3
MALDI profiling of cancerous vesicles in culture media:
Application to liquid biopsies
Methods
• Two colon cancer cell lines corresponding to a primary
colon cancer (CCL-228) and colon cancer cells derived
from the metastasis cell line (CCL-227) were used in this
study. Cell lines were obtained from the ATCC
(Rockville, MD, USA).
Cell lines were cultured in RPMI-1640 (Gibco,
Invitrogen, Germany) at 37ºC.
• Resistant sub-clones of CCL-227 were generated by
continuous exposure of tumour cells to increasing
concentrations of 5-fluorouracil (FU) over a time period
of more than 2 years. Starting with the addition of 1μM
FU under standard cell culture conditions, this
concentration was gradually enhanced when the
growth rate of the exposed cells was similar to that of
FU naive cells. Escalation to the most resistant cell lines
included cultivation periods at 5, 10, 25, 75 and 125μM
FU; 15–31 cell passages were necessary before
proceeding to the next higher-dose level.
• Culture media was collected with the corresponding
cells for analysis. After the separation of the cells, cell
culture media was subjected to ultracentrifugation and
the pellets were retained. Particle measurements
showed a sample related size distribution of 100-200
nm. Proteins were obtained by solvent-based extraction
from the samples, dried under vacuum and stored at
-80ºC before analysis.
• Mass spectra were obtained using CHCA in the mass
range between 2,000-20,000 m/z. Protein extracts were
prepared in 0.1% TFA: acetonitrile 70:30 (v/v) and 0.5
μL was spotted with 0.5 μL of 10mg/ml CHCA matrix.
Once dry, the samples were analysed using a
MALDI-8020 benchtop MALDI-ToF mass spectrometer
(Shimadzu).
• Partial least squares data analysis (PLS-DA) was
performed using Mass++ DA (Shimadzu)).
Figure 3 - Normalised spectra of the cancerous cells. No obvious differences observed.
