Kinetics-Based Measurement of Hypoxia in Living Cells and Animals Using an Acetoxymethyl Ester Chemiluminescent Probe

Abstract

Oxygenation and tissue hypoxia play critical roles in mammalian biology and contribute to aggressive phenotypes in cancerous tumors, driving research to develop accurate and easy-to-implement methods for monitoring hypoxia in living cells and animal models. This study reports the chemiluminescent probe HyCL-4-AM, which contains a nitroaromatic sensing moiety and, importantly, an acetoxymethyl (AM) ester that dramatically improves operation in cells and animals. HyCL-4-AM provides a selective 60 000-fold increase in luminescence emission in the presence of rat liver microsomes (RLM). For cellular operation, the chemiluminescence response kinetics is sharply dependent on oxygen levels, enabling highly significant and reproducible measurement of hypoxia in living cells. Whole animal imaging experiments in muscle tissue and tumor xenografts show that HyCL-4-AM can differentiate between well oxygenated muscle tissue and hypoxic tumors, demonstrating potential for monitoring tumor reoxygenation via hyperoxic treatment.

Keywords: 1,2-dioxetanes; chemiluminescence; hypoxia; in vivo imaging; nitroreductase.

Figure 1.

Response of HyCL-3 and HyCL-4-AM. Chemiluminescence emission spectra of (A) 10 μM HyCL-3 (blue trace) before and (red trace) after treatment with 1 μg mL⁻¹ NTR and 0.2 mM NADH and (D) 10 μM HyCL-4-AM (blue trace) before and (red trace) after treatment with 1 μg mL⁻¹ NTR, 0.2 mM NADH, and 1 U mL⁻¹PLE. (C) Integrated emission intensity of 10 μM HyCL-4-AM (red trace) or HyCL-3 (blue trace) alone or with 200–1000 ng mL⁻¹ NTR and 0.2 mM NADH. PLE (1 U mL⁻¹) was added for HyCL-4-AM. (D) Time-course of the chemiluminescence emission of 10 μM HyCL-4-AM alone (blue trace) or with 200, 400, 600, 800, and (red trace) 1000 ng mL⁻¹ NTR, 0.2 mM NADH, and 1 U mL⁻¹ PLE. All experiments were performed in 20 mM PBS (pH 7.4) containing ≤1% DMSO. Error bars are ± SD.

Figure 2.

Selectivity of HyCL-3 ad HyCL-4-AM. Response of (A) 20 μM HyCL-3 in the (blue trace) absence and (red trace) presence of 200 μg mL⁻¹ rat liver microsomes (RLM) and 50 μM NADPH and (B) 20 μM HyCL-4-AM in the (blue trace) absence and (red trace) presence of 200 μg mL⁻¹ rat liver microsomes (RLM) and 50 μM NADPH. Response of (C) 20 μM HyCL-3 and (D) 20 μM HyCL-4-AM to the indicated analytes from 0 to (red bars) 50 at 10 min increments. All experiments were performed in 10 mM PBS (pH 7.4) containing ≤1% DMSO. Error bars are ± SD.

Figure 3.

Measuring hypoxia in living A549 cells. (A–C) Time-course of the chemiluminescence emission of A549 cells incubated with 40 μM HyCL-4-AM at 1% O₂ or 20% O₂ in the presence or absence of DPI. (D) Chemiluminescence emission intensity at 300 min of A549 cells incubated with 40 μM HyCL-4-AM at 1% O₂ or 20% O₂. (E) Measured rate constants for the cellular response of HyCL-4-AM. (F) Time-course of the chemiluminescence emission of A549 cells incubated with 40 μM HyCL-4-AM-Cont at 20% O₂ in the presence or absence of DPI. All experiments were performed in F12K containing 10% FBS and ≤1% DMSO. Error bars are ± SD. Statistical significance was assessed using a two-tailed Student’s t test. **p < 0.005 (n = 3–4 biological replicates), ***p < 5 × 10⁻⁶ (n =9–12 wells across 3–4 biological replicates).

Figure 4.

Measuring oxygenation in healthy muscle tissue and tumor xenografts. (A–C) Time course of the chemiluminescence emission of 30 of 120 μM HyCL-4-AM in 20 mM PBS (pH 7.4) containing 2.4% DMSO in athymic nude mice after intramuscular (IM) injection into the flank, intratumoral injection while mice were breathing 16% O₂ (IT, 16% O₂), and intratumoral injection while mice were breathing 100% O₂ (IT, 100%). (D) Photon flux and (E) chemiluminescence images of athymic nude mice 60 min after IM injection, IT injection while mouse breathed 16% O₂, and after IT injection while mouse breathed 100% O₂. Error bars are ± SE from n = 3–6 mice. Statistical significance was assessed using a two-tailed Student’s t test. *p < 0.05.

Scheme 1.

Probe Designs and Sensing Reactions for HyCL-3, HyCL-4-AM, and HyCL-4-AM-Cont).

Scheme 2.

Synthesis of HyCL-3, HyCL-4-AM, and HyCL-4-AM-Cont