Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA356235

Local Sample ID:	CD_A549_TW_CTL3_rep3
Subject ID:	SU003406
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Not applicable

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Subject:

Subject ID:	SU003406
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Not applicable

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
CD_A549_TW_CTL3_rep3	SA356235	FL041574	Non-small cell lung cancer cells	Sample source
CD_A549_TW_CTL3_rep3	SA356235	FL041574	A549	Cell line
CD_A549_TW_CTL3_rep3	SA356235	FL041574	Indirect Co-culture	Culture Type
CD_A549_TW_CTL3_rep3	SA356235	FL041574	No treatment	Immune checkpoint inhibitor

Collection:

Collection ID:	CO003399
Collection Summary:	Using Nivolumab (anti-PD1) and Ipilimumab (anti-CTLA4), we pharmacologically modulated non-small cell lung cancer (NSCLC) cell lines in co-culture with CD8+ T lymphocytes (T CD8+) obtained from healthy blood donors. Two different modalities of co-cultures were established: direct and indirect (with T CD8+ separated from the cancer cells by a Boyden chamber with 1.0 μm pores). 1H-NMR spectroscopy was conducted to characterize the impact of the ICI in the metabolic profile of the immune and cancer cells, as well as the influence of immune cells in the cancer cells exometabolome. Therefore, the media obtained from the established cultures were centrifuged at 135 × g for 5 minutes and the supernatants were collected, stored at -80⁰C and later explored and defined by NMR.
Sample Type:	Non-small cell lung cancer

Treatment:

Treatment ID:	TR003415
Treatment Summary:	For the experimental conditions, cells were exposed to Nivolumab (10 mg/mL, OPDIVO), anti-PD1) or Ipilimumab (5 mg/mL, YERVOY, anti-CTLA-4) for 48 h. The Nivolumab and Ipilimumab concentrations were chosen in accordance to the posology indications from the European Medicines Agency (EMA) and using as reference a male adult with 80 kg and 5 L of blood. The conditions were tested in monoculture or in co-culture with TCD8+.

Treatment ID:

TR003415

Treatment Summary:

For the experimental conditions, cells were exposed to Nivolumab (10 mg/mL, OPDIVO), anti-PD1) or Ipilimumab (5 mg/mL, YERVOY, anti-CTLA-4) for 48 h. The Nivolumab and Ipilimumab concentrations were chosen in accordance to the posology indications from the European Medicines Agency (EMA) and using as reference a male adult with 80 kg and 5 L of blood. The conditions were tested in monoculture or in co-culture with TCD8+.

Sample Preparation:

Sampleprep ID:	SP003413
Sampleprep Summary:	The supernatant samples were diluted in Milli-Q water to make up 540 μL (150 μL of water was added to 390 μL of supernatant). A volume of 30 µL of phosphate buffer was added to the samples along with 30 μL of a solution of 2.2 mM 3-(trimethylsilyl) propionic 2,2,3,3-d4 acid (TSP) in D2O. TSP was used as an internal reference for quantification and chemical shift.

Analysis:

MB Sample ID:	SA356235
Analysis ID:	AN005383
Analysis Type:	NMR
Num Factors:	27
Num Metabolites:	28
Units:	nanomoles

NMR:

NMR ID:	NM000285
Analysis ID:	AN005383
Instrument Name:	Bruker Ultrashield 500 Plus
Instrument Type:	FT-NMR
NMR Experiment Type:	1D-1H
NMR Comments:	Spectra were acquired in a magnetic field of 500 MHz in an Ultrashied 500 Plus spectrometer (Bruker) with a TCI-Z probe at Instituto de Tecnologia Química e Biológica da Universidade NOVA de Lisboa (ITQB-UNL), Oeiras. The spectra analysis was made in TopSpin 4.1 software, according to Chenomx NMR suite 8.11 and Human Metabolome Database (HMDB).
Spectrometer Frequency:	500 MHz
NMR Probe:	5 mm prodigy TCI-z
NMR Solvent:	10% D2O/90% H20
NMR Tube Size:	5 mm