Protocol to secretome investigation of tumor 3D co-culture model
ANNA MARIA AP FERNANDES, Giulia Carli Mendes, Alex Rosini, Andrea Corazzi Pelosi, Leonardo Maciel, Luísa F Bueno, Lívia Maria F Silva, Rafael F Bredariol, Maycon Giovani Santana, Andreia de Melo Porcari, Denise G. Priolli
Abstract
Three-dimensional (3D) cell culture technologies, which more closely mimic the complex microenvironment of tissue, are being increasingly evaluated as a tool for the preclinical screening of clinically promising new molecules, and for the study of tissue metabolism. Studies of metabolites released into the extracellular space (secretome) allow understanding the metabolic dynamics of tissues and changes caused by therapeutic interventions. Although quite advanced in the field of proteomics, studies on the secretome of low molecular weight metabolites (< 1500 Da) are still very scarce.
We present an untargeted metabolomic protocol based on the hybrid technique of high-performance liquid- chromatography coupled with high-resolution mass spectrometry for the analysis of low-molecular-weight metabolites released into the culture medium by 3D cultures and coculture (secretoma model). For that, we analayzed HT-29 human colon carcinoma cells and 3T3-L1 preadipocytes in 3D-monoculture and 3D-coculture.
This protocol represents a possibility to list metabolites released in the extracellular environment in a comprehensive and untargeted manner, opening the way for the generation of metabolic hypotheses that will certainly contribute to the understanding of tissue metabolism, tissue-tissue interactions, and metabolic responses to the most varied interventions. Moreover, it brings potential to determine novel pathways and identify accurate biomarkers in cancer and other diseases. The metabolites indicated in our study have a close relationship with the tumor microenvironment in accordance with the literature review.
For the 3D cell culture by levitation we used Bio-Assembler™ system (Bio Science 662840, Greiner One Bio, Americana, Brazil) in the n3D Biosciences and adapted protocol published by:
- HAISLER, W. L. et al. Three-dimensional cell culturing by magnetic levitation. Nature protocols , v. 8, n. 10, p. 1940-1949, 2013. https://doi.org/10.1038/nprot.2013.125
Steps
2D CELL CULTURE
Use human colon carcinoma (HT-29) and pre-adipocytes cells (3T3-L1) (Banco de Células do Rio de Janeiro (BCRJ; Duque de Caxias, Brazil).
Thaw HT-29 and 3T3-L1 cells and propagate in culture using Modified Dulbecco Eagle Medium (DMEM - Sigma D-5648, São Paulo, Brazil), supplement with 100millimolar (mM) sodium pyruvate (Gibco -11- 360, Thermo Fisher Scientific, Waltham, Massachusetts, USA), 10% (v/v) fetal bovine serum (Gibco 2010-09, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 1% (v/v) antibiotics (100U/ml of penicillin and 10mg/mL of streptomycin (Gibco 15140-122, Thermo Fisher Scientific, Waltham, Massachusetts, USA).
Culture cells in a humidified chamber with5% (v/v) CO2(HeraCELL 150) at 37°C.
Incubate cell cultures with 3mL trypsin-EDTA 0.25% (v/v) (Gibco 25 200, Fisher Scientific, Waltham, Massachusetts, USA) at 37°C for three minutes to allow cell disaggregation and propagation.
Use DMEM plus 10% FBS to inactivate trypsin. Transfer the cell pellet to a new 75 cm3 flask (T75) containing 10mL DMEM.
Change the culture medium according to the cell doubling time.
Determine cell viability in a Neubauer chamber using Trypan Blue (Gibco 15250061, Thermo Fisher Scientific, Waltham, Massachusetts, USA)
3D CELL CULTURE
Use Bio-Assembler™ system (Bio Science 662840, Greiner One Bio, Americana, Brazil) in the n3D Biosciences 24-well configuration (HAISLER et al., 2013).
Prepare the magnetic nanoparticles by removing it from the refrigerator and thawing it at room temperature 20-25°C for about 15 min.
Culture HT- 29 cells (passage 12th) in monolayer culture at T75 flask. Determine the cell viability (>75%) in a Neubauer chamber, when the cells confluence reaches 80%-90%.
Add 1µL of magnetic nanoparticles (Nanoshuttle™- PL, Greiner) in the single cell suspension flask, homogenize gently the suspension and centrifuge 1500rpm.
Resuspend the cells and fill each well of cell-repellent 24-well plate with an amount of solution necessary to reach 7.5 x 103cells, after centrifugation.
