Phenotypic stability of early passage stromal cell lines isolated from prostatic adenocarcinoma
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Introduction: The thorough characterization of markers and phenotypic stability in early passage cell lines is necessary for the proper interpretation of data generated by their use. In our research group, 20 early passage stromal cell lines from prostatic adenocarcinoma (PCa) have recently been isolated. As an initial characterization, the transcript levels of known reactive stromal markers VIM1, TNC and CD90 were measured at three time points to evaluate the phenotypic stability of these novel cell lines. Method: One cell line of cancer-associated stromal cells (CAFs) and one of benign prostatic hyperplasiaassociated stromal cells (HAFs) from each of 10 patients were seeded in triplicate. RNA was isolated at first confluency from one of the triplicate wells (p0), while the other two wells were harvested at confluency in passages 1 (p1) and 2 (p2). Following cDNA-synthesis, we used reverse transcription real-time polymerase chain reaction to evaluate transcript levels of markers VIM1, TNC and CD90. Intra- and interplate normalization was performed using GAPDH as endogenous control and a calibrator sample included in all plates. Results: CD90 was significantly upregulated in CAFs compared with HAFs both at p0 and at p1 and p2. The most sizeable difference was found in the p0 cells. No significant difference was found for VIM1 or TNC. CD90 levels increased with passage, TNC showed no obvious pattern, while VIM1 was stable from p0 to p1, but showed a marked increase at p2. Discussion: The higher CD90 expression in CAFs is in accordance with other studies, and indicates that our cells are representative of in vivo CAFs. However, the lack of difference between CAFs and HAFs for VIM1 and TNC renders our findings inconclusive. The expression levels of all three markers varied with passage, suggesting a lack of phenotypic stability. Conclusion: The data indicate that our cell lines are representative of the in vivo situation. However, it would not be advisable to passage them before study, as the differences between CAFs and HAFs appear to decrease over time, and transcript levels vary considerably with passage.