Figure 5.

Association of tumor-initiating cell (TIC) activity and mitochondrial membrane potential (MMP). (a) Top: Heterogeneous MMP staining (Mitotracker) of spheroid cells assessed by flow cytometry (representative plots shown). Colored cell populations indicate sorted fractions. Bottom: Heterogeneous MMP staining pattern in tumor cells. Axis scale numbers are representative for all plots. PDX, patient-derived xenograft. (b) Spheroid-forming cell (SFC) frequencies of MMP sorted spheroid cells (P1, P4: n = 3; P5: n = 2; P2, P10: n = 1) determined by in vitro limiting dilutions. SFC frequencies were calculated based on sphere formation 5–7 days after seeding and normalized to bulk (P1, P4, P5: n = 2; P2, P10: n = 1). (c) Experimental layout of co-cultivation experiments. x- and y-axis are displayed biexponentially. Results for a representative spheroid culture (P1) are shown. Axis scale numbers are representative for all plots. EGFP, enhanced green fluorescent protein. (d,e) Composition of (d) MMP^low and MMP^high cells or (e) EGFP^– and EGFP⁺ cells in the MMP^low and MMP^high subpopulations over time. t₀: before, t₁: directly after, t₂: 21 days after sort. (f) Results of limiting dilutions in vivo. Left: Overview of dose (D) and response (R). Right: TIC frequencies of MMP sorted spheroid cells. TIC frequencies were calculated based on tumor formation seven weeks (P1: n = 42 mice) or five weeks (P4: n = 48 mice) after transplantation. Freq, frequency; T, tested.