TITLE: Protocol to secretome investigation of tumor 3D co-culture model
AUTHORS: 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

[DESCRIPTION]
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


[GUIDELINES]
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]
SECTION: 2D CELL CULTURE
1. Use human colon carcinoma (HT-29) and pre-adipocytes cells (3T3-L1) (Banco de Células do Rio de Janeiro (BCRJ; Duque de Caxias, Brazil).
SECTION: 2D CELL CULTURE
2. 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 100 millimolar (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 (100 U/ml of penicillin and 10 mg/mL of streptomycin (Gibco 15140-122, Thermo Fisher Scientific, Waltham, Massachusetts, USA).

SECTION: 2D CELL CULTURE
3. Culture cells in a humidified chamber with5 % (v/v) CO2 (HeraCELL 150) at 37 °C.

SECTION: 2D CELL CULTURE
4. Incubate cell cultures with 3 mL 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 10 mL DMEM.

SECTION: 2D CELL CULTURE
5. Change the culture medium according to the cell doubling time.
SECTION: 2D CELL CULTURE
6. Determine cell viability in a Neubauer chamber using Trypan Blue (Gibco 15250061, Thermo Fisher Scientific, Waltham, Massachusetts, USA)

SECTION: 3D CELL CULTURE
7. Use the Bio-Assembler™ system (Bio Science 662840, Greiner One Bio, Americana, Brazil) in the n3D Biosciences 24-well configuration (HAISLER et al., 2013).
 

SECTION: 3D CELL CULTURE
8. Prepare the magnetic nanoparticles by removing it from the refrigerator and thawing it at room temperature 20-25 °C  for about 15 min. 

SECTION: 3D CELL CULTURE
9. 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%.
SECTION: 3D CELL CULTURE
10. Add  1 µL of magnetic nanoparticles (Nanoshuttle™- PL, Greiner)  in the single cell suspension flask, homogenize gently the suspension and centrifuge 1500 rpm, 5 min.

SECTION: 3D CELL CULTURE
11. Resuspend the cells and fill each well of cell-repellent 24-well plate with an amount of solution necessary to reach 7.5 x 103 cells, after centrifugation.

SECTION: 3D CELL CULTURE
12. Complete with 250 µLof supplemented medium Modified Dulbecco Eagle Medium (DMEM - Sigma D-5648, São Paulo, Brazil), supplement with 100 millimolar (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 (100 U/ml of penicillin and 10 mg/mLof streptomycin) (Gibco 15140-122, Thermo Fisher Scientific, Waltham, Massachusetts, USA) to a volume of 250 µL

SECTION: 3D CELL CULTURE
13. Place a magnetic coupling driver under the plate for 300 minand incubate it in the humidified chamber with5 % (v/v) CO2 (HeraCELL 150) at 37 °C.

SECTION: 3D CELL CULTURE
14. Close the plate and place the levitation drive atop the intermediate lid to levitate the cells. 

 

SECTION: 3D CELL CULTURE
15. 
Keep the magnetic coupling drive for 7 days. 


SECTION: 3D CELL CULTURE
16. 
Use the field microscopy to verify the cohesion of the structures formed.
SECTION: 3D CELL CULTURE
17. 
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.
SECTION: 3D CELL CULTURE
18. Culture 3T3-L1 cells600 µL  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/  humidity and monitore until the aggregates have formed and differentiated to adipocytes.

SECTION: 3D CELL COCULTURE
19. 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.
SECTION: EXTRACTION OF SAMPLES
20. 
Using a pipet, take  200 µL  of the culture medium of each well and place it into a microtube. Then, add 50 µL of iced isopropanol and keep the microtube at  -20 °C   . Prepare each sample in triplicate. Additionally, prepare a pooled quality control (QC) sample. For that, an aliquot (20 µL)  of each sample must be collected and pooled together in a microtube to generate a QC sample containing the chemical composition representative of the sample set. Homogenize this sample and split it into different vials, then prepare them following the same extraction protocol. Use QC samples for system suitability before batch analysis. Also, inject a QC sample after 10 samples to investigate and filter instrumental variation. Blank samples (N=3) must also be prepared using the culture medium without adding any cell or culture and following the complete protocol for sample preparation. Samples must be organized by starting with QC samples, followed by blank samples, and then the set of test samples randomly organized. A QC sample must be placed every 10 samples. The final sample must be a QC sample.

