Experimental Verification on the Hypothesis about the Possibility of Molecular Diagnostics of Local Tumor Spread on the Lewis Lung Carcinoma Model

Abstract

Expression of tumor-associated protein beta-III tubulin (TUBB3) was evaluated quantitatively by immunofluorescence analysis using flow cytometry in lung tissue of intact animals and on day 11 after the Lewis lung carcinoma transplantation as well in lung metastasis tissue and in visually “normal” surrounding tissue. It was shown that the tumor is characterized by a high TUBB3 expression, while also the expression of the marker was detected in both variants of visually normal lung tissue: it was lower than in tumor tissue but significantly higher than in lung tissue of intact animals. The detection of TUBB3 outside the tumor indicates that the tissue appeared to be normal has already involved in malignancy and this supports the hypothesis that tumor-associated protein TUBB3 can be used as a molecular marker of local tumor spread.

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“…TUBB3 expression was evaluated by immunofluorescence assay and flow cytometry [ 33 ] . The cell suspensions at a concentration of 4 × 10 5 cells/ml were incubated for 1.5 h with primary monoclonal anti-TUBB3 antibody (clone EP1569Y, Abcam, UK) at room temperature in the dark; the suspensions were then incubated for 1.5 h with secondary anti-rabbit antibody conjugated with DyLight®650 (ab98510, Abcam, UK) at + 4°С in the dark.…”

“…Secondary antibodies to rabbit Ig conjugated with horseradish peroxidase were used for the detection (Jackson ImmunoResearch). ECL detection reagents for analysis were prepared according to Mruk and Cheng's protocol [32] . ImageQuant LAS 4000 imager (GE Healthcare) was used to detect signals.…”