Figure 3.
Time-Dependent Decrease in EGFP Expression in Initially 100% EGFP(+) Cells Indicates Gradual Loss of Stemness Observed In Vitro and In Vivo
(A) Example of EGFP(+) and EGFP(−) ZL55-SO cells in a cell culture dish initially plated with FACS-sorted ZL55-SOhigh cells. At 80 days in vitro many non-green EGFP(−) cells are observed, often arranged in clusters (marked by yellow dotted lines) and EGFP(+) (examples marked by red dotted lines).
(B) Decrease of the fraction of EGFP(+) cells starting from sorted ZL55-SOhigh cells (initially 78.6% EGFP(+) cells) to 68.7% after culturing without puromycin selection for 35 days (ten passages). Note the appearance of a distinct population of cells with lower EGFP fluorescence (arrow).
(C) Tumor tissue derived from a mouse injected with RN5-SOhigh cells for 8 weeks in vivo. In the tumor mass identified by pan-cytokeratin (CK) immunohistochemistry (red fluorescence), clusters of cells with weak-to-none EGFP staining (green fluorescent image; left) are seen. Staining with DAPI (blue) allows better identification of individual cells. In the merged image, the presence of clusters of yellow cells (co-localization of CK and EGFP), but also of cells without EGFP (CK only) demonstrates loss of EGFP expression over time in vivo.
(D) RN5-SOhigh cells additionally expressing NLS-mCherry (red fluorescent nuclei) formed tumors in vivo as demonstrated after 8 weeks. Tumor-derived cells were cultivated in vitro and revealed that a fraction of NLS-mCherry(+) cells had lost EGFP expression (cells marked by arrows). In the merged image these cells appear as red only.
(E) Cells shown in (D) were maintained in culture in vitro for an additional 57 days and analyzed by FACS for green (EGFP) and red (NLS-mCherry) fluorescence. While the fraction of NLS-mCherry(+) cells only marginally decreased from 99.8% to 99.0%, the fraction of EGFP(+) cells decreased from 99.6% to 77.8%. Note the left shift of the population of EGFP(+) cells: blue cloud at t1; red cloud at t57. See also Figure S2.