Characterization of naturally occurring floating cells and adherent cells within the CEA-producing UP-LN1 carcinoma cell line in vitro: Identification of cancer stem cells with metastatic potential

[[abstract]]Previously we have documented an initial characterization of a cell line (UP-LN1) from a lymph node metastatic lesion of the neck of a male patient with unknown primary. This tumor was poorly differentiated, and was likely of gastrointestinal tract origin, based on the immunophenotype, CK7-/CK20+/CEA+/SCCA- detected in the original lymph node metastatic lesion and cultured cells. The persistent appearance of floating (designated F) cells along with attachment (A) cells in culture prompted us to investigate into the nature of these two cell types in this study. We found that (i) F and A cells could be isolated and maintained in culture independently; (ii) F cells grew faster and larger than A cells as xenografts in nude mice when injected subcutaneously; and (iii) F cells expressed greater amounts of mRNAs of selected cancer stem cell (CSC) and stem-like cell marker genes, including PSCA (prostate stem cell antigen), ALDH1A1 (aldehyde dehydrogenase 1 family, member A1) and ABCA7 (ABC transporters related drug resistant gene). We hypothesized that F cells contain a higher proportion of CSCs. RT-PCR analysis showed that in consistence with the DNA microarray results, mRNAs of a number of CSC related genes including other drug resistance genes, CD133, ABCC1, ABCC4 and ABCG2 were expressed by F cells in greater quantities. Interestingly, upon treatment with IFN-\#947; at a concentration as low as 150 U/ml, CXCR4 (a chemokine receptor) could be induced in about 20% subpopulation in F cells but not in A cells. It has been reported that CD133+ cancer cells when induced to co-express with CXCR4 acquire potent invasive and metastatic potential. Moreover, F cells but not A cells were able to form aggregates with platelets, suggesting that F cells are capable of forming hematogenous metastasis. The aggregation could be inhibited by anti-CD44 mAb. Taken together, our data suggest that F cells in the UP-LN1 cell line may contain a higher percentage of CSCs, which can be induced to become metastatic CSCs. Experiments are underway to confirm the in vitro observations and to determine whether our in vitro results could be extrapolated into in vivo situations using a NOD-SCID mice xenograft model