Summary of Study ST003657

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002267. The data can be accessed directly via it's Project DOI: 10.21228/M8KV62 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003657
Study TitleLactate-coated polyurea-siRNA dendriplex: a gene therapy-directed and metabolism-based strategy to impair glioblastoma (GBM)
Study SummaryGlioblastoma (GBM) is a highly lethal disease with limited treatment options due to its infiltrative nature and the lack of efficient therapy able to cross the protective blood-brain barrier (BBB). GBMs are metabolically characterized by increased glycolysis and glutamine dependence. This study explores a novel metabolism-based therapeutic approach using a polyurea generation 4 dendrimer (PUREG4) surface functionalized with lactate (LA) (PUREG4-LA12), to take advantage of glucose-dependent monocarboxylate transporters (MCTs) overexpression, loaded with selenium-chrysin (SeChry) and temozolomide (TMZ) or complexed with anti-glutaminase (GLS1) siRNAs to abrogate glutamine dependence. The anti-GLS1 siRNA dendriplex with PUREG4-LA12 (PUREG4-LA12-anti-GLS1-siRNA) knocked down GLS1 in the GBM cell lines. In two in vitro BBB models, these dendriplexes successfully crossed the BBB, decreased GLS1 expression and altered the exometabolome of GBM cell lines, concomitantly with autophagy activation. Our findings highlight the potential of targeting glucose and glutamine pathways in GBM using dendrimer-based nanocarriers, overcoming the BBB and disrupting key metabolic processes in GBM cells.
Institute
ITQB NOVA
Last NameGonçalves
First NameLuís
AddressAvenida Republica, Oeiras, 2780-157 Oeiras, Portugal
Emaillgafeira@itqb.unl.pt
Phone214469464
Submit Date2025-01-08
Raw Data AvailableYes
Raw Data File Type(s)fid
Analysis Type DetailNMR
Release Date2025-04-08
Release Version1
Luís Gonçalves Luís Gonçalves
https://dx.doi.org/10.21228/M8KV62
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002267
Project DOI:doi: 10.21228/M8KV62
Project Title:Lactate-coated polyurea-siRNA dendriplex: a gene therapy-directed and metabolism-based strategy to impair glioblastoma (GBM)
Project Summary:Glioblastoma (GBM) is a highly lethal disease with limited treatment options due to its infiltrative nature and the lack of efficient therapy able to cross the protective blood-brain barrier (BBB). GBMs are metabolically characterized by increased glycolysis and glutamine dependence. This study explores a novel metabolism-based therapeutic approach using a polyurea generation 4 dendrimer (PUREG4) surface functionalized with lactate (LA) (PUREG4-LA12), to take advantage of glucose-dependent monocarboxylate transporters (MCTs) overexpression, loaded with selenium-chrysin (SeChry) and temozolomide (TMZ) or complexed with anti-glutaminase (GLS1) siRNAs to abrogate glutamine dependence. The anti-GLS1 siRNA dendriplex with PUREG4-LA12 (PUREG4-LA12-anti-GLS1-siRNA) knocked down GLS1 in the GBM cell lines. In two in vitro BBB models, these dendriplexes successfully crossed the BBB, decreased GLS1 expression and altered the exometabolome of GBM cell lines, concomitantly with autophagy activation. Our findings highlight the potential of targeting glucose and glutamine pathways in GBM using dendrimer-based nanocarriers, overcoming the BBB and disrupting key metabolic processes in GBM cells.
Institute:ITQB NOVA
Last Name:G Gonçalves
First Name:Luís
Address:Avenida Republica, Oeiras, 2780-157 Oeiras, Portugal
Email:lgafeira@itqb.unl.pt
Phone:214469464
Contributors:Filipa Martins; Renata Arada; Hélio Barros, Paulo Matos, José Ramalho, Valentín Ceña, Vasco D.B. Bonifácio, Luís G. Gonçalves, Jacinta Serpa

Subject:

