Evaluation of Tumor Interstitial Fluid-Extraction Methods for Proteome Analysis: Comparison of Biopsy Elution versus Centrifugation

The analysis of tumor
interstitial fluid (TIF) composition is a
valuable procedure to identify antimetastatic targets, and different
laboratories have set up techniques for TIF isolation and proteomic
analyses. However, those methods had never been compared in samples
from the same tumor and patient. In this work, we compared the two
most used methods, elution and centrifugation, in pieces of the same
biopsy samples of cutaneous squamous cell carcinoma (cSCC). First,
we established that high G-force (10 000g)
was required to obtain TIF from cSCC by centrifugation. Second, we
compared the centrifugation method with the elution method in pieces
of three different cSCC tumors. We found that the mean protein intensities
based in the number of peptide spectrum matches was significantly
higher in the centrifuged samples than in the eluted samples. Regarding
the robustness of the methods, we observed higher overlapping between
both methods (77–80%) than among samples (50%). These results
suggest that there exists an elevated consistence of TIF composition
independently of the method used. However, we observed a 3-fold increase
of extracellular proteins in nonoverlapped proteome obtained by centrifugation.
We therefore conclude that centrifugation is the method of choice
to study the proteome of TIF from cutaneous biopsies.