Brazil (where 69% of cases with sequence data were the P.2 variant) and in South Africa (where 95% of cases with sequence data were the B.1.351 variant). In the current study in macaques, B.1.351 infection led to a higher magnitude of and more prolonged viral replication in the upper and lower respiratory tracts than did WA1/2020. Nevertheless, Ad26.COV2.S provided robust protection against both viruses, although levels of virus in BAL and nasal swabs were higher after B.1.351 challenge than after WA1/2020 challenge.Our data are consistent with findings in humans in a recent phase III clinical trial of Ad26.COV2.S that was conducted in the USA, Latin America  and South Africa22.To the best of our knowledge, this is the first report of a SARS-CoV-2 vaccine evaluated for efficacy against a SARS-CoV-2 variant of concern in macaques. Several SARS-CoV-2 vaccines have previously been reported to protect against homologous WA1/2020 challenges, but have not yet been reported against B.1.351 challenges. Our study does not define mechanistic correlates of protection against SARS-CoV-2 variants, but it has previously been reported that IgG was sufficient for protection against homologous SARS-CoV-2 challenge in macaques and that CD8 T cell responses also contributed to protection if antibody titres were subprotectiveIn conclusion, Ad26.COV2.S induced cross-reactive humoral and cellular immune responses and provided robust protection against the heterologous SARS-CoV-2 variant B.1.351 in rhesus macaques. Future studies will determine whether Ad26.COV2.S, as well as other vaccines, protect against other SARS-CoV-2 variants of concern.No statistical methods were used to predetermine sample size. Macaques were randomized into groups. All immunological, virological and histopathological studies were performed blinded.Macaca mulatta) (3–11 years old) were randomly allocated to groups. All macaques were housed at Bioqual. Macaques received a single immunization of 5 × 1010 viral particles of Ad26.COV2.S (n = 12) or sham (n = 12) by the intramuscular route without adjuvant at week 0. At week 6, all macaques were challenged with 5 × 105 TCID50 SARS-CoV-2 from strains USA-WA1/2020  (which was grown in VeroE6 cells and deep sequenced as previously described16) or B.1.351 . The B.1.351 stock was grown in Calu-3 cells and was deep-sequenced, which confirmed the expected sequence identity with no mutations in the S greater than 2.5% frequency and no mutations elsewhere in the virus at greater than 13% frequency. Virus was administered as 1 ml by the intranasal route (0.5 ml in each nare) and 1 ml by the intratracheal route. All immunological, virological and histopathological studies were performed blinded. Animal studies were conducted in compliance with all relevant local, state and federal regulations and were approved by the Bioqual Institutional Animal Care and Use Committee.Twenty-four outbred Indian-origin adult male and female rhesus macaques , luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene) and S expressing pcDNA3.1-SARS CoV-2 SΔCT of variants were co-transfected into HEK293T cells by lipofectamine 2000 (ThermoFisher). Pseudoviruses of SARS-CoV-2 variants were generated by using WA1/2020 strain , D614G mutation, B.1.1.7 variant (GISAID accession identifier EPI_ISL_601443) or B.1.351 variant (GISAID accession identifier EPI_ISL_712096). The supernatants containing the pseudotype viruses were collected 48 h after transfection, and were purified by centrifugation and filtration with a 0.45-μm filter. To determine the neutralization activity of the plasma or serum samples from participants, HEK293T cells expressing human ACE2 (HEK293-hACE2 cells) were seeded in 96-well tissue culture plates at a density of 1.75 × 104 cells per well overnight. Threefold serial dilutions of heat-inactivated serum or plasma samples were prepared and mixed with 50 μl of pseudovirus. The mixture was incubated at 37 °C for 1 h before adding to HEK293T-hACE2 cells. Forty-eight hours after infection, cells were lysed in Steady-Glo Luciferase Assay (Promega) according to the manufacturer’s instructions. SARS-CoV-2 neutralization titres were defined as the sample dilution at which a 50% reduction in relative light units (RLU) was observed relative to the average of the virus control wells.The SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were generated essentially as previously described19. One day before the assay, Vero E6 USAMRID cells were plated at 20,000 cells per well in clear-bottom black-walled plates. Cells were inspected to ensure confluency on the day of assay. Serum samples were tested at a starting dilution of 1:20 and were serially diluted threefold up to nine dilution spots. Serially diluted serum samples were mixed in equal volume with diluted virus. Antibody–virus and virus-only mixtures were then incubated at 37 °C with 5% CO2 for 1 h. After incubation, serially diluted sera and virus-only controls were added in duplicate to the cells at 75 plaque-forming units at 37 °C with 5% CO2. Twenty-four hours later, the cells were lysed, and luciferase activity was measured via Nano-Glo Luciferase Assay System (Promega) according to the manufacturer specifications. Luminescence was measured by a Spectramax M3 plate reader (Molecular Devices). Virus neutralization titres were defined as the sample dilution at which a 50% reduction in RLU was observed relative to the average of the virus control wells.Full-length SARS-CoV-2 WA1/2020, B.1.351 and B.1.1.7, viruses were designed to express nanoluciferase (nLuc) and were recovered via reverse genetics17. In brief, 96-well plates were coated with 0.5 μg ml−1 RBD protein in 1× DPBS and incubated at 4 °C overnight. After incubation, plates were washed once with wash buffer (0.05% Tween-20 in 1× DPBS) and blocked with 350 μl casein block per well for 2–3 h at room temperature. After incubation, block solution was discarded and plates were blotted dry. Serial dilutions of heat-inactivated serum diluted in casein block were added to wells and plates were incubated for 1 h at room temperature, before three further washes and a 1-h incubation with a 1 μg ml−1 dilution of anti-macaque IgG HRP (Nonhuman Primate Reagent Resource) at room temperature in the dark. Plates were then washed three times, and 100 μl of SeraCare KPL TMB SureBlue Start solution was added to each well; plate development was halted by the addition of 100 μl SeraCare KPL TMB Stop solution per well. The absorbance at 450 nm was recorded using a VersaMax microplate reader. For each sample, ELISA endpoint titre was calculated in GraphPad Prism software, using a four-parameter logistic curve fit to calculate the reciprocal serum dilution that yields an absorbance value of 0.2 at 450 nm. log10-transformed endpoint titres are reported.WA1/2020, B.1.1.7 and B.1.351 RBD-specific binding antibodies were assessed by ELISA essentially as previously described20. The antigens included were WA1/2020, B.1.1.7, P.1, and B.1.351 S and RBD. The plates were blocked with 50 μl of blocker A (1% BSA in MilliQ water) solution for at least 30 m at room temperature shaking at 700 rpm with a digital microplate shaker. During blocking, the serum was diluted 1:5,000 in diluent 100. The plates were then washed 3 times with 150 μl of the MSD kit wash buffer, blotted dry, and 50 μl of the diluted