Subject ID:SU003787
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Condition Method of exposure
SA398938RA_S96h_15_240524U251 anti-GLS1 siRNA Transfection
SA398939RA_S96h_14_240524U251 anti-GLS1 siRNA Transfection
SA398940RA_S96h_13_240524U251 anti-GLS1 siRNA Transfection
SA398888RA_SI_127_240826U251 Control Direct exposure
SA398889RA_SI_126_240826U251 Control Direct exposure
SA398890RA_SI_02_240710U251 Control Direct exposure
SA398891RA_SI_01_240710U251 Control Direct exposure
SA398892RA_SI_128_240826U251 Control Direct exposure
SA398893RA_S96h_10_240524U251 Control Transfection
SA398894RA_S96h_12_240524U251 Control Transfection
SA398895RA_S96h_11_240524U251 Control Transfection
SA398896RA_Psi_11_240607U251 Control Transwell b.End3
SA398897RA_Psi_13_240607U251 Control Transwell b.End3
SA398898RA_Psi_25_240607U251 Control Transwell b.End3
SA398899RA_Psi_12_240607U251 Control Transwell b.End3
SA398900RA_SI_TW_71_240710U251 Control Transwell b.End3 + iNHA
SA398901RA_SI_TW_65_240710U251 Control Transwell b.End3 + iNHA
SA398902RA_SI_TW_66_240710U251 Control Transwell b.End3 + iNHA
SA398903RA_SI_108_240826U251 Control Transwell b.End3 + iNHA
SA398904RA_SI_109_240826U251 Control Transwell b.End3 + iNHA
SA398905RA_S96h_17_240524U251 Lipofectamine Transfection
SA398906RA_S96h_18_240524U251 Lipofectamine Transfection
SA398907RA_S96h_16_240524U251 Lipofectamine Transfection
SA398908RA_SI_132_240826U251 PUREG4-LA12-anti-GLS1 siRNA Direct exposure
SA398909RA_SI_134_240826U251 PUREG4-LA12-anti-GLS1 siRNA Direct exposure
SA398910RA_SI_133_240826U251 PUREG4-LA12-anti-GLS1 siRNA Direct exposure
SA398911RA_SI_05_240710U251 PUREG4-LA12-anti-GLS1 siRNA Direct exposure
SA398912RA_SI_06_240710U251 PUREG4-LA12-anti-GLS1 siRNA Direct exposure
SA398913RA_Psi_28_240607U251 PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3
SA398914RA_Psi_2_240529U251 PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3
SA398915RA_Psi_16_240607U251 PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3
SA398916RA_Psi_17_240607U251 PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3
SA398917RA_Psi_29_240607U251 PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3
SA398918RA_SI_113_240826U251 PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3 + iNHA
SA398919RA_SI_TW_69_240710U251 PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3 + iNHA
SA398920RA_SI_112_240826U251 PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3 + iNHA
SA398921RA_SI_TW_70_240710U251 PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3 + iNHA
SA398922RA_SI_TW_75_240710U251 PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3 + iNHA
SA398923RA_SI_131_240826U251 PUREG4-LA12 Direct exposure
SA398924RA_SI_03_240710U251 PUREG4-LA12 Direct exposure
SA398925RA_SI_04_240710U251 PUREG4-LA12 Direct exposure
SA398926RA_SI_130_240826U251 PUREG4-LA12 Direct exposure
SA398927RA_SI_129_240826U251 PUREG4-LA12 Direct exposure
SA398928RA_Psi_1_240529U251 PUREG4-LA12 Transwell b.End3
SA398929RA_Psi_14_240607U251 PUREG4-LA12 Transwell b.End3
SA398930RA_Psi_15_240607U251 PUREG4-LA12 Transwell b.End3
SA398931RA_Psi_27_240607U251 PUREG4-LA12 Transwell b.End3
SA398932RA_Psi_26_240607U251 PUREG4-LA12 Transwell b.End3
SA398933RA_SI_TW_67_240710U251 PUREG4-LA12 Transwell b.End3 + iNHA
SA398934RA_SI_TW_68_240710U251 PUREG4-LA12 Transwell b.End3 + iNHA
SA398935RA_SI_TW_73_240710U251 PUREG4-LA12 Transwell b.End3 + iNHA
SA398936RA_SI_110_240826U251 PUREG4-LA12 Transwell b.End3 + iNHA
SA398937RA_SI_111_240826U251 PUREG4-LA12 Transwell b.End3 + iNHA
SA398885RA_S96h_6_240524U-87MG anti-GLS1 siRNA Transfection
SA398886RA_S96h_5_240524U-87MG anti-GLS1 siRNA Transfection
SA398887RA_S96h_4_240524U-87MG anti-GLS1 siRNA Transfection
SA398832RA_SI_117_240826U-87MG Control Direct exposure
SA398833RA_SI_116_240826U-87MG Control Direct exposure
SA398834RA_SI_115_240826U-87MG Control Direct exposure
SA398835RA_SI_114_240826U-87MG Control Direct exposure
SA398836RA_SI_A_240710U-87MG Control Direct exposure
SA398837RA_S96h_1_240524U-87MG Control Transfection
SA398838RA_S96h_2_240524U-87MG Control Transfection
SA398839RA_S96h_3_240524U-87MG Control Transfection
SA398840RA_Psi_18_240607U-87MG Control Transwell b.End3
SA398841RA_Psi_20_240607U-87MG Control Transwell b.End3
SA398842RA_Psi_30_240607U-87MG Control Transwell b.End3
SA398843RA_Psi_19_240607U-87MG Control Transwell b.End3
SA398844Ra_SI_90_240820U-87MG Control Transwell b.End3 + iNHA
SA398845RA_SI_103_240826U-87MG Control Transwell b.End3 + iNHA
SA398846Ra_SI_84_240820U-87MG Control Transwell b.End3 + iNHA
SA398847Ra_SI_85_240820U-87MG Control Transwell b.End3 + iNHA
SA398848Ra_SI_91_240820U-87MG Control Transwell b.End3 + iNHA
SA398849RA_SI_102_240826U-87MG Control Transwell b.End3 + iNHA
SA398850RA_S96h_9_240524U-87MG Lipofectamine Transfection
SA398851RA_S96h_7_240524U-87MG Lipofectamine Transfection
SA398852RA_S96h_8_240524U-87MG Lipofectamine Transfection
SA398853RA_SI_C_240710U-87MG PUREG4-LA12-anti-GLS1 siRNA Direct exposure
SA398854RA_SI_122_240826U-87MG PUREG4-LA12-anti-GLS1 siRNA Direct exposure
SA398855RA_SI_123_240826U-87MG PUREG4-LA12-anti-GLS1 siRNA Direct exposure
SA398856RA_SI_124_240826U-87MG PUREG4-LA12-anti-GLS1 siRNA Direct exposure
SA398857RA_SI_125_240826U-87MG PUREG4-LA12-anti-GLS1 siRNA Direct exposure
SA398858RA_Psi_33_240607U-87MG PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3
SA398859RA_Psi_34_240607U-87MG PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3
SA398860RA_Psi_24_240607U-87MG PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3
SA398861RA_Psi_23_240607U-87MG PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3
SA398862RA_Psi_6_240529U-87MG PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3
SA398863RA_SI_106_240826U-87MG PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3 + iNHA
SA398864Ra_SI_88_240820U-87MG PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3 + iNHA
SA398865Ra_SI_89_240820U-87MG PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3 + iNHA
SA398866Ra_SI_94_240820U-87MG PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3 + iNHA
SA398867RA_SI_107_240826U-87MG PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3 + iNHA
SA398868Ra_SI_95_240820U-87MG PUREG4-LA12-anti-GLS1 siRNA Transwell b.End3 + iNHA
SA398869RA_SI_120_240826U-87MG PUREG4-LA12 Direct exposure
SA398870RA_SI_121_240826U-87MG PUREG4-LA12 Direct exposure
SA398871RA_SI_119_240826U-87MG PUREG4-LA12 Direct exposure
SA398872RA_SI_B_240710U-87MG PUREG4-LA12 Direct exposure
SA398873RA_SI_118_240826U-87MG PUREG4-LA12 Direct exposure
SA398874RA_Psi_21_240607U-87MG PUREG4-LA12 Transwell b.End3
SA398875RA_Psi_31_240607U-87MG PUREG4-LA12 Transwell b.End3
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Collection:

