ABSTRACT: In order to metastasize, few cells need to detach from the primary tumor, and transition through the circulation to distant sites to establish new solid tumor metastases. The gene expression changes occurring between these transitions are poorly understood, and controversial findings have been reported regarding transitions between epithelial (E) and mesenchymal (M) cell states. To model the metastasis process in vitro, here we examined the dynamics of gene expression changes and respective cell fates that are occurring in cells that are transitioning from adhesion culture to suspension 3D culture (mammospheres) and back to adhesion culture in vitro. We found that mammosphere and replating cultures of epithelial (E) cell lines (HP cells and E clones) led to enrichment for mesenchymal (M) signatures characteristic of the mesenchymal cell lines, indicative of an epithelial to mesenchymal transition (EMT), but retainment of E-specific signatures (Grosse-Wilde et al., 2015). This co-expression of E and M signatures was stably maintained between the mammosphere state in suspension and in replated cells, indicating a stable E/M gene expression state by partial EMT in E cell lines, which has been associated with a stem-like state (Grosse-Wilde et al., 2015; Jolly et al., 2014; Lu et al., 2014; Zhang et al., 2014). Interestingly, mammosphere suspension culture of mesenchymal M clones also resulted in co-expression of E and M signatures generated by partial mesenchymal to epithelial transition (MET). However, in stark contrast to E clones and HP cells, immediately upon replating M cell-derived mammospheres to adhesion lost the intermediate E/M state and converted back to stable expression of M signatures indicative of rapid EMT. This suggests that the intermediate state in M cells is very unstable. Together, these replating data suggest that prolonged detachment of epithelial-derived cells leads to loss of expression of E signatures and EMT. Life-threatening macrometastases typically recapitulate the gene expression of the primary tumor, which requires expression of E signatures, while the mesenchymal micrometastases are frequent but not-proliferative and harmless. Since all HMLER cell lines independent of their initial E or M state converged to an M state upon expansion in suspension, our data suggest that induction of EMT in suspended HMLER cells may be one reason for the observed benign non-metastatic character of HMLER cells when used in mouse xenograft experiments, and for their metastatic inefficiency. Our datasets of the replating kinetic may be useful to identify which pathways are involved in the processes enabling the mixed HP population to maintain a stable E/M expression signature and a more stem-like state, which we have identified as caused by cell-cell cooperation between phenotypically different E and M cell-types. By contrast, M cells can only transiently assume this E/M gene expression state, and not generate a stable phenotypic heterogeneity and therefore absence of cooperation between E and M cell-types. Instability of the intermediate stem-like E/M state in M cell-types and rapid conversion to the M state can explain why 1) pure M populations are often phenotypically stable and are not undergoing MET in vitro (Schmidt et al., 2015; Zhang et al., 2014), 2) why pure M populations are less aggressive resulting in less metastases than mixed cooperating E and M cell populations (Ocaña et al., 2012; Tsuji et al., 2008) or 3) why complete EMT results in more benign less metastasizing tumors than incomplete EMT (Tran et al., 2014; Tsai et al., 2012) and finally 4) why expression of M traits in primary tumors are associated with good patient outcomes and less metastases than E signatures (Kim et al., 2011; Mylona et al., 2008; Tan et al., 2014). 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Tsuji, T., Ibaragi, S., Shima, K., Hu, M.G., Katsurano, M., Sasaki, A., and Hu, G. -f. (2008). Epithelial-Mesenchymal Transition Induced by Growth Suppressor p12CDK2-AP1 Promotes Tumor Cell Local Invasion but Suppresses Distant Colony Growth. Cancer Res. 68, 10377–10386. Zhang, J., Tian, X.-J., Zhang, H., Teng, Y., Li, R., Bai, F., Elankumaran, S., and Xing, J. (2014). TGF- -induced epithelial-to-mesenchymal transition proceeds through stepwise activation of multiple feedback loops. Sci. Signal. 7, ra91–ra91. For these analyses we used the heterogeneous parental HMLER (HP) cell line, with isogenic epithelial (E) and mesenchymal (M) cell-types (Elenbaas et al., 2001), and stable clonal HMLER-derived E and M cell lines, which are normally passaged under adhesion conditions (_adh, GSE66527). We cultured cell lines under 3D suspension conditions resembling mammosphere growth conditions that are typically used to enrich for stem-like cells (Dontu et al., 2003), and examined gene expression of cells under mammosphere conditions (_sus, GSE66527), or after replating (_re) in a kinetic examining gene expression from 6, 24, 96, and 240 hrs after cells were allowed to readhere to normal dishes for the heterogeneous but mostly epithelial (HP) cell lines and mesenchymal (M4) cell lines. To validate our findings we also examined gene expression of cells replated after mammosphere culture for about two weeks from three different E (E3, E4, E5) and M clones (M3, M4, M1). All gene expression samples were taken from biological replicates (at least duplicates) of cells grown in different wells or even at different times. Gene expression of replated (_re) cells was compared to the respective cell lines grown under adhesion conditions (_adh), as well as to the respective cell line grown under suspension mammosphere condition (_sus) which have been deposited previously under accession number GSE66527. HP replicates (re): HP_re_AGW120913, HP_re_RK12026, HP_re_AGW1209_5, HP_re_AGW1209_6, HP_re_AGW1209_7, HP_re_AGW1209_8, HP_re_AGW1209_9, HP_re_0607_1, HP_re_0607_2 E3 replicates (re): E3_re_RK1202, E3_re_RK12028; E4 replicates: E4_re_0622, E4_re_0613; E5 replicates: E5_re_0613_1, E5_re_0613_2 M1 replicates (re): M1_re_0607_1, M1_re_0607_2, M2 replicates: M2_re_RK1202, M2_re_RK12029, M2_re_AGW1209_3, M2_re_AGW1209_4, M2_re_AGW1209_5, M3 replicates: M4_re_0607_1, M4_re_0607_2 HP replicates (adh): HP_adh_0629, HP_adh_2210, HP_adh_0607sc, HP_adh_0607c E3 replicates (adh): E3_adh_0629, E3_adh, E3_adh_0826. E4 replicates (adh): E4_adh_0607, E4_adh_0210, E5 replicates (adh): E5_adh_0210, E5_adh_0607 M1 replicates (adh): M1_adh_0210, M1_adh_0607, M4 replicates (adh): M4_adh_0607, M4_adh_0210, M3 replicates (adh): M3_adh, M3_adh_2210, M3_adh_2810 HP replating kinetic (adh): HP_adh_0607c (GSE66527), HP_adh_0607sc (GSE66527), HP (mammospheres, suspension): HP_sus_0603_2 (GSE66527), HP_sus_0603_1 (GSE66527), HP (replated 6 hrs): HP_re_6_0603_1, HP_re_6_0603_2, HP (replated 24hrs): HP_re_24_0607_1, HP_re_24_0607_2, HP (replated 96 hrs): HP_re_96_0607_1, HP_re_96_0607_2, HP (replated 240hrs): HP_re_0607_1, HP_re_0607_2, M4 replating kinetic (adh): M4_adh_0210 (GSE66527), M4_adh_0607 (GSE66527), M4 (mammospheres, suspension): M4_sus_0603_2 (GSE66527), M4_sus_0603_1 (GSE66527), M4 (replated 24 hrs): M4_re_24_0603_1, M4_re_24_0603_2, M4 (replated 96 hrs), M4_re_96_0607_1, M4_re_96_0607_2, M4 replated 240hrs): M4_re_0607_1, M4_re_0607_2