samples were added in duplicate to the plates and set to shake at 700 rpm at room temperature for at least 2 h. The plates were again washed 3 times and 50 μl of SULFO-tagged anti-human IgG detection antibody  diluted to 1× in diluent 100 was added to each well and incubated shaking at 700 rpm at room temperature for at least 1 h. Plates were then washed 3 times and 150 μl of MSD GOLD read buffer B was added to each well and the plates were read immediately after on a MESO QuickPlex SQ 120 machine. MSD titres for each sample are reported as RLUs, which were calculated as sample RLU minus blank RLU for each spot for each sample. The limit of detection was defined as 1,000 RLU for each assay.ECLA plates  were designed and produced with up to nine antigen spots in each well, and assays were performed essentially as previously described21. In brief, fluorescent beads (LifeTechnologies) were coupled via carboxy-coupling, and plasma were added, allowing immune complex formation, excess antibodies were washed away, followed by the addition of THP1 monocytes, primary neutrophils or guinea pig complement, individually, respectively. The level of phagocytosis and complement deposition was assessed by flow cytometry.Fc functional profiling included the assessment of antibody-dependent monocyte phagocytosis and antibody-dependent complement deposition17. Peptide pools consisted of 15 amino acid peptides overlapping by 11 amino acids spanning the SARS-CoV-2 S from the WA1/2020 strain or variant strains. ELISPOT plates were coated with mouse anti-human IFNγ monoclonal antibody from BD Pharmigen at 5 μg per well and incubated overnight at 4 °C. Plates were washed with DPBS wash buffer (DPBS with 0.25% Tween-20), and blocked with R10 medium (RPMI with 10% heat-inactivated FBS with 1% of 100× penicillin–streptomycin) for 1–4 h at 37 °C. SARS-CoV-2 peptides  were prepared and plated at a concentration of 1 μg per well, and 200,000 cells per well were added to the plate. The peptides and cells were incubated for 18–24 h at 37 °C. All steps after this incubation were performed at room temperature. The plates were washed with ELISPOT wash buffer (11% 10× DPBS and 0.3% Tween-20 in 1 l MilliQ water) and incubated for 2 h with rabbit polyclonal anti-human IFNγ biotin from U-Cytech (1 μg ml−1). The plates were washed a second time and incubated for 2 h with streptavidin–alkaline phosphatase from Southern Biotech (2 μg ml−1). The final wash was followed by the addition of nitor-blue tetrazolium chloride or 5-bromo-4-chloro 3′ indolyphosphate p-toludine salt (NBT/BCIP chromagen) substrate solution for 7 min. The chromagen was discarded and the plates were washed with water and dried in a dim place for 24 h. Plates were scanned and counted on a Cellular Technologies Limited Immunospot Analyzer.Pooled peptide ELISPOT assays were performed essentially as previously described17. Peptide pools consisted of 15 amino acid peptides overlapping by 11 amino acids spanning the SARS-CoV-2 S from the WA1/2020 strain or variant strains. Then, 106 PBMCs per well were resuspended in 100 μl of R10 medium supplemented with CD49d monoclonal antibody (1 μg ml−1). Each sample was assessed with mock , peptide pools (2 μg ml−1), or 10 pg ml−1 phorbol myristate acetate and 1 μg ml−1 ionomycin (Sigma-Aldrich)  and incubated at 37 °C for 1 h. After incubation, 0.25 μl of GolgiStop and 0.25 μl of GolgiPlug in 50 μl of R10 was added to each well and incubated at 37 °C for 8 h and then held at 4 °C overnight. The next day, the cells were washed twice with DPBS, stained with near-IR live/dead dye for 10 min and then stained with predetermined titres of monoclonal antibodies against CD279 , CD38 , CD28 , CD4 , CD95  and CD8 , for 30 min. Cells were then washed twice with 2% FBS in DPBS buffer and incubated for 15 min with 200 μl of BD CytoFix/CytoPerm Fixation/Permeabilization solution. Cells were washed twice with 1× Perm Wash buffer (BD Perm/Wash Buffer 10× in the CytoFix/CytoPerm Fixation/Permeabilization kit diluted with MilliQ water and passed through a 0.22-μm filter) and stained with intracellularly with monoclonal antibodies against Ki67 , CD69 , IL-10 , IL-13 , TNF , IL-4 , IFNγ , CD45 , IL-2  and CD3 , for 30 min. Cells were washed twice with 1× Perm Wash buffer and fixed with 250 μl of freshly prepared 1.5% formaldehyde. Fixed cells were transferred to a 96-well round-bottom plate and analysed by BD FACSymphony system. Central memory T cells were defined as CD28+CD95+ T cells. Data were analysed with FlowJo v.9.9.Multi-parameter pooled-peptide intracellular cytokine staining assays were performed essentially as previously describedE gene sgRNA was assessed by RT–PCR using primers and probes as previously described24. A standard was generated by first synthesizing a gene fragment of the subgenomic E gene23. The gene fragment was subsequently cloned into a pcDNA3.1+ expression plasmid using restriction site cloning (Integrated DNA Techonologies). The insert was in vitro-transcribed to RNA using the AmpliCap-Max T7 High Yield Message Maker Kit (CellScript). log dilutions of the standard were prepared for RT–PCR assays ranging from 1 × 1010 copies to 1 × 10−1 copies. Viral loads were quantified from BAL fluid and nasal swabs. RNA extraction was performed on a QIAcube HT using the IndiSpin QIAcube HT Pathogen Kit according to manufacturer’s specifications (Qiagen). The standard dilutions and extracted RNA samples were reverse-transcribed using SuperScript VILO Master Mix (Invitrogen) following the cycling conditions described by the manufacturer, 25 °C for 10 min, 42 °C for 1 h, then 85 °C for 5 min. A Taqman custom gene expression assay (Thermo Fisher Scientific) was designed using the sequences targeting the E gene sgRNA23. The sequences for the custom assay were as follows, forward primer, sgLeadCoV2.Fwd: CGATCTCTTGTAGATCTGTTCTC, E_Sarbeco_R: ATATTGCAGCAGTACGCACACA, E_Sarbeco_P1 (probe): VIC-ACACTAGCCATCCTTACTGCGCTTCG-MGB. These primers and probes were equally reactive for both variants. Reactions were carried out in duplicate for samples and standards on the QuantStudio 6 and 7 Flex Real-Time PCR Systems (Applied Biosystems) with the thermal cycling conditions, initial denaturation at 95 °C for 20 s, then 45 cycles of 95 °C for 1 s and 60 °C for 20 s. Standard curves were used to calculate sgRNA copies per ml or per swab; the quantitative assay sensitivity was 50 copies per ml or per swab.SARS-CoV-2 2. Medium was aspirated and replaced with 180 μl of DMEM with 2% FBS and gentamicin. Serial dilution of samples as well as positive (virus stock of known infectious titre) and negative (medium only) controls were included in each assay. The plates are incubated at 37 °C, 5.0% CO2 for 4 days. Cell monolayers were visually inspected for cytopathic effect. The TCID50 was calculated using the Read–Muench formula.Vero TMPRSS2 cells (obtained from A. Creanga) were plated at 25,000 cells per well in DMEM with 10% FBS and gentamicin, and the cultures were incubated at 37 °C, 5.0% COLungs on day 10 after SARS-CoV-2 challenge were evaluated by histopathology. At the time of fixation, lungs were suffused with 10% formalin to expand the alveoli. All tissues were fixed in 10% formalin and blocks sectioned at 5 μm. Slides were incubated for 30–60 min at 65 °C then deparaffinized in xylene and rehydrated through a series of graded ethanol to distilled water. Sections were stained with haematoxylin and eosin. Blinded evaluation and scoring was performed by a board-certified veterinary pathologist (A.J.M.).P values of less than 0.05 were considered significant.Comparisons of virological, immunological and histopathological data were performed using GraphPad Prism 8.4.2 (GraphPad Software). Comparison of data between groups was performed using two-sided Wilcoxon rank-sum tests. Correlation analyses were performed using two-sided Spearman rank-correlation tests. Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-021-03732-8.Supplementary Figure 1Sample Raw Flow Cytometry Data.Reporting Summary


