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

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)

Use the 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.

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.

Using a pipet, take 200µL of the culture medium of each well and place it into a microtube. Then, add 50µL 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 (20µL) 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.

Centrifuge the samples for 12880x g,4°C.

Remove a medium aliquot of 150µLof each sample and dry under nitrogen pressure.

Resuspend the dry extracts in 150µL of a solution composed ofat 200micromolar (µM) in: 1:1 v/v).

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) .

Set the flow rate to 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 to 30°C, while the injection volume must be 5µLfor the positive and 2µLfor the negative ionization modes.

Perform the analyses using the XEVO-G2XSQTOF (Waters) instrument equipped with an electrospray ion source.

Use a 0.5millimolar (mM) sodium formate solution for the instrument calibration.

Perform the analyses in the positive (+) and negative (-) ionization modes.

Optimize the source parameters for better performance. Suggestion of initial parameters:

A	B	C
Parameters	Positive Ion Mode	Negative Ion Mode
Source temperature (°C)	140	140
Desolvation temperature (°C)	550	550
Desolvation flow (L/h)	900	900
Capillary (kV)	3	2.5
Sampling cone (kV)	30	40
Cone gas flow (L/h)	10	50

Acquire the spectra under the acquisition range of 50-1200 Da, using the MS^E 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:1:1 v/v) as the lockmass, infused at 25 μL/min.

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 MS^E -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.

A	B	C	D	E	F	G	H	I	J
Compound	Compound ID	Adducts	Formula	Score	Fragmentation Score	Mass Error (ppm)	Isotope Similarity	Description	Annotation Confidence Level*
Mode Negative
4.58_245.0920m/z	HMDB0004259	M-H2O-H	C13H16N2O4	38,4	0	-4,29	96,9	Acetyl-N-formyl-5-methoxykynurenamine	3
8.13_301.2162m/z	HMDB0011134	M-H2O-H	C20H32O3	39,9	5,12	-3,30	98,4	5-HETE	2
9.00_305.2475m/z	HMDB0002925	M-H	C20H34O2	38,7	0	-3,67	97,8	8,11,14-Eicosatrienoic acid	3
8.59_329.2473m/z	HMDB0001976	M-H	C22H34O2	38,8	0	-4,08	98,8	Docosapentaenoic acid (22n-6)	3
8.49_303.2320m/z	HMDB0001043	M-H	C20H32O2	40,3	5,73	-3,27	99,6	Arachidonic acid	2
0.61_232.0824m/z	HMDB0012150	M+FA-H	C8H13NO4	40	11,7	-1,46	90,2	2-Keto-6-acetamidocaproate	2
0.61_134.0460m/z	HMDB0000056	M+FA-H	C3H7NO2	39,8	4,66	1,51	96,0	beta-Alanine	2
0.54_802.6697m/z	HMDB0013408	M-H	C46H94NO7P	37,1	0	0,29	85,9	PC(o-16:0/22:0)	3
1.67_291.0973m/z	HMDB0011741	M-H2O-H	C14H18N2O6	38,7	0	-4,25	98,5	gamma-Glutamyltyrosine	3
3.52_241.1184m/z	HMDB0011170	M-H2O-H	C11H20N2O5	41,1	13,4	-3,83	96,4	gamma-Glutamylisoleucine	2
0.75_151.0247m/z	HMDB0000139	M+FA-H	C3H6O4	39,5	3,17	-0,62	95,0	Glyceric acid	2
Mode Positive
0.56_251.1008n	HMDB0000101	M+Na, M+K, M+H, M+H-H2O	C10H13N5O3	39	5,71	-4,30	94,5	Deoxyadenosine	2
0.54_139.0743n	HMDB0012234	M+H-H2O, M+H	C6H9N3O	39,6	10,2	-2,20	90,2	Histidinal	2
4.43_407.1214m/z	HMDB0062198	M+ACN+H	C12H19N3O8S	38,9	3,93	-4,58	96,0	2-S-glutathionyl acetate	2
1.39_298.0970m/z	HMDB0001173	M+H	C11H15N5O3S	43,5	19	0,59	98,9	5'-Methylthioadenosine	2
0.77_153.0402m/z	HMDB0000292	M+H	C5H4N4O2	39,6	4,75	-3,13	97,2	Xanthine	2
0.65_152.0566m/z	HMDB0000132	M+H	C5H5N5O	40,6	4,18	-0,46	99,4	Guanine	2
0.50_364.2445m/z	HMDB0011531	M+ACN+Na	C17H32O4	38,8	0,505	-4,36	98,6	MG(0:0/14:1(9Z)/0:0)	2

• Annotation Confidence Level. (1) Reference standard confirmed structure; (2) exact mass, isotopic pattern, retention time, and MS/MS spectrum matched to an in-house spectral database or literature spectra; (3) putative ID assignment based only on elemental formula match with exact mass and isotopic pattern, and (4) unknown compound.

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.

Rank the relevant molecular features according to the false discovered ratio (FDR) and log2 fold change (FC) values.

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).