Ed cells from a primary implanted tumour, circulating cells in the peripheral blood stream and cells within established metastases in Human parathyroid hormone-(1-34) web newborn scid mice from the same, individual animal could be studied. In addition expression patterns could be compared with those generated in adult scid mice either as primary tumours as lung colonies since spontaneous metastases are not formed in this animal population. We followed the CD44 VE expression changesCD44 Alternative Splicing Pattern of MelanomaFigure 7. Relative quantitative expression of CD44 variable exons in cell cultures from metastatic (newborn) and non-metastatic [adult (AP)] human xenograft model (Real-Time PCR measurement) of HT168M1, a human melanoma cell line of originally high variable exon expression level. A. The relative expression level of all variable exons is raised in certain lung metastasis (NM), when remaines low or even decreases in other lung metastases resulting in large error bars. It should be noted that the expression level of the primaries from different localisations [newborn primary (NP), adult primary (AP) and intravenously implanted lung colony (IVLC)] are all comparable, although the slightly higher expression observed in the adult primary is an unexpected finding. B. We used the lung metastasis from the newborn animal with the highest CD44 variable exon expression level (NM = S1T2) for subcutaneous re-implantation into another newborn animal. The expression level in the primary ASP-015K tumour (PNM) and its metastases (MPNM) was 24 times lower on average. C. The liver metastases (LMIVLC) from the intravenously implanted lung colonies (IVLC) showed a decrease in expression, when compared to the lung colonies (IVLC). doi:10.1371/journal.pone.0053883.gduring tumour progression of two human 10457188 melanomas, that express CD44 VEs at different orders of magnitude, in this experimental animal model. We found that CD44 VE expression and 
metastasis formation showed inverse correlation, similarly to our recent findings in colorectal carcinomas [37]. The adult primary tumour, newborn primary tumour and lung colony of HT199, the human melanoma cell line with low base CD44 VE expression level, all expressed the variable exons in the same order of magnitude and within the error bar. However, as only a limited number of cells within the primary tumour are capable of forming metastasis, the above finding is not surprising. The dramatic increase in CD44 VE expression level seen in the metastatic tumours means, that the variants most likely play a role in metastasis formation. The extreme high level of CD44 VE expression in the circulating tumour cells of the newborn animals seems to indicate, that their role is mainly in getting into and/or surviving in the circulation. It seems, that for the new population of metastatic cells in the target organ, CD44 VE expression level is not as much or even not at all important.The CD44 VE expression level in HT168M1, which has a high base CD44 VE expression, varies within the metastases, ranging from barely detectable to approaching the level detected in circulating tumour cells. When a population with extreme high CD44 VE level is then re-implanted, the expression level dramatically decreases in the metastases while the qualitative picture (fingerprint) remains unchanged. From these results, we suggest that predicting the role of CD44 variable exon expression in tumour progression is more complex than previously anticipated. Our qualitative studies id.Ed cells from a primary implanted tumour, circulating cells in the peripheral blood stream and cells within established metastases in newborn scid mice from the same, individual animal could be studied. In addition expression patterns could be compared with those generated in adult scid mice either as primary tumours as lung colonies since spontaneous metastases are not formed in this animal population. We followed the CD44 VE expression changesCD44 Alternative Splicing Pattern of MelanomaFigure 7. Relative quantitative expression of CD44 variable exons in cell cultures from metastatic (newborn) and non-metastatic [adult (AP)] human xenograft model (Real-Time PCR measurement) of HT168M1, a human melanoma cell line of originally high variable exon expression level. A. The relative expression level of all variable exons is raised in certain lung metastasis (NM), when remaines low or even decreases in other lung metastases resulting in large error bars. It should be noted that the expression level of the primaries from different localisations [newborn primary (NP), adult primary (AP) and intravenously implanted lung colony (IVLC)] are all comparable, although the slightly higher expression observed in the adult primary is an unexpected finding. B. We used the lung metastasis from the newborn animal with the highest CD44 variable exon expression level (NM = S1T2) for subcutaneous re-implantation into another newborn animal. The expression level in the primary tumour (PNM) and its metastases (MPNM) was 24 times lower on average. C. The liver metastases (LMIVLC) from the intravenously implanted lung colonies (IVLC) showed a decrease in expression, when compared to the lung colonies (IVLC). doi:10.1371/journal.pone.0053883.gduring tumour progression of two human 10457188 melanomas, that express CD44 VEs at different orders of magnitude, in this experimental animal model. We found that CD44 VE expression and metastasis formation showed inverse correlation, similarly to our recent findings in colorectal carcinomas [37]. The adult primary tumour, newborn primary tumour and lung colony of HT199, the human melanoma cell line with low base CD44 VE expression level, all expressed the variable exons in the same order of magnitude and within the error bar. However, as only a limited number of cells within the primary tumour are capable of forming metastasis, the above finding is not surprising. The dramatic increase in CD44 VE expression level seen in the metastatic tumours means, that the variants most likely play a role in metastasis formation. The extreme high level of CD44 VE expression in the circulating tumour cells of the newborn 
animals seems to indicate, that their role is mainly in getting into and/or surviving in the circulation. It seems, that for the new population of metastatic cells in the target organ, CD44 VE expression level is not as much or even not at all important.The CD44 VE expression level in HT168M1, which has a high base CD44 VE expression, varies within the metastases, ranging from barely detectable to approaching the level detected in circulating tumour cells. When a population with extreme high CD44 VE level is then re-implanted, the expression level dramatically decreases in the metastases while the qualitative picture (fingerprint) remains unchanged. From these results, we suggest that predicting the role of CD44 variable exon expression in tumour progression is more complex than previously anticipated. Our qualitative studies id.