Trastuzumab emtansine (T-DM1) is a second-line standard therapy for patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. Evidence regarding post–T-DM1 treatments is currently lacking. We evaluated the effectiveness of post–T-DM1 drug therapy in patients with HER2-positive, unresectable and/or metastatic breast cancer.In this multicenter, retrospective, observational study, real-world clinical data of female patients with HER2-positive breast cancer who had a history of T-DM1 treatment were consecutively collected from five sites in Japan. We investigated the effectiveness of post–T-DM1 therapy by evaluating the real-world progression-free survival (rwPFS), time to treatment failure (TTF), overall survival (OS), objective response rate (ORR), and clinical benefit rate (CBR). Tumor response was assessed by investigators according to Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) guidelines. Subgroup and exploratory analyses according to background factors were also undertaken.Of the 205 patients who received T-DM1 treatment between 1 January 2014 and 31 December 2018, 128 were included in this study. Among the 128 patients analyzed, 105 (82%) patients received anti-HER2 therapy and 23 (18%) patients received regimens without anti-HER2 therapy. Median  rwPFS, TTF, and OS were 5.7 (4.8–6.9) months, 5.6 (4.6–6.4) months, and 22.8 (18.2–32.4) months, respectively. CBR and ORR (95% CI) were 48% (38.8–56.7) and 23% (15.1–31.4), respectively. Cox-regression analysis showed that an ECOG PS score of 0, a HER2 immunohistochemistry score of 3+, recurrent type, ≥12 month duration of T-DM1 therapy, and anti-HER2 therapy were independent variables for rwPFS. An exploratory subgroup analysis of regimens after T-DM1 showed that those with anti-HER2 therapy had a median rwPFS of 6.3 and those without anti-HER2 therapy had a median rwPFS of 4.8 months.In the real-world setting in Japan, several post–T-DM1 regimens for patients with unresectable and/or metastatic HER2-positive breast cancer, including continuation of anti-HER2 therapy, showed some effectiveness; however, this effectiveness was insufficient. Novel therapeutic options are still needed for further improvement of PFS and OS in later treatment settings.UMIN000038296; registered on 15 October 2019.The online version contains supplementary material available at 10.1186/s12885-021-08504-1. HER2) is a growth factor receptor gene that is amplified in approximately 15–20% of breast cancers, and HER2 protein overexpression on the plasma membrane of tumor cells reportedly correlates with a poor prognosis ), and 65% (n = 83) were hormone receptor-positive. Recurrent disease was reported for 64% (n = 82) of patients , and 36% (n = 46) had de novo disease . Regarding prior anti-HER2 therapies before T-DM1 , trastuzumab, pertuzumab, and lapatinib had been received by 94% (n = 120), 56% (n = 72), and 28% (n = 36) of patients, respectively. Regarding prior chemotherapy, 84% (n = 108) had received taxane-based therapy and 50% (n = 64) had received anthracycline-based therapy (Table Table n = 89), skin/subcutaneous soft tissue/lymph node , bone , and CNS . Overall, characteristics in the population with measurable lesions were similar to those in the total analysis population.At the start of post–T-DM1 treatment, 67 patients (52%) had an ECOG PS score of 0, 25 patients (20%) had a score of 1, nine patients (7%) had a score of ≥2, and 27 patients (21%) had an unknown score. The metastatic sites at post–T-DM1 treatment initiation  were as follows: visceral  are shown in Table The median (range) follow-up time was 15.5 (0.5–57.4) months. The median rwPFS  was 5.7 months  Fig. A and theSubgroup analysis revealed that rwPFS was longer (per the 95% CI) in patients with the following characteristics: an ECOG PS score of 0 (6.7 months [95% CI: 5.4–7.4]) vs an ECOG PS score of ≥1 (3.9 months [95% CI: 2.1–5.8]), HER2 IHC 3+ (6.2 months [95% CI: 5.1–7.1]) vs IHC 2+/ISH+ and IHC unknown/ISH+ (3.9 months [95% CI: 2.1–6.7]), no history of pertuzumab treatment (7.1 months [95% CI: 5.7–7.9]) vs history of pertuzumab treatment (4.9 months [95% CI: 4.0–5.8]), and anti-HER2 therapy (6.3 months [95% CI: 5.1–7.2]) vs regimens without anti-HER2 therapy (4.8 months [95% CI: 1.9–5.9]). Other subgroup outcomes of TTF, OS, ORR, and CBR are shown in Additional File p = 0.004, Fig. p = 0.058) , recurrent type , a HER2 immunohistochemistry score of 3+ , more than 12 months duration of T-DM1 treatment , and anti-HER2 therapy  were identified as independent variables for rwPFS in both analyses  the proportion of patients with liver metastasis, which is considered high risk at the time of initial metastasis diagnosis, was high; and (2) a high proportion of patients had brain metastasis at the time that post–T-DM1 treatment was initiated. Our study did not collect data related to tumor size or other factors, so further exploration of this is needed.An ECOG PS of 0 (vs ≥ 1) and tumors that were IHC3+ for HER2 (vs IHC2+/ISH+ and IHC unknown/ISH+) have been reported as prognostic factors , 37. It This study had several limitations. As with any retrospective observational study, we relied on accurate record-keeping from treating physicians. Additionally, the sample size was limited and the study only included data from Japanese patients at five study sites; therefore, the results may not be entirely representative of the general population, potentially limiting their generalizability. However, as mentioned above, these data were collected at core cancer treatment hospitals. The data presented herein are specific to the real-world setting in Japan and these findings should be interpreted carefully in the case of real-world settings in other countries. Larger studies would be useful to confirm these findings and to expand on the subgroup analyses.We conclude that the results of this study represent the real-world treatment patterns and outcomes of post–T-DM1 therapy in Japan for patients with unresectable and/or metastatic HER2-positive breast cancer. Our results showed that continuation of anti-HER2 treatment and higher expression of HER2 were important factors for longer rwPFS, even in later lines of therapy for patients with HER2-positive breast cancer. However novel therapeutic options are still needed to further improve both PFS and OS in the real world. Future studies clarifying the real-world treatment situation for new anti-HER2 therapies for HER2-positive breast cancer are expected.Additional file 1: Supplementary Text 1. Study outcomes. Supplementary Table 1. Patient demographic characteristics . Supplementary Table 2. Analysis of outcomes according to subgroup.