Complete with 250µLof supplemented medium Modified Dulbecco Eagle Medium (DMEM - Sigma D-5648, São Paulo, Brazil), supplement with 100millimolar (mM) sodium pyruvate (Gibco -11- 360, Thermo Fisher Scientific, Waltham, Massachusetts, USA), 10% (v/v) fetal bovine serum (Gibco 2010-09, Fisher Scientific, Waltham, Massachusetts, USA) and 1% (v/v) antibiotics (100U/ml of penicillin and 10mg/mLof streptomycin) (Gibco 15140-122, Thermo Fisher Scientific, Waltham, Massachusetts, USA) to a volume of 250µL
Place a magnetic coupling driver under the plate for 5h 0m 0sand incubate it in the humidified chamber with5% (v/v) CO2(HeraCELL 150) at 37°C.
Close the plate and place the levitation drive atop the intermediate lid to levitate the cells.
Keep the magnetic coupling drive for 7 days.
Use the field microscopy to verify the cohesion of the structures formed.
Collect the culture medium whenever the exchange is necessary. To do it, use the holding drive to hold the 3D culture down while aspirating the liquid.
Culture 3T3-L1 cells 600µL in monolayer to 90% confluence and cell viability (>75%), and incubate in a supplemented medium in repellent hanging drop plate at 37°C/5% (v/v) CO2/95 humidity and monitore until the aggregates have formed and differentiated to adipocytes.
3D CELL COCULTURE
Add 3T3-L1 spheroid suspension in each well of cell repellent HT-29 wells plate using a magnetic pen, 21 days after the beginning of HT-29 spheroid formation and keep it for 7 days.
EXTRACTION OF SAMPLES
Add an aliquot of iced isopropanol 50mg to the culture medium 200µL and maintain at -20°C, for secretome analysis.
Centrifuge the samples for 12500rpm,4°C.
Remove a medium aliquot of 150µLof each sample and dry under nitrogen pressure.
Resuspend dry extracts in 150µL of an internal fluoride-phenylalanine standard solution at200micromolar (µM).
LIQUID CHROMATOGRAPHY ANALYSES
Perform chromatography analyses using a UPLC H-class (Waters), with an ACQUITY CSH C18 column (Waters) with dimensions 2.1 × 100 mm x 1.7 μm using a mobile phase:
Phase A)0.1% (v/v)
Phase B)
Set the flow rate at 0.4 mL/min.
Apply the segmented gradient as follows:
| A | B | C | D |
|---|---|---|---|
| Time (min) | Flow (ml/min) | A (%) | B (%) |
| Initial | 0.400 | 90.0 | 10.0 |
| 2.00 | 0.400 | 90.0 | 10.0 |
| 7.00 | 0.400 | 10.0 | 90.0 |
| 9.00 | 0.400 | 10.0 | 90.0 |
| 11.00 | 0.400 | 90.0 | 10.0 |
| 13.00 | 0.400 | 90.0 | 10.0 |
Set the temperature at 20°C, while the injection volume is 5µLfor positive and 2µLfor negative modes.
MASS SPECTROMETRY ANALYSES
Perform the analyses using XEVO-G2XSQTOF (Waters) equipment equipped with an electrospray source.
Perform the analyses in the mode of positive (+) and negative (-) ionization.
Optimize the font parameters for better performance. Initially:
| A | B | C |
|---|---|---|
| Parameters | Mode Positive | Mode Negative |
| Source temperature (°C) | 140 | 140 |
| Desolvation temperature (°C) | 550 | 550 |
| Desolvation flow (L.h-1) | 900 | 900 |
| Capillary (kV) | 3 | 2.5 |
| Sampling cone (kV) | 30 | 40 |
| Cone gas flow (L.h-1) | 10 | 50 |
Acquire the spectra under the acquisition mass range of 50-1200 Da, using the MSE approach (6 V for low energy and a 15-30 V ramp for high energy scanning).
Use Leucine encephalin 200Parts per Million (PPM) in an
Use a 0.5millimolar (mM) sodium formate solution for instrument calibration.
DATA PROCESSING AND POTENTIAL IDENTIFICATION OF COMPOUNDS
Process the raw data with Progenesis IQ (Waters) software for peak detection, alignment, integration, deconvolution, data filtering, ion annotation, and MSE based putative identification of compounds.
Use the following databases to suggest the identifications : Lipid Maps (http://www.lipidmaps.org/), LipidBlast (https://fiehnlab.ucdavis.edu/projects/LipidBlast) and Human Metabolome Database (http://www.hmdb.ca/metabolites).
Use the following parameters for identification : mass error of the precursor ≤ 5 ppm, mass error of the fragment ≤ 10 ppm, mass precision, and isotopic similarity.
STATISTICAL ANALYSES
Perform the volcano plot statistical test between all culture conditions and in the presence and absence (white samples) of the cells.
Rank the relevant molecular features according to the false discovered ratio (FDR) and log2 fold change (FC) values.
Consider only the molecular features that presented FDR value < 0.05 and log2 FC>0 when compared to the secretome of the culture medium (white), In the assignment of cell secretomes
