Longitudinal and multimodal in vivo imaging of tumor hypoxia and its downstream molecular events

Abstract

Tumor hypoxia and the hypoxia-inducible factors (HIFs) play a central role in the development of cancer. To study the relationship between tumor growth, tumor hypoxia, the stabilization of HIF-1alpha, and HIF transcriptional activity, we have established an in vivo imaging tool that allows longitudinal and noninvasive monitoring of these processes in a mouse C51 allograft tumor model. We used positron emission tomography (PET) with the hypoxia-sensitive tracer [(18)F]-fluoromisonidazole (FMISO) to measure tumor hypoxia over 14 days. Stabilization of HIF-1alpha and HIF transcriptional activity were assessed by bioluminescence imaging using the reporter constructs HIF-1alpha-luciferase and hypoxia response element-luciferase, respectively, stably expressed in C51 cells. Interestingly, we did not observe any major change in the level of tumor hypoxia throughout the observation period whereas HIF-1alpha levels and HIF activity showed drastic temporal variations. When comparing the readouts as a function of time we found a good correlation between HIF-1alpha levels and HIF activity. In contrast, there was no significant correlation between the [(18)F]-FMISO PET and HIF readouts. The tool developed in this work allows for the longitudinal study of tumor hypoxia and HIF-1alpha in cancer in an individual animal and will be of value when monitoring the efficacy of therapeutical interventions targeting the HIF pathway.

Fig. 1.

Assessment of tumor hypoxia by measuring the uptake of [¹⁸F]-FMISO. (A) Four consecutive PET images representing cross-sections of the same tumor-bearing mouse on days 7, 10, 12, and 14 are shown. Tumor regions are indicated by the black dotted line and show increased uptake of [¹⁸F]-FMISO. (B) Tumor volume as function of time relative to the values measured on day 6; mean ± SEM (n = 6). (C) Average tumor-to-muscle retention signal (hypoxic TMRR) for the hypoxic fraction in the tumor defined by a TMRR ≥1.4 (10); mean ± SEM (black), individual animals (gray).

Fig. 2.

In vivo monitoring of the stabilization of HIF-1α using bioluminescence imaging. (A) pcDNA3.1-mHIF-1α-luciferase (HIF-1α-luc) and pcDNA3.1-luciferase (luciferase) reporter constructs. (B) Immunofluorescence staining of firefly luciferase in MEF cells transiently transfected with pcDNA3.1-mHIF-1α-luciferase. Nuclei were stained with DAPI. (Magnification: × 300.) (C) Assessment of oxygen-dependent regulation and transcriptional activity of the HIF-1α-luciferase fusion construct. MEF-Hif1a^+/+ or MEF-Hif1a^−/− were cotransfected with pcDNA3.1 mHIF-1α-luciferase, and the reporter plasmid pH3SVB, which drives the expression of β-galactosidase from a HIF responsive promoter. β-Galactosidase activity was assessed. Data are representative of 2 independent experiments. (D) Bioluminescent images of a mouse carrying a HIF-1α-luciferase C51 tumor in the neck. Four consecutive images of the same animal are shown. Color bar indicates total photon counts. (E) Normalized bioluminescence photon counts (mean ± SEM) relative to day 5 values for both the HIF1α-luciferase and the luciferase control tumors. Values indicated by * significantly (P ≤ 0.05) differ from values measured in control groups.

Fig. 3.

Measurement of HIF transcriptional activity reporter. (A) Reporter construct pGL(P2P)95bp (HRE-luciferase). (B) Immunoblot analysis of stably transfected C51 cells cultured at different oxygen concentrations or treated with DMOG. Unspec. refers to an unspecific band on the Western blots used as loading control. (C) In vitro luciferase activity measurement. MEF-Hif1a^+/+ and MEF-Hif1a^−/− cells were transiently transfected with the HRE-luciferase reporter construct and exposed to normoxic or hypoxic conditions. (D) Luciferase activity (photon counts) normalized to total amount of protein in HRE-luciferase C51 reporter cells exposed to varying oxygen concentrations. (E) Normalized bioluminescence photon counts (mean ± SEM) relative to day 5 values for both the HRE-luciferase and the luciferase control tumors. * indicate significant differences (P ≤ 0.05).

Fig. 4.

Immunohistochemical analyses of tumor sections. (A) Immunohistochemical stainings of tumor sections extracted on days 7, 8, 9, 11, and 14. Images of whole tissue sections are shown. (Magnification: × 0.8.) (B) Tumor regions of a section from a tumor isolated on day 14 are shown with high magnification to confirm the subcellular localization of the detected antigens. (Scale bars: 20 μm for H&E, pimonidazole, HIF-1α, and GLUT1 stainings; 50 μm for the CD31 image.) (C) Immunofluorescence stainings. (i) Pimonidazole staining. (ii) CD31 staining. (iii) Hoechst 33342 (perfusion marker). (iv) Overlay of i–iii. (Scale bar: 100 μm.)

Fig. 5.

Comparisons of in vivo tumor hypoxia, HIF-1α stability, and HIF activity measurements. (A) Tumor hypoxia as assessed by [¹⁸F]-FMISO PET, HIF-1α stability, and HIF activity readouts as a function of time. HIF-1α stability and HIF activity reporter values were normalized to the values measured on day 5 (left y axis). Tumor hypoxia is given by the hypoxic TMRR (right y axis). For each readout, mean ± SEM values of the 2 groups measured are displayed. (B–D) Quantitative correlations of the different hypoxia readouts. (B) Spearman r = 0.6364, P = 0.05. (C) Spearman r = −0.5357, P = 0.24. (D) Spearman r = 0.2143, P = 0.62. For each readout, mean ± SEM values of the 2 groups measured are displayed.