Simultaneous Probing of Metabolism and Oxygenation of Tumors In Vivo Using FLIM of NAD(P)H and PLIM of a New Polymeric Ir(III) Oxygen Sensor

Abstract

Tumor cells are well adapted to grow in conditions of variable oxygen supply and hypoxia by switching between different metabolic pathways. However, the regulatory effect of oxygen on metabolism and its contribution to the metabolic heterogeneity of tumors have not been fully explored. In this study, we develop a methodology for the simultaneous analysis of cellular metabolic status, using the fluorescence lifetime imaging microscopy (FLIM) of metabolic cofactor NAD(P)H, and oxygen level, using the phosphorescence lifetime imaging (PLIM) of a new polymeric Ir(III)-based sensor (PIr3) in tumors in vivo. The sensor, derived from a polynorbornene and cyclometalated iridium(III) complex, exhibits the oxygen-dependent quenching of phosphorescence with a 40% longer lifetime in degassed compared to aerated solutions. In vitro, hypoxia resulted in a correlative increase in PIr3 phosphorescence lifetime and free (glycolytic) NAD(P)H fraction in cells. In vivo, mouse tumors demonstrated a high degree of cellular-level heterogeneity of both metabolic and oxygen states, and a lower dependence of metabolism on oxygen than cells in vitro. The small tumors were hypoxic, while the advanced tumors contained areas of normoxia and hypoxia, which was consistent with the pimonidazole assay and angiographic imaging. Dual FLIM/PLIM metabolic/oxygen imaging will be valuable in preclinical investigations into the effects of hypoxia on metabolic aspects of tumor progression and treatment response.

Keywords: bioimaging; in vitro; in vivo; oxygen sensing; phosphorescence lifetime imaging; phosphorescent polymeric iridium(III) complexes; tumor.

Figure 1

Chemical structures of the polymeric probes PIr1–PIr3.

Figure 2

Absorption spectra of PIr1–PIr3 in CH₂C_l2 solution (A,C) and in H₂O solution (B,D).

Figure 3

Photoluminescence spectra of PIr1–PIr3 in (A,B,E) CH₂C_l2 solution and (C,D,F) aqueous solution at room temperature. λ_ex = 360 nm.

Figure 4

Stern–Volmer oxygen quenching plot for PIr3 in aqueous solution.

Figure 5

Phosphorescence lifetime measurements for PIr1, PIr2 and PIr3 in degassed and aerated aqueous solutions. (A) PLIM images of the tubes with solutions. Image size is 16 × 16 mm. (B) Phosphorescence decay curves of PIr1–PIr3 probes. On the phosphorescence decay curves, photons are indicated by dots (black—21% O₂; blue—0% O₂), and the lines indicate the mono-exponential phosphorescence decay curve (the red line is 21% O₂; the green line is 0% O₂).

Figure 6

MTT assay for viability of CT26 cells after incubation with PIr1, PIr2, and PIr3 polymer probes. Mean ± SD. *, **, # p ≤ 0.05 vs. controls (0 µM). n = 3 repetitions.

Figure 7

Cellular uptake and intracellular distribution of PIr3 in CT26 cells. (A) Bright-field, phosphorescence intensity and lifetime images obtained by laser scanning microscopy in the course of incubation with PIr3. (B) Phosphorescence intensity of PIr3 in cells. Mean ± SD, n = 70 cells. (C) Phosphorescence lifetime of PIr3 in cells. Mean ± SD, n = 50 cells; (D) Intracellular localization of PIr3. Phosphorescence of PIr3 is shown in red. Lysosomes were stained with LysoTracker Yellow HCK-123 (yellow), plasma membrane was stained with CellMask Green dye (green), and mitochondria were visualized by autofluorescence of NAD(P)H cofactor. Scale bar = 50 μm.

Figure 8

Simultaneous probing of oxygen and metabolism of CT26 cells in normoxic and hypoxic conditions. (A) PLIM images of cells with PIr3 and FLIM images of NAD(P)H from the same cells upon hypoxia induction. Scheme of the experiment on modeling hypoxia using cover glass. Scale bar = 50 μm. For FLIM: ex. 750 nm, reg. 450–490 nm. For PLIM: ex. 750 nm, reg. 570–640 nm. (B) Dependence of the phosphorescence lifetime of PIr3 in cells on the level of oxygenation. (C) Relative contribution of free NAD(P)H (a₁, %) at different levels of oxygenation. Mean ± SD, n = 30 cells. p-values are indicated on the diagram. (D) Correlation between τ PIr3 and a₁ NAD(P)H. The Pearson coefficient r is shown on the scatter plot. Blue dots are the measurements from individual cells.

Figure 9

Simultaneous oxygen PLIM and metabolic FLIM of CT26 tumors in vivo. (A) PLIM and FLIM images of tumors of different sizes. Tumor volume is indicated above the images. Scale bar = 50 μm. For FLIM: ex. 750 nm, reg. 450–490 nm. For PLIM: ex. 750 nm, reg. 570–640 nm. (B) Phosphorescence decay curves of the polymer probe PIr3 in the zones of normoxia (red line) and hypoxia (green line). Black or blue dots are the experimental data. (C) Scatter plot for cell measurements of PIr3 phosphorescence lifetime (τ PIr3) and NAD(P)H fluorescence lifetime (a₁, %). The Pearson correlation r is shown. (D) Phosphorescence lifetime of the PIr3 polymer probe and the relative contribution of free NAD(P)H (a₁, %) in the individual tumors numbered from 1 to 8. The median and the quartiles Q1 and Q3 are shown. Dots are the measurements in the cytoplasm of individual cells. (E) Comparisons of τ PIr3 and a₁ NAD(P)H between tumors of different volumes. Box shows the median and the quartiles Q1 and Q3, whiskers show minimum and maximum. n = 8–10 fields of view from 2–4 tumors. (F) Dendrogram of hierarchical clustering showing the presence of two clusters in a cell population corresponding to the normoxic (green) and hypoxic (red) cells.

Figure 10

In vivo assessment of tumor microvascular network using OCT-A. (A) Representative structural OCT and OCT-A images of tumors. Scale bar = 0.5 mm. Tumors are shown by dashed circles. (B) Perfused vessel density in the tumors. Mean ± SD, n = 3–5. p-values are indicated on the diagram.

Figure 11

Histopathology and immunohistochemistry of CT26 tumors. (A) Upper row: representative histological slices of tumors, hematoxylin/eosin (HE) staining, initial magnification ×10. Scale bar = 270 μm. Bottom row: Immunohistochemical staining for hypoxia with pimonidazole. Scale bar = 20 μm. Initial magnification ×10. (B) Quantification of hypoxic fraction. Mean ± SD.

Figure 12

Intravital simultaneous PLIM of the polymer probe PIr3 and FLIM of NAD(P)H in a mouse tumor. (A) Mouse preparation for PLIM/FLIM microscopy. (B) Photographs of CT26 tumor on the ear on day 14 after implantation and the anesthetized mouse at the microscopy stage. (C) Phosphorescence intensity and lifetime images of the tumor without PIr3 probe (control) and with PIr3 probe injected locally. Scale bar = 50 µm.