SECTION: EXTRACTION OF SAMPLES
21. Centrifuge the samples  for 12880 x g, 10 min, 4 °C.

SECTION: EXTRACTION OF SAMPLES
22. Remove a medium aliquot of 150 µLof each sample and dry under nitrogen pressure.
SECTION: EXTRACTION OF SAMPLES
23. Resuspend the dry extracts in 150 µL of a solution composed of at 200 micromolar (µM) in :  1:1 v/v).
SECTION: LIQUID CHROMATOGRAPHY ANALYSES
24. 
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)  plus  0.1 % (v/v)  ..   
Phase B)  . 

SECTION: LIQUID CHROMATOGRAPHY ANALYSES
25. Set the flow rate to 0.4 mL/min.

SECTION: LIQUID CHROMATOGRAPHY ANALYSES
26. Apply the segmented gradient as follows: 
 


SECTION: LIQUID CHROMATOGRAPHY ANALYSES
27. Set the temperature to 30 °C, while the injection volume must be 5 µLfor the positive and 2 µLfor the negative ionization modes.

SECTION: MASS SPECTROMETRY ANALYSES
28. Perform the analyses using the XEVO-G2XSQTOF (Waters) instrument equipped with an electrospray ion source.
SECTION: MASS SPECTROMETRY ANALYSES
30. Perform the analyses  in the positive (+) and negative (-) ionization modes. 
SECTION: MASS SPECTROMETRY ANALYSES
31. Optimize the source parameters for better performance. Suggestion of initial parameters:

 

SECTION: MASS SPECTROMETRY ANALYSES
32. Acquire the spectra  under the acquisition range of 50-1200 Da, using the MSE approach (6 V for low-energy, and a 15-30 V ramp for high-energy scanning).

SECTION: MASS SPECTROMETRY ANALYSES
33. Use Leucine encephalin 200 Parts per Million (PPM)  in an : 1:1 v/v)  as the lockmass, infused at 25 μL/min.

SECTION: MASS SPECTROMETRY ANALYSES
29. Use a  0.5 millimolar (mM) sodium formate solution  for the instrument calibration.

SECTION: DATA PROCESSING AND POTENTIAL IDENTIFICATION OF COMPOUNDS
34. Process the .RAW files obtained after LC-ESI-MS analysis by using the Progenesis QI software 2.4.69.11 (Nonlinear Dynamics, Newcastle, UK)). Choose to use centroid data and the mass resolution of 40,000. Perform peak alignment based on the QC samples. Select the following adduct species: [M+H]+, [M+Na]+, [M+K]+, [M+ACN+Na]+, [M+ACN+H]+, [M+H -H2O]+, [M+H-2H2O]+ for the positive ion mode, and [M-H]-, [M+FA-H]-, [M+Na-2H]-, [M-H2O-H]-, [M +Cl]-, for the negative ion mode. Progenesis QI generates a table containing the intensity of the ions listed according to their nominal masses for each sample. This software also generates MSE -based putative identification of compounds.
SECTION: DATA PROCESSING AND POTENTIAL IDENTIFICATION OF COMPOUNDS
35. 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). 
SECTION: DATA PROCESSING AND POTENTIAL IDENTIFICATION OF COMPOUNDS
36. 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. 


 

SECTION: STATISTICAL ANALYSES
37. 
The statistical analyses were performed using the Metaboanalyst web platform (version 5.0 - https://www.metaboanalyst.ca/). For the negative ionization mode: data was normalized by the  IS (  ), log-transformed, and Pareto scaled. The same procedure was used for the positive ionization mode, but the cubic transformation was applied instead of the log.

Perform multiple volcano plots to test between the compared culture conditions and also to blank samples.
SECTION: STATISTICAL ANALYSES
38. Rank the relevant molecular features  according to the false discovered ratio (FDR) and log2 fold change (FC) values.
SECTION: STATISTICAL ANALYSES
39. For the assignment of cell secretomes, consider only the molecular features that present FDR value < 0.05 and log2 FC>0 when compared to the secretome of the culture medium (blank sample).