The tracer was produced with a decay corrected yield of 41.7 ± 16.5% and had a molar activity of 65.4 ± 30.3 GBq/μmol. The tracer was shown to be stable in mouse serum at 60 min. To test the new tracer, HCT116 colorectal carcinoma cells were engineered to overexpress human AF. In vitro evaluation revealed 3.5-fold higher uptake in HCT116AF cells compared to HCT116 controls (26.4 ± 7.8 vs. 7.5 ± 1.0 kBq/106 cells). Static PET scans 50 min post injection revealed 2.5-fold higher tracer uptake in the HCT116AF tumors (3.0 ± 0.8%ID/cc (n = 6)) compared with the controls (1.2 ± 0.8 (n = 5)). Dynamic scans showed higher uptake in the HCT116AF tumors at all time-points (n = 2). Ex vivo analysis of the tumors, utilizing fluorescent DDK2 antibodies, confirmed the expression of human AF in the HCT116AF xenografts. We have developed a novel PET tracer to image AF in vivo and will now apply this to relevant disease models.The acidic hydrolase α-fucosidase (AF) is a biomarker for maladies such as cancer and inflammation. The most advanced probes for α-fucosidase are unfortunately constrained to ex vivo or in vitro applications. The in vivo detection and quantification of AF using positron emission tomography would allow for better discovery and diagnosis of disease as well as provide better understanding of disease progression. We synthesized, characterized, and evaluated a radiolabeled small molecule inhibitor of AF based on a known molecule. The radiosynthesis involved the  Tools that can non-invasively detect metabolic alterations in vivo provide us with unique and profound opportunities in personalized medicine and offer insight into the cause, progression, and treatment of disease. Positron emission tomography (PET) is an imaging modality that offers high sensitivity and good spatial resolution, relying on the administration of radiolabeled biologically active molecules that can be detected in trace quantities. Through the use of PET probes, biological processes can be interrogated without effecting changes in the system being evaluated.Helicobacter pylori infection CH3I to produce an isotopically labeled probe with proven pharmacology. There are many reported antagonists of AF A with vahibitors ,9,10. Athibitors . The imp11C]3 and evaluate its potential for detecting AF expression in vivo using PET.Given the wide range of pathologies associated with changes in AF expression, a radiolabeled small molecule inhibitor of AF was envisaged, which could be used to probe AF expression in preclinical and clinical disease models. Compared to a substrate, which is useful for quantifying enzyme activity, the radiolabeled inhibitor binds tightly to its associated enzyme, allowing one to not only show localization of AF, but also provide quantitative information about enzyme expression at the protein level. The aim of this study was to establish a robust and rapid synthesis of +(theor.) = 238.31). The removal of the acetonide protection has been described as problematic, requiring a slow hydrolysis in 1 M HCl . Owing totection A. The de’ method . Applyin’ method , this waonitrile A, followonitrile B reveale11CH3I methylation was nontrivial, with many conventional bases being found to be unsuitable for the radiolabeling. Cs2CO3, K2CO3, and 1,8-Diazabicyclo(5.4.0)undec-7-ene (DBU) all yielded none of the desired radiolabeled intermediate 11, while NaOH produced only trace amounts. Although phenols are readily methylated, this was not found to be the case with our precursor. Instead of directly labeling the phenol, our approach relied on methylating an in situ generated phenoxide 9, which was produced through room temperature ultrasonication of 10 with NaH in acetonitrile for 5 min . To avoid the associated time penalties and hassle of further concentration steps, we aimed to find a biocompatible HPLC eluent so that the tracer could be used directly for imaging. Luckily, this was made possible by using a 10% solution of ethanol in PBS (buffered to pH 6.0). This afforded clean separation of the tracer from the precursor, which had respective retention times of 3.3 and 6.3 min. The tracer was produced with an overall decay-corrected yield of 41.7 ± 16.5% (n = 5), calculated from the MeI delivered to the reactor of the FX M module. The molar activity was determined to be 65.4 ± 30.3 GBq/μmol (n = 14). The identity of the tracer was confirmed by comparing its HPLC retention with that of non-radioactive 3 ) compared with the controls (1.2 ± 0.8 (n = 5)). Furthermore, dynamic scans of [11C]3 (n = 2).Although this work was primarily focused on tracer development, we evaluated the tracer in BALB/c nu/nu mice bearing HCT116AF (right) and HCT116 (left) xenografts. The xenograft model was complicated by the slow growth of the HCT116AF xenografts; static PET scans 50 min post injection revealed visibly higher activity in the transfected tumors compared to the controls A. Quantif [11C]3 C were meOwing to the complexity of the in vivo tumor growth and the primary focus on novel chemistry, blocking experiments (to assess specificity toward AF) were only performed in two mice D. Althou13C) spectrometer . NMR experiments were carried out in chloroform-d. Chemical shifts (δ) were recorded relative to residual chloroform (δ = 7.26 in 1H NMR and δ = 77.00 in 13C NMR). All chemical shifts are reported in ppm. High resolution mass spectroscopy (HRMS) was performed using electron spray ionization (ESI) and a MAXI4G spectrometer . The accuracy, as designated in PPM, must be <2.00 from predicted mass. LCMS was performed using an Agilent 1200 series system and a 6120-quadrupole mass spectrometer , with a reverse-phase C18(2) analytical column  100u, 250 × 4.6 mm, 5 μm). The mobile phase comprised CH3CN and water (+0.1% trifluoroacetic acid) and the flow rate was 1 mL/min. The standard gradient program started with 5% CH3CN for 2 min. The CH3CN component was then increased to 100% over 15 min, where it was maintained for a further 6 min. Mass spectra were obtained using ESI + APCI in positive and negative modes. All reagents and solvents for synthesis, unless otherwise stated, were purchased from Sigma-Aldrich . Solvents and reagents were at least of analytical grade and were used without further purification. Dried solvents were obtained from Sigma-Aldrich and were stored under inert gas. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Advance III 600 -2-(4-Methoxyphenyl)-5-methylpyrrolidine-3,4-diol, 3 dioxolopyrrol-4-yl)phenol, 12 +(theor.) = 278.37, Measured = 278.0.enol, 12 . Compoun-5-Benzyl-4-(4-(benzyloxy)phenyl)-2,2,6-trimethyltetrahydro-4H-dioxolopyrrole, 7b. The glycosylamine 5  was prepared according to the method of Kotland et. al. + (theor.) = 430.23767, measured = 430.23848Mesyl chloride  was added slowly to an ice-cooled solution of the intermediate material  in pyridine (30 mL), under an argon atmosphere. After 30 min at 0 °C, the ice bath was removed, and the reaction was stirred for a further 6 h at room temperature. TLC analysis revealed the consumption of the starting material and the formation of a less polar product. The mixture was quenched with saturated aqueous NaHCO-5-Benzyl-4-(4-(benzyloxy)phenyl)-2,2,6-trimethyltetrahydro-4H-dioxolopyrrole, 8b: To a 3-neck flask, charged with argon and cooled in an ice bath, was added Pd/C . The catalyst was carefully solvated with EtOH (35 mL), after which 7b  in DCM (5 mL) was added. After repeatedly degassing the system (5×), the solution was placed under a H2 atmosphere and stirred overnight at room temperature. TLC analysis revealed the spot-to-spot conversion of the starting material to a more polar product. The solution was degassed, placed under an argon atmosphere, and filtered through Celite® to remove the catalyst. The solvent was removed under reduced pressure, and the resulting colorless oil 8b was used without further purification . TLC: Rf = 0.35 (EtOAc:PE = 1:1); 1H NMR  δ = 7.08 , 6.66 , 4.91 , 4.67 , 4.47 , 3.48 , 3.42 , 1.59 , 1.38 , 1.34 ; 13C NMR  δ = 156.29, 130.09, 128.75 (2 × ArC), 115.86 (2 × ArC), 111.68, 86.34, 81.71, 66.27, 56.72, 25.95, 24.01, 