Figure 7.

Two-photon FLIM of NAD(P)H reveals a glycolytic switch in HCT116 spheroids with “direct” and “inverted” oxygen gradients. (AB) 3D reconstructions of phasor-FLIM in 3D of early and mature spheroids with “inverted” gradient phenotype. The “glycolytic core” (demarcated by a purple dashed line) demonstrated local homogeneity of shorter NAD(P)H lifetime and was surrounded by a zone with longer lifetime distribution. Combined phasor plots of sections from 3D stack of spheroid from the “inverted” gradient group display a characteristic shift toward monoexponential free-NAD(P)H lifetime (0.45 ns) (magenta arrow). Scale bar is 100 μm. (C) Comparison of distances (d) between the theoretical position of phasor plot of free-NAD(P)H and centroids of free-NAD(P)H phasor plots of spheroids from “forward” and “inverted” gradient groups. Analyzed phasor plots corresponded to individual XYZ sections of spheroids imaged within ∼100–200 μm depth from the top of the spheroid. T-test (P < 0.05; n “forward” gradient spheroids is 7; n “inverted” gradient spheroids is 9) revealed a significant difference between groups, with the shift of phasor coordinates from “inverted” gradient group spheroids toward the free-NAD(P)H lifetime. Boxes represent standard deviation, and whiskers represent 10 and 90 percentiles. (D) Linear fitting of phasor plot centroids and free NAD(P)H theoretical coordinates demonstrated accurate linear alignment with R² = 0.96. (E) Hypothetical metabolic rearrangements in spheroids during the formation of “inverted” O₂ gradients.