Collection ID:CO003780
Collection Summary:Cell culture Two commercial GBM cell lines, U-87MG (HTB-14, American Type Culture Collection – ATCC, Manassas, Virginia, USA) and U251 (09063001, European Collection of Authenticated Cell Cultures – ECACC, Salisbury, United Kingdom) were used. The cell lines were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 Ham (DMEM/F-12; 11330-032, Gibco, Life Technologies, Grand Island, New York, USA), supplemented with 10% fetal bovine serum (FBS; P40-37500, PAN Biotech, Aidenbach, Germany), 1% Antibiotic Antimycotic (AA; P06-07300, PAN Biotech) and 50 µg/mL gentamicin (15750-060, Gibco, Life Technologies). Direct exposition Cells were exposed to the PUREG4-LA12-anti-GLS1 siRNA. The culture media of three conditions were analysed: without dendriplex (control), with only the dendriplex (PUREG4-LA12) and with the siRNA complexed on the dendriplex (PUREG4-LA12-anti-GLS1 siRNA). Transfection with anti-GLS1 siRNA Cells were transfected with anti-GLS1 siRNA in lipofectamine 2000 (11668019, Invitrogen). Lipofectamine 2000 and siRNA were incubated separately with DMEM/F-12 without FBS for 5 min, 37 ºC and then the two suspensions were mixed and incubated for 20 min, 37 ºC. After incubation, cells were exposed to the mix, in a final concentration of 30 nM siRNA, according to the manufacturer’s protocol. In the case of the transfection, the culture media of three conditions were analysed: without transfection (control), transfected without siRNA (lipofectamine) and transfected with the siRNA (anti-GLS1 siRNA). Transwell experiment For the BBB (transwell) in vitro model, mouse BALB/c brain endothelioma cell line b.End3 was used (CRL.2299, ATCC). This cell line was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose (11965092, Gibco) supplemented with 10% FBS, 1% AA and 50 μg/mL Gentamicin. A normal human astrocyte cell line was also used, iNHA (NHA variant immortalized with SV40) (CC-2565, ATCC). This cell line was cultured in DMEM/F-12 supplemented with 10% FBS, 1% AA and 50 μg/mL Gentamicin. A BBB in vitro model was established. In this model, a transwell insert (9321002, cellQART 24-Well Insert 1.0 µm pore PET translucent, Northeim, Germany) with a microporous semipermeable membrane was inserted in a 24-well plate and separated the upper (luminal, blood side) and the lower (abluminal, brain side) chambers. In the BBB model with iNHA (human astrocyte cell line NHA variant immortalized with SV40), the bottom of the inserts was precoated with 0.001% Poly-L-Lysine (L 7240, Biochrom AG, Darmstadt, Germany) for 45 min at room temperature. iNHA were plated on the lower side of the insert, which was upside down until cells’ adherence. Then, b.End3 cells were seeded on the inserts after 1 h coating with 2% matrigel DMEM without FBS and antibiotics, at 37 ºC. After 3 days in culture, 550 nM of hydrocortisone (H4001, Sigma-Aldrich), was added. On the 6th day after seeding, 5 × 104 cells/well GBM cells were seeded in the 24-well plate, on the 7th day the inserts with b.End3 cells were placed on top of the 24-well plate with the GBM cells, and experimental conditions were added. Two types of experiments were performed one with the only the brain endothelioma cell line b.End3 (Transwell b.End3) and the other with also the astrocyte cell line (Transwell b.End3 + iNHA). The culture media of three conditions were analysed: without dendriplex (control), with only the dendriplex (PUREG4-LA12) and with the siRNA complexed on the dendriplex (PUREG4-LA12-anti-GLS1 siRNA).
Sample Type:Culture Media