12.70; HRMS (ESI): [M + H]+ (theor.) = 250.14377, measured = 250.14399.tert-Butyl -4-(4-hydroxyphenyl)-2,2,6-trimethyltetrahydro-5H-dioxolo-pyrrole-5-carboxylate, 10: Boc2O  in THF (1 mL) was added drop-wise to an ice-cooled solution of 8b , Et3N , and NaHCO3  in THF (5 mL) and water (5 mL). The reaction was allowed to warm to room temperature over 1 h and was then stirred as such overnight. TLC analysis revealed the spot-to-spot conversion of the starting amine to the less polar carbamate. The solution was diluted with water (20 mL) and extracted into EtOAc (4 × 20 mL). The combined organic layers were washed with water (50 mL), dried over MgSO4, and concentrated under reduced pressure. Purification using silica gel column chromatography, which involved an increasing gradient of EtOAc in PE (20–50%), yielded the product 10 as a colorless syrup . TLC: Rf = 0.53 (EtOAc:PE = 1:1); 1H NMR  δ = 6.92 , 6.72 , 4.84 , 4.81–4.73 , 4.50 , 4.27–4.18 , 4.00–3.91 , 1.54–1.10 ; HRMS (ESI): [M + Na]+ (theor.) = 372.17814, measured = 372.1782312 , acetonitrile (1 mL), and HCl  was heated at 140 °C for 5 min in a sealed flask. Upon cooling, the solution was analyzed using LCMS. (ESI-APCI): 13 (9.5 min), [M + H]+(theor.) = 278.37, Measured = 278.0; 13 (5.3 min), [M + H]+(theor.) = 238.31, measured = 238.1.Acid hydrolysis of acetonide: A solution of 11C was produced through the 14N11C nuclear reaction as [11C]CO2, using a 16.5 MeV PETtrace cyclotron , from N2 that contained 0.5% (v/v) O2. The beam current was 70 μA. [11C]CH3I was produced using an automated Tracerlab FX-MeI synthesis module . The automated synthesis was performed by an FX-M synthesis module . Radio-HPLC was performed using a 1260 Infinity HPLC system (Agilent) with a thallium activated sodium iodide scintillation detector. The column and gradient were the same as described for LCMS. Centrifugation for serum stability was performed using an Eppendorf mini-spin .10 (10 mg) in anhydrous CH3CN was prepared and ultra-sonicated for 5 min at room temperature. This solution was then transferred directly to the reactor of the FX-M synthesis module, and cooled to −20 °C. The [11C]CH3I was transferred through a PTFE delivery line and trapped in the reactor at −20 °C. Upon completion of delivery, the reactor was heated to 110 °C for 5 min and then cooled to 40 °C. Next, 6.0 M HCl (200 μL) was added, and the reactor was heated to 140 °C for 5 min to allow for the removal of the carbamate and hydrolysis of the acetonide protection. The reactor was then cooled to 40 °C, after which a solution comprising 2.0 M NH4OAc (1.1 mL) and 6.0 M NaOH (0.2 mL) was added. The product was purified with semi-preparative HPLC , 100u, 250 × 10.0 mm, 5 μm), using 10% EtOH in PBS (pH 6.0) as eluent. The identity of the tracer was confirmed using radio-HPLC, by spiking with the authentic nonradioactive standard 3 that was synthesized according to the method outlined by Kotland et al. [3 at 254 nm was used to calculate the molar activity. A mixture of tracer (200 μL) and mouse serum  was incubated with gentle agitation at 37 °C for 60 min. Cold acetonitrile (800 μL) was added, after which the mixture was vortexed and placed on ice for 5 min. Centrifugation at 10,000 rpm for 5 min yielded a supernatant that was analyzed by radio-HPLC.A suspension of NaH (5 mg) and the precursor ® standard cell culture flasks  at 37 °C in a humidified atmosphere of 5% CO2. HCT116 cells were cultured in McCoy’s 5A medium . Growth medium was supplemented with penicillin (100 U/mL), streptomycin (100 mg/L), and 10% fetal calf serum (FCS), and cells were regularly tested for mycoplasma using Venor GeM Mycoplasma Detection Kit.Cells were grown in CELLSTAR® 3000 (ThermoFisher) and treated in accordance with the manufacturer’s guidelines, using G418 selection reagent (ThermoFisher). Clones were isolated by selective trypsination and screened individually for FUCA1 expression by Western blot.pCMV6 mammalian vector containing cDNA of human α-fucosidase (FUCA1) was purchased from OriGene . The plasmid was amplified in NEB Turbo cells, harvested using a Midi-Prep Plasmid kit , and verified by Sanger sequencing. Cells were stably transfected using Lipofectamineg) for 5 min at 4 °C. A mixture of the lysate (20 μL) and 2× sample loading buffer (20 μL) was heated to 95 °C for 5 min and then loaded onto an Expedeon RunBlue—20% precast gradient gel . The separation was effected by running at 60 V for 10 min, followed by 150 V for 40 min. The proteins were transferred to a PVDF membrane using a Trans-Blot® Transfer system . The membrane was incubated for 1 h in Odyssey PBS blocking buffer . Protein bands were stained with primary mouse anti-DDK2  and rabbit anti-β-actin  antibodies. After incubation with IRDye 680RD goat  anti-mouse  and IRDye 800CW Goat  anti-rabbit  secondary antibodies, the readout was performed using the Odyssey Sa imaging system (Li-Cor).Cells or tissue were suspended in a mixture comprising lysis buffer (200 μL) and cOmplete™ protease inhibition buffer . The mixture was mechanically triturated at 0 °C for 5 min, after which it was centrifuged  and HCT116 were grown to 70–80% confluence. The medium was removed, and cells washed with PBS. They were incubated with 10 MBq tracer in PBS containing 0.1% FCS (10 mL)) for 20 min at 37 °C. The cells were washed with PBS, detached with trypsin, and counted. One million cells were then aliquoted into each gamma-counter tube. Uptake was quantified using a standard curve with known activity with the data of the gamma counter Wizard2 2480  after decay corrected activity detection. All data were normalized to the number of cells in each tube.® 555 Conjugate . Nuclear counterstaining was performed by incubating the samples in 300 nM DAPI (ThermoFischer Scientific) for 5 min and washing thoroughly with PBS (3 × 5 mL). The slides were carefully cleaned, mounted with Mowiol® 4-88 mounting medium (Sigma Aldrich), and examined under a Axiovert 200 inverted fluorescence microscope .Tumor cryosections (10 μm) on microscope slides were fixed for 10 min at room temperature in 3.7% paraformaldehyde, after which they were washed with PBS (2 × 5 mL). The samples were then incubated for 5 min in PBS containing Triton X-100  to permeablize the cells. Blocking was performed by incubating the samples for 20 min in 1% BSA in PBS, which was followed by washing with PBS (2 × 5 mL). Staining involved the same primary antibodies used for Western blot. Thus, the slides were incubated first with the primary anti-DDK antibody (1:1000) for 1 h at room temperature, followed by a 1 h incubation with the secondary antibody anti-mouse IgG Alexa Fluor6 HCT116AF (left) and HCT116 (right) cells. For in vivo experiments, tumors achieved a volume of approximately 250–300 mm³. A calibrated vaporizer  was used to anesthetize the mice at 1.5% (v/v) isoflurane  in oxygen. Anesthesia was monitored by measuring the respiratory frequency, and the body temperature was kept at 37 °C using a heating pad. The radiolabeled AF inhibitor (12.8 ± 1.7 MBq) was injected into the tail vein. PET data were acquired in list-mode, histogrammed, and reconstructed by using an iterative ordered subset expectation maximization (OSEM) algorithm. No attenuation correction was applied. MR imaging was performed on a 7 T small animal MR tomograph , obtaining anatomical information for organ delineation. A T2WI 3D space sequence  was used for whole-body imaging. PET images were normalized to each other, subsequently fused to the respective MR images, and analyzed by using Inveon Research Workplace software . Regions of interest (ROIs) were drawn around the whole tumor and reference (muscle) tissue based on the anatomical information from the MR images. Absolute quantification of the PET data was expressed as percentage of the injected dose per cubic centimeter (%ID/cc).Animal studies were conducted according to approved animal use and care protocols in accordance with the German Animal Protection Law (Regierungspräsidium Tübingen). For in vivo experiments, tumors were induced in the flank of BALB/c nu/nu mice by subcutaneous injection of 1 × 103 in PBS 10 min prior to administering the tracer.We performed a preliminary blocking experiment using just 2 mice that had a similar size and stage of tumor. Blocking was performed by injecting 5 μL of a 3.5 mg/mL solution of non-radioactive compound n > 2) groups relied on the Kruskal–Wallis test when data were not normally distributed. When data sets passed the normality test, analysis of variance (ANOVA) was used. Comparing the differences between 2 groups relied on either a two-tailed t test or Mann–Whitney test. Differences were deemed significant when p < 0.05 and highly significant when p < 0.01.All statistics were performed using Graphpad Prism 7. All data sets were checked for normality using the D’Agostino-Pearson test. The comparison of multiple (11C methylation and rapid deprotection. We further produced an AF overexpressing cell model, which could be translated for preclinical imaging. Our results evidence a probe that has the ability to identify and visualize the expression of AF both in vitro and in vivo. Future work will focus on applying this imaging probe to various disease models, including arthritis and cancer.In our efforts to develop a probe for AF, we successfully synthesized a precursor that is suitable for late-stage


Alisma canaliculatum extracts (ACE) were identified in AGS gastric cancer cells. Our results showed that ACE inhibited the growth of AGS cells, increased the proportion of sub-G1 phase cells, and depolarized the membrane potential of mitochondria. ACE-induced gastric cancer cell death was associated with Bcl-2, survivin and Bax level changes, and it activated caspase-3 and -9. In addition, it was involved in the activation of MAPKs and increased the reactive oxygen species (ROS). These results suggest that ACE induces apoptosis in AGS gastric cancer cells, and therefore, ACE may have the potential to treat gastric cancer.The anti-cancer effects of  Gastric cancer places a heavy burden on people worldwide and is becoming the most common cancer globally Alisma canaliculatum is a commonly used traditional medicine in China, Japan, and Korea A. canaliculatum on gastric cancer cells have not yet been studied. Thus, we investigated the mechanisms underlying the anti-cancer effects of A. canaliculatum extract (ACE) on AGS gastric cancer cells.Apoptosis plays a vital role in maintaining the balance between cell death and cell division. Apoptosis induced abnormalities can result in uncontrolled cell division, leading to diseases such as cancer A. canaliculatum extract (ACE) was purchased from Korea Plant Extract Bank . Alisol B monoacetate was obtained from Chemfaces . High-performance liquid chromatography (HPLC) grade acetonitrile (ACN) and distilled water were purchased from Thermo Fisher Scientific .TM C18 column (4.6 × 250 mm × 5 μm). The mobile phase consisted of a mixture of (A) 0.1% phosphoric acid in distilled water and (B) ACN. The gradient running condition was as follows: 40-40% A for 0-10 min, 40-30% A for 10-20 min, 30-20% A for 20-30 min, 20-40% A for 30-35 min, with an assay determination run time of 35 min.A stock solution of ACE and alisol B monoacetate was prepared by separately dissolving each of them in methanol. Standard calibration solutions were prepared, ranging from 6.25 to 100 μg/mL. The HPLC analysis of the extract was performed using a JASCO HPLC system equipped with a binary pump, an auto-sampler, a column oven, and a UV detector. An aliquot containing 20 μL of standard or ACE sample solutions was directly injected onto a Symmetry3005 cells/well. Cell viability was determined using MTT assay.AGS cells were propagated in RPMI-1640 medium  supplemented with 10% heat-inactivated fetal bovine serum  containing 1% penicillin/streptomycin  at 37°C and seeded onto 12-well plates at a density of 3 × 10AGS cells were treated with ethyl alcohol and vortexed. Samples were centrifuged for 5 min and the supernatant was discarded. Cell pellets were resuspended in propidium iodide (PI) staining solution containing RNase and incubated for 40 min in the dark at room temperature. Samples were analyzed using a fluorescence-activated cell sorter .AGS cells were treated with 50 nM tetramethylrhodamine methyl ester  for 30 min. Fluorescence intensities were measured using a BD FACSCANTO II  at the excitation and emission wavelengths of 510 and 580 nm, respectively.The amount of protein extracted from cells was measured using Bradford method. An equal amount of protein (20 μg) from the samples was separated by 8% or 10% SDS-PAGE and probed with specific antibodies. Antibodies against survivin (#2808), ERK (#9102), pERK (#9106), JNK (#9252), pJNK (#9251), p38 (#9212), and pp38 (#9216) were purchased from Cell Signaling Technology , and antibodies against Bcl-2 (#sc-783), Bax (#sc-493), caspase-3 (#sc-7148), caspase-9 (#sc-7885), PARP (#sc-7150), β-actin (#sc-47778), and GAPDH (#sc-32233) were procured from Santa Cruz Biotechnology .Caspase-3 and -9 assay kits  were used. After resuspending the cells in ice-cold cell lysis buffer, the supernatant was removed. Supernatant samples were incubated with caspase substrate  at 37°C and then, samples were read at 405 nm.ROS levels were determined with 2',7'-dichlorodihydrofluorescein diacetate . Fluorescence was measured using FACS , at excitation/emission wavelengths of 488/525 nm.post hoc comparison was used for multiple comparisons. The analysis was performed using Prism 6.0  and Origin 8.0  software. Data were expressed as the mean ± standard error of the mean (SEM), and P values < 0.05 were considered statistically significant.One-way ANOVA with Tukey's A previous study reported that alisol B monoacetate is a major component in ACE p< 0.01) at 200 µg/ml, 26.1 ± 6.4 % (p< 0.01) at 300 µg/ml, 16.1 ± 6.6 % (p< 0.01) at 400 µg/ml, and 8.8 ± 2.0 % (p< 0.01) at 500 µg/ml of ACE at 24 h  at 100 µg/ml, 41.5 ± 7.6 % (p< 0.01) at 200 µg/ml, 25.3 ± 2.2 % (p< 0.01) at 300 µg/ml, 18.3 ± 0.5 % (p< 0.01) at 400 µg/ml, and 3.4 ± 0.2 % (p< 0.01) at 500 µg/ml of ACE at 48 h  at 100 µg/ml, 35.2 ± 1.1 % (p< 0.01) at 200 µg/ml, 23.2 ± 1.2 % (p< 0.01) at 300 µg/ml, 7.3 ± 0.3 % (p< 0.01) at 400 µg/ml, and 3.3 ± 0.1 % (p< 0.01) at 500 µg/ml of ACE at 72 h  at 200 µg/ml, 38.8 ± 2.7 % (p< 0.01) at 300 µg/ml, 27.9 ± 2.9 % (p< 0.01) at 400 µg/ml, and 16.5 ± 1.4 % (p< 0.01) at 500 µg/ml  polymerase (PARP) functions as a sign of apoptosis To investigate the effects of ACE on the MAPK signaling pathway, we investigated the phosphorylation of MAPK proteins , JNK, and p38) with western blotting. The phosphorylation of p38 increased with ACE treatment (200 μg/ml) Fig. . These rSince ROS also play a key role in apoptosis, we investigated whether ACE increased ROS levels in AGS cells. Flow cytometry results showed that ACE increased ROS levels Fig. . These rA. canaliculatum is a plant used as a traditional medicine in Asia Traditional medicine is an ancient form of therapy that treats not only an illness or ailment but also the entire body holistically Apoptosis is mediated in eukaryotic cells through extrinsic and intrinsic pathways, and caspase cascade plays a key role in this process. The extrinsic pathway is initiated by the activation of caspase-8, which is triggered by the combination of death ligand to the death receptor present in the cell membrane, followed by the activation of caspase-3 and -7 Ion channels play an important role in the formation and death of cancer. They are related with cancer cell growth, migration, and metastasis. Ion channels are also known to be critical components in apoptosis MAPKs are related with different cellular mechanisms including apoptosis In conclusion, ACE inhibits the growth of AGS cells, induces the increase proportion of sub-G1 phase, and depolarizes the membrane potential of mitochondria. We found ACE-induced apoptosis to be associated with BCl-2 and survivin decreases and Bax increase levels; it also activated caspase-3 and -9 and MAPKs. Moreover, ACE increased the production of ROS. Thus, the present study on ACE may markedly contribute to the development of gastric cancer treatments.