Treatment:

Treatment ID:TR003796
Treatment Summary:Direct exposition Cells were subject to the dendriplex PUREG4-LA12 with anti-GLS1 siRNA. As controls are used only the dendriplex without siRNA and without dendriplex addition. Transfection with anti-GLS1 siRNA The cells transfect with siRNA, cells transfected without siRNA and without conditions are grown in the conditions described before. Transwell experiment In the two different transwell models, cells are subject to the dendriplex PUREG4-LA12 with anti-GLS1 siRNA. As controls are used only the dendriplex without siRNA and without dendriplex addition.

Sample Preparation:

Sampleprep ID:SP003794
Sampleprep Summary:After exposure to experimental conditions for 72 h, supernatants were collected and stored at -80 ºC, to be analyzed by 1H-NMR spectroscopy. To 60 µL solution of 1.5 M potassium phosphate buffer (KPi, pH 7.4), NaN3 2 mM and 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP) 0.16 mM in D2O was added to 540 µL of supernatant sample.

Analysis:

Analysis ID:AN006007
Analysis Type:NMR
Analysis Protocol File:LG_GBM_Protocol.pdf
Num Factors:24
Num Metabolites:40
Units:mM

NMR:

NMR ID:NM000302
Analysis ID:AN006007
Instrument Name:Bruker Avance II+ 800 MHz
Instrument Type:FT-NMR
NMR Experiment Type:1D-1H
Spectrometer Frequency:800
NMR Probe:5 mm TCI cryoprobe
NMR Solvent:10 % D2O/ 90 % H20
NMR Tube Size:5 mm
Shimming Method:Topshim
Pulse Sequence:noesygppr1d
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