Choriocarcinoma is one of the most aggressive gestational trophoblastic neoplasias (GTN). Methotrexate (MTX) resistance is the main cause of treatment failure in choriocarcinoma. However, the mechanism of MTX resistance in choriocarcinoma is poorly known. This study aims to explore the function of Lectin galactoside-binding soluble 3 binding protein (LGALS3BP) in MTX-resistance in choriocarcinoma cells. Gradual dose escalation of MTX was used to establish MTX-resistant choriocarcinoma cells (JAR-MTX and JEG3-MTX cell lines). RNA-sequencing was used to explore the differentially expressed genes. Plasmids or SiRNA transfection was used to regulate the expression of LGALS3BP. ELISA was used to detect the concentrations of LGALS3BP in the serum of MTX-sensitive and MTX-resistant patients. qRT-PCR, Western blot, and CCK-8 assay were used to determine the effects of LGALS3BP on MTX-resistance in JAR and JEG3 cells. The results showed the relative resistance index (RI) of MTX is 791.50 and 1040.04 in JAR-MTX and JEG3-MTX, respectively. LGALS3BP was up-regulated in MTX-resistant cells compared to original cells in both RNA and protein level. The concentrations of LGALS3BP were higher in the sera of MTX-resistant patients than in MTX-sensitive patients. Knocking down LGALS3BP can reverse the MTX-resistance in JAR-MTX and JEG3-MTX cells. In summary, we preliminarily established two MTX-resistant cells, and performed RNA-sequencing, and found LGALS3BP may play important role in MTX-resistance. Our work not only provides a research tool (MTX-resistant cells) for other researchers, but gives some hint on how MTX resistance is regulated. Among tThis study focused on the relationship between LGALS3BP expression and MTX-resistance in choriocarcinoma to verify whether LGALS3BP can regulate the sensitivity of choriocarcinoma cell lines toward MTX. In order to uncover the mechanisms involved in MTX-resistance in choriocarcinoma and the effect of LGALS3BP on the effectiveness of MTX treatment, we established MTX-resistant choriocarcinoma cells by gradual dose escalation of MTX, and found LGALS3BP was up-regulated in MTX-resistant cells compared to original cells. Meanwhile, we found that the sera concentration of LGALS3BP was higher in MTX-resistant patients than MTX-sensitive patients. Moreover, knocking down of LGALS3BP can significantly reverse the resistance of MTX while overexpressing of LGALS3BP will lead to MTX-resistance in choriocarcinoma.2.2.1.2 and detached from the culture plates using trypsin/EDTA solution  [50) of MTX, the IC50 of MTX was 0.44 μM that as the base concentration, then gradually increasing dose of MTX. When the concentration of the drug was increasing, the cells cultured for 24 h, then washed with PBS  for 3–5 times, and cultured in medium without MTX. Surviving cells were collected and the above protocol was repeated 3–5 times, until the cells could grow stably in 88 μM MTX [Two human choriocarcinoma cancer cell lines, JAR and JEG3, were obtained from the American Type Culture Collection , maintained in Women’s Hospital, School of Medicine, Zhejiang University, and authenticated and tested for Mycoplasma contamination. Cells used in this study were not passaged continuously for more than 3 months. JAR cells were cultured in RPMI-1640 medium , and JEG3 cells were cultured in EBSS-MEM medium . All cell lines were supplemented with 10% fetal bovine serum (FBS) , maintained at 37°C in 5% CO, China) . MTX was8 μM MTX .2.2.To compare the cell morphology of MTX-resistant JAR and JEG3 cells, with that of original JAR and JEG3 cells, cells in the logarithmic phase were replaced with fresh culture medium and photographed with an inverted microscope.2.3.3 per well into 96-well plates. After 24, 48, 72 and 96 hours of culture, a 10 μL CCK8 solution was added to each well and incubated for 1 hour. The absorbance at 490 nm was measured with a Varioskan Flash microplate reader  [Cell proliferation was measured by CCK8 kit . Briefly, cells were seeded 5 × 10MA, USA) .2.4.3 cells per well. After one day of growth, the cells were treated with different concentrations of Etoposid (VP – 16)  5-Fluorouracil (5 – FU) , Methotrexate (MTX) , Paclitaxel (TAXOL) , or ActD  and incubated at 37°C for 72 hours. Cell viability was detected by CCK – 8 viability assay at 72 hours after drug treatment [Cells were seeded into 96-well plates at a density of 5 × 10The concentrations of drugs used in MTX-resistant cells were listed below:VP-16:0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20 ng/ml;5-FU:0.05, 0.1, 0.5, 1, 5, 10, 20, 50 μg/ml;TAXOL:0.06, 0.1, 0.3, 0.6, 1, 3, 6, 10, 30 nM;MTX:1, 4, 10, 40, 100, 400, 1000 μM;ActD:0.1, 0.4, 0.8, 1, 4, 8, 10, 40 μg/ml.The concentrations of drugs used in original cells were listed below:VP-16:0.005, 0.01, 0.05, 0.1, 0.5, 1, 2 ng/ml;5-FU:0.05, 0.1, 0.5, 1, 5, 10, 20 μg/ml;TAXOL:0.06, 0.1, 0.3, 0.6, 1, 3, 6, 10, 30 nM;MTX:0.01, 0.04, 0.1, 0.4, 1, 4, 10, 40 μM;ActD:0.1, 0.4, 0.8, 1, 4, 8, 10, 40 μg/ml.(Note: It is converted into a uniform concentration unit for subsequent statistics)2.5.6 cells/ml. 100 μL cell suspension of each sample was fixed in ice-cold 70% ethanol overnight and incubated with 50 μg/ml PI at 4°C for 30 minutes [Cell cycle analysis was performed using Propidium Iodide Stain (PI)  following manufacturer’s instructions. Briefly, cells were harvested, washed with PBS, and resuspended in binding buffer at a concentration of 1 × 10 minutes . The sta2.6.−ΔΔCt method and normalized to GAPDH expression.Total RNA was isolated using an RNA extraction kit  and 1000 ng RNA was used to reverse-transcribed into cDNA by the reverse transcription cDNA kit . Quantitative real-time PCR was performed with 20 ng of cDNA using the SYBR® Premix ExTaq™ . The mix was added to 96-well plate detected by a Biosystems 7900HT fast real-time PCR system . The real-time PCR cycling condition is 40 cycles of 95°C 5 sec, 60°C 30 sec . The seq2.7.Cells were lysed with RIPA lysis buffer  supplemented with PMSF inhibitor . 20 ug protein lysates were loaded and separated by 10% SDS-containing polyacrylamide gel  and transferred to 0.22 μm polyvinylidene fluoride (PVDF) membranes  in a condition of 120 V 60 min. The membranes were then blocked with Tris buffered saline Tween20 (TBST) containing 5% nonfat milk for 1 hour at room temperature, and incubated with primary antibodies overnight at 4°C. The membranes were then washed twice with TBST for 10 min each and incubated with HRP-linked secondary antibody for 1 hour, followed by washing three times in TBST for 10 min per wash. The bands were visualized using an enhanced chemiluminescence (ECL) kit  in Image quant LAS400 mini . Antibodies against the following proteins were used in the experiments: LGALS3BP , Actin .2.8.2 package (1.10.1). The resulting P-values were adjusted using Benjamini and Hochberg approach for controlling the false discovery rate (FDR). Genes with an adjusted P− value < 0.05, as identified by DESeq2, were considered to be differentially expressed.The differentially expressed genes between MTX-resistant and original cell lines were identified by RNA-seq analysis , whose results were obtained by removing reads containing adapter, poly-N, and low-quality reads from the raw data. Next, the Q20, Q30, and GC contents of the clean data were calculated. Paired-end clean reads were aligned to the reference genome using STAR. Read numbers mapped to each gene were counted by HTSeq v0.6.0. Differential expression analysis of two conditions was performed using the DESeq2.9.Serum of patients with gestational trophoblastic neoplasia (GTN) which have different effects to MTX was collected at the Women’s Hospital, School of Medicine Zhejiang University between January 2016 and December 2019. The samples were classified according to drug resistance criteria  as eitheTwenty samples were classified as MTX-sensitive and twenty samples were classified as MTX-resistant. When samples preparation was completed, the concentration of LGALS3BP was determined with the Human Galectin-3 Binding Protein ELISA Kit .2.10.LGALS3BP and negative control plasmids were produced by GeneChem . Small-interfering RNAs (siRNAs) and negative controls were purchased from RiboBio. Transfections were performed using X-treme GENE HP DNA transfection reagent  following the manufacturer’s instructions .2.11.Statistical analysis was performed with the SPSS 20.0 software package . All experiments were repeated 3 times. All data are presented as the means ± SD. Shapiro-Wilk test was used to determine whether or not a sample comes from a normal distribution. The two tailed Student’s t test or one-way analysis of variance test was used to determine the significant differences. P < 0.05 was considered as statistically significant.3.This study aimed to analyze the effect of LGALS3BP on the effectiveness of MTX treatment in choriocarcinoma. After establishing two MTX-resistant choriocarcinoma cell lines by gradual dose escalation of MTX, we performed RNA-sequencing to explore the possible genes associated with MTX-resistance, and found LGALSBP was up-regulated in MTX-resistant choriocarcinoma cell lines in both RNA and protein level. Meanwhile, the sera concentration of LGALS3BP was higher in MTX-resistant patients than MTX-sensitive patients indicating LGALS3BP may be a molecular mark to predict MTX-resistance in choriocarcinoma. In order to further understand the function of LGALS3BP in MTX-resistance in choriocarcinoma, we overexpressed LGALS3BP in original cells and knocked down LGALS3BP in MTX-resistant cells and found overexpressing LGALS3BP can promote MTX-resistance, while knocking down LGALS3BP can reverse the MTX-resistance in choriocarcinoma.3.1.50 of MTX in JAR-MTX, JEG3-MTX cells are significantly higher than their original cells . The ICal cells ), The ceal cells ; Table 2The resistance of MTX-resistant cells to different chemotherapeutic agents was analyzed by cell viability assay. The results showed that MTX-resistant cells were resistant to MTX, as well as VP-16, 5-FU, TAXOL and ActD, the RI of MTX is 791.50 and 1040.04, concurrently it has cross resistance to VP-16, 5-FU, TAXOL and ActD, compared with JAR, JEG3 . After d3.2.Total RNA was extracted from the paired-comparison groups JAR VS JAR-MTX and JEG3 VS JEG3-MTX.RNA-sequence  was used to screen for differentially expressed genes between MTX-resistant JAR, JEG3 cells and their counterparts. The heat map showed JAR-MTX VS JAR have 492 different genes, JEG3-MTX VS JEG3 have 220 different genes, that with significantly differential expression (fold change ≥2 or≤0.5) by DeCyderMS™2.0 analysis, as shown in 3.3.After RNA-sequencing, we then explored the expression of six up-regulated genes was detected by qRT-PCR ). We foc3.4.50 compared to control cells . Choriocarcinoma cancer cells were transfected with plasmid for 48 H; expression of LGALS3BP was detected by q-PCR and Western blot , which sol cells ). MTX-reol cells , e), whice cells ). These 4.Choriocarcinoma characterized by human chorionic gonadotropin hormone elevation, is one of the most aggressive gestational trophoblastic neoplasias (GTN). Although it is rare in western country, 1 in 2882 pregnant women will develop choriocarcinoma in China. Although chemotherapy especially MTX treatment has been considered as the first-line therapy in treating choriocarcinoma due to the high sensitivity of choriocarcinoma to chemotherapy, a part of patients may fail to achieve complete remission after single-agent MTX treatment because of MTX resistance in clinical practice. Meanwhile, the mechanism of MTX resistance in choriocarcinoma still remains poorly understood. It is reported that activation of interferon signaling, inhibition of cell death/apoptosis, involvement of cancer stem cells, dysregulation of long non-coding RNAs, up-regulation of drug target gene and drug efflux may all contribute to the drug resistance in choriocarcinoma ,20–23. CLGALS3BP was initially identified as a heavily glycosylated protein in tumor tissue and serum from breast cancer patients and lung cancer cell lines ,26. It iAs we can see in In summary, we preliminarily established two MTX-resistant cells, and performed RNA-seq, and found LGALS3BP may play important role in MTX-resistance. We also used siRNA and plasmids to regulate the expression of LGALS3BP and found LGALS3B can regulate MTX sensitivity. Our work not only provides a research tool (MTX-resistant cells) for other researchers, but gives some hint on how MTX resistance is regulated.5.Our data indicate that LGALS3BP contributes to MTX-resistance in choriocarcinoma. Our findings suggest the potential of LGALS3BP as a useful prognostic marker and a promising therapeutic strategy for development of more effective choriocarcinoma therapies.


The cation is disordered and was refined as two equivalent forms with occupancies of 0.750 (4)/0.250 (4), while the chloride anion is triply disordered with occupancies of 0.774 (12), 0.12 (2), and 0.11 (2). Unusually, the bond angles around the C=O unit range from 127.2 (6) to 115.2 (3)° and similar angles have been found in other structures containing a 6-oxo-3,6-dihydropyrimidin-1-ium cation, including the monclinic polymorph of the title compound, which crystallizes in the monoclinic space group P21/c [Kawai et al.  and R32(9) rings. Theoretical calculations seem to indicate that the reason for the unusual angles at the sp2 C is the electrostatic interaction between the oxygen atom and the adjacent N—H hydrogen.The title compound, C al. 1973. Cryst.  Heterocycles containing the pyrimidine moiety are of great interest because they constitute an important class of natural and non-natural products, many of which exhibit useful biological activities and clinical applications , 118.2 (8), and 115.7 (8)° for 2.The title compound, , and C—D and N—D distances of 0.95 and 0.88 Å, respectively. The deuterium atoms for the methyl substituent were refined isotropically.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698902100270X/ru2073sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698902100270X/ru2073Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698902100270X/ru2073Isup3.cmlSupporting information file. DOI: 2069792CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: