A Bioluminescent Probe for Tumor Hypoxia ... - ACS Publications

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A Bioluminescent Probe for Tumor Hypoxia Detection via CYP450 Reductase in Living Animals Yuqi Gao, Yuxing Lin, Tingting Liu, Hui Chen, Xiaofeng Yang, Chengsen Tian, Lupei Du, and Minyong Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03597 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on October 27, 2017

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Analytical Chemistry

A Bioluminescent Probe for Tumor Hypoxia Detection via CYP450 Reductase in Living Animals Yuqi Gao,† Yuxing Lin,† Tingting Liu,† Hui Chen,† Xiaofeng Yang,§ Chengsen Tian,¶ Lupei Du,† and Minyong Li†,‡,* †

Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong University, Jinan, Shandong 250012, China. Tel./fax: +86-531-8838-2076; E-mail: [email protected] § School of Chemistry and Chemical Engineering, University of Jinan, Jinan, Shandong 250022, China ¶ School of Chemistry and Chemical Engineering, Qilu Normal University, Jinan, Shandong 250200, China ‡ State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, China ABSTRACT: Hypoxia is a pathogenic characteristic of solid tumors owing to absent or abnormal vasculature in the tumor microenvironment and essential in tumor progression, angiogenesis, metastasis, invasion and resistance to immune system and therapy. In hypoxic environments, CYP450 enzymes are more efficient than in normoxia. Herein, based on the reductive capacity of CYP450 enzymes/NADPH system, we managed to cage aminoluciferin developing a reaction-based bioluminescent probe as well as an imaging method for the hypoxia detection. Exhibiting enhanced about 3-fold total flux in big (1.2 cm-diameter) tumors, Hypoxia BioLuminescent probe (HBL) can afford potential utility for in cellulo and in vivo hypoxia imaging in tumor model mice.

Introduction It is well known that the tumor microenvironment (TME) is not the same with normal tissues, including a highly reducing status, a low pH, and hypoxia. Hypoxia, which is defined as lower-than-normal oxygen conditions, is a pathogenic characteristic of solid tumors owing to absent or abnormal vasculature in the tumor microenvironment.1,2 Compared to the physiologic oxygenation level (5%–10% O2), the oxygen level is recognized to be less than 1.3% O2 in most solid tumor tissues. Developing hostile microenvironments such as hypoxia, solid tumors typically exhibit unregulated growth.3-5 In order to adapt to hypoxia, cancer cells promote survival pathways mediated through protein stabilization of hypoxia inducible factor (HIF) subunits (HIF1α, HIF2α and HIF3α) which are regulated by prolyl hydroxylase domain (PHD) and factor inhibiting HIF1 (FIH-1) enzymes. Hydroxylated by PHD and FIH-1, polyubiquitinated and degraded by the proteasome, HIFα subunits stay inactive in oxygenated cells. While in hypoxia, HIFα dimerized with HIFβ subunit which is constitutively expressed, after translocated into the nucleus. As a result, the dimer binds to DNA to initiate gene transcription, supporting cancer cells survival. Recently, cytochrome P450 enzymes were discovered to perform a more powerful reductive capacity in cells under hypoxia conditions through investigation on the reduction of azide-containing compounds under different oxygen levels.6 Consequently, hypoxia, as an important tumor microenvironmental factor, has a pivotal role in tu-

mor progression,3 angiogenesis,1 metastasis,6,7 invasion,5,8 and resistance to immune system2 and therapy.8-10 Awareness of resistant to chemotherapy and radiotherapy caused by overexpressed HIF1 promote explorations, improvements and innovation in cancer diagnosis and therapies.11-14 Considering that hypoxia emerges when the diameter of solid tumor increases to approximate 350 μm in the body, hypoxia imaging is essential in diagnosis of cancer in early stage.15,16 Different kinds of imaging system have been explored and developed for hypoxia detection, including positron emission tomography (PET) and/or magnetic resonance imaging (MRI) probes,17,18 phosphorescent probes,19,20 fluorescent probes,21-26 bioluminescent probes27 and chemiluminescent probes,28 many of which could be employed for hypoxia imaging in living cells and animals. In recent decade, bioluminescent imaging (BLI), a reliable, sensitive, convenient, and noninvasive imaging method, has been extensively applied in pathogen detection, tumor imaging, therapeutic drug screening, and many other aspects in life science. Usually, firefly luciferin or aminoluciferin associated with firefly luciferase is employed in BLI system, transmitting light in the presence of adenosine triphosphate (ATP), O2, and Mg2+. When 6’hydroxy (or 6’-amino) group of D-luciferin (or aminoluciferin) converted into an inactive “caged” form chemically, it cannot be recognized by luciferase, forbidding light emission ultimately. However, the capability could recover once luciferin (or aminoluciferin) released from its

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caged form after reaction with specific reagents or in specific environment.29,30 Both the Nagano and Qian groups reported that probes bearing azo group are sensitive to hypoxia.21-23 The results manifested that the N=N bond performs as a novel and effective moiety in designing a probe for hypoxia detection. In order to construct a promising approach for hypoxia imaging in living animals, we decided to take advantage of firefly bioluminescence imaging (BLI) as a noninvasive alternative on account of its ability to afford high-sensitivity in vivo imaging. In the current exploration, we managed to introduce an azo group into firefly luciferin to develop a hypoxia bioluminescent probe HBL (Figure 1) with a high sensitivity and fine resolution in living cells and animals using the “caging” strategy30, referencing the previous works in our group27,31-34.

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generated HBL at ambient temperature. The details for the preparation of the compounds and their NMR and HR-MS spectra can be found in the Supplementary Materials. Bioluminescence measurements in vitro Measurements for sensitive assays were performed in Tris-HCl buffer (10 mM, pH 7.44). Hypoxia bioluminescence probe was incubated with various concentrations of cytochrome P450 reductase at various concentrations (0 to 10 μg/mL) and NADPH tetrasoldium at 37 °C for 60 min. After that, 50 μL of Tris buffer containing firefly luciferase (20 μg/mL), ATP (2 mM) and Mg2+ (10 mM) was added, and bioluminescence intensity was measured immediately with an acquisition time of 0.5 s by IVIS Kinetic imaging system equipped with a cooled CCD camera. Measurements for selectivity assays were also performed in Tris-HCl buffer (10 mM, pH 7.44). Hypoxia bioluminescence probe was incubated with various concentrations of reductive species at 37 °C for 60 min. After that, 50 μL of firefly luciferase (20 μg/mL) in Tris buffer containing ATP (2 mM) and Mg2+ (10 mM) was added, and bioluminescence intensity was measured immediately with an acquisition time of 0.5 s by IVIS Kinetic imaging system equipped with a cooled CCD camera.

Biolu-

Bioluminescence measurements in cellulo Figure 1. Proposed mechanism of bioluminescent probe HBL for the detection of hypoxia.

Experiments All reagents and solvents available from commercial sources were used without further purified unless otherwise noted. The reactions were visually monitored with TLC analysis performed on glass-backed silica plates associated with UV light (or I2). Water used for the bioluminescence studies was doubly distilled and further purified with a Milli-Q filtration system. HPLC analysis was undertaken with a 1260 Infinity HPLC (Agilent Technologies, Santa Clara, CA). For bioactivity evaluation of HBL, bioluminescence intensity was measured by IVIS Kinetic imaging system (Caliper Life Sciences, Hopkinton, Massachusetts, USA) instrument equipped with a cooled charge-coupled device (CCD) camera. Using Living Image software, the data were quantified and reported as total photon flux within a circular region of interest (ROI) in photons per second. Sythesis Our hypoxia probe can be prepared with an efficient and easy-operated approach as depicted in Scheme S1. In breif, 6-aminobenzo[d]thiazole-2-carbonitrile (1) reacted with sodium nitrite in diluted hydrochloric acid (6 M) in ice-salt bath forming the diazonium salt which further reacted with N, N-diethylaniline then to obtain pure azo intermediate 2 after neutralization and column chromatography separation. A subsequent cross-coupling reaction of the intermediates 2 with D-cysteine hydrochloride

To qualify HBL for hypoxia assay in living cells, luciferase-transfected human ovarian cancer cell lines ES-2 (ES2-luc) were used. ES-2-luc cells were passed and plated (4×104 cells per well) in 96-well plates (Corning, 3603). After incubated overnight, the medium was replaced with of cobalt chloride solution of various concentrations (0 to 2000 μM) dissolved in FBS free medium. After 4 hourincubation at 37 °C, the medium containing CoCl2 was discarded and FBS free medium was added. Incubated for 30 min, the medium was removed and HBL in saline solution was added. The bioluminescence intensity was measured immediately with an acquisition time of 60 s by IVIS Kinetic imaging system equipped with a cooled CCD camera. ES-2-luc cells were passed and plated (4×104 cells per well) in two 96-well plates (Corning, 3603). After incubated overnight, one of them was transferred into a tri-gas incubator with 1% pO2 while the other remained in normoxic condition (20% pO2). After 6 hour-incubation at 37 °C, the medium was removed and HBL in saline solution of various concentrations (0 to 100 μM) was added. The bioluminescence intensity was measured with an acquisition time of 60 s by an IVIS Kinetic imaging system equipped with a cooled CCD camera. Bioluminescence imaging in vivo All animal studies were approved by the Ethics Committee and IACUC of Cheeloo College of Medicine, Shandong University, and were conducted in compliance with European guidelines for the care and use of laboratory animals. BALB/c nude mice were purchased from the Animal Center of the China Academy of Medical Sciences


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(Beijing, China). To investigate hypoxia imaging of HBL in vivo, ES-2-luc cells (approximate 1.5×106) were grafted subcutaneously under the right forelimb armpit of BALB/c nude mice, aged 6 weeks. After about 3 weeks, tumors were harvested and cut into pieces, and then 10mg or 5-mg tumor pieces were implanted subcutaneously into the right armpit region of another group of nude mice. Tumor xenografts with diameters of around 10 and 13 mm were formed respectively after two weeks. The mice were anesthetized by single intraperitoneal injected chloral hydrate (8%) before imaging. Then, 50 μL of HBL (1 mM) in saline. Photon emission was collected using a cooled CCD camera with an acquisition time of 20 s.

combined with beneficial characteristics of the bioluminescence technique, can afford potential utility for in cellulo and in vivo hypoxia imaging.

RESULTS AND DISCUSSION Initial experiments focused on the confirmation of our bioluminescent probe’s mechanism. As azo compounds is liable to reductants, HBL can be reduced to aminoluciferin as the reduced product. To test the liability of HBL to chemical reductant, the probe in aqueous solution was reacted with stannous chloride in strong acidic condition at room temperature. The color of the solution changed from orange to yellow distinctly, after reaction for about 30 min at room temperature. Moreover, cytochrome P450 reductase (CYP450 reductase), a common reductase in endoplasmic reticulum and mitochondria of animals’ cells, was selected to examine the reducible ability of HBL to bio-reductant. A similar phenomenon was observed when HBL was reacted with cytochrome P450 reductase and nicotinamide adenine dinucleotide phosphate (NADPH) in Tris-HCl (10 mM) buffered to pH 7.44 at 37 ºC for 5 h. The previously conjected mechanism of the probe was confirmed with HPLC by comparing the reduction sample of HBL by stannous chloride or cytochrome P450 reductase with amino-luciferin. When reacted with reductants, absorbance at 20.6 min (the retention time of HBL) declined, while new peaks appeared at 3.6 min, as the retention time of aminoluciferin (Figure S1). Luciferin/luciferase system is highly efficient and sensitive to afford a significant enhancement of bioluminescent signal for cell and animal imaging, despite only a trace amount of aminoluciferin generated though the reduction procedure. 27,32,33,35 Therefore, further investigations involving performance of HBL towards hypoxia were carried out. Selectivity of HBL (50 µM) for cytochrome P450 reductase (5 µg/mL) against a range of reductive species existing in tumor cells, including nitroreductase (NTR) (5 µg/mL), NADPH (100 µM), Vitamin C (1000 µM), reduced glutathione (GSH) (1000 µM) and HS- (1000 µM), was assessed consequently in aqueous system. As illustrated in Figure 2, when cytochrome P450 reductase and NADPH was added simultaneously, an evident enhancement in bioluminescence intensity displayed, compared with NTR and other reductants. That means our bioluminescent probe is more liable to CYP450 reductase/NADPH system, which is a primary and oxygen-dependent reductive system in metabolism.6 These response and selectivity data suggested that HBL with fine sensitivity and selectivity,

Figure 2. Response and selectivity assay of HBL in vitro. (A) Bioluminescence imaging of selectivity of HBL towards various relevant reductants which may exist in cells (cytochrome P450 reductase, NADPH, NTR, Vitamin C, GSH, HS-) with luciferase, ATP and Mg2+ added in a 96well plate. (B) Quantification of bioluminescence intensity of HBL for each condition. Afterwards, the ability of HBL to image hypoxia in living in cellulo was investigated. To qualify HBL for hypoxia assay in living cells, cobalt chloride was utilized to induce hypoxia in luciferase-transfected human ovarian cancer cell lines ES-2 (ES-2-luc).27,36,37 As illustrated in Figure 3, bioluminescence intensity increased continually with the gradually increasing concentrations (0−800 µM) of cobalt chloride with the peak value (293% relative increase) at 800 µM CoCl2. The results expose that Co2+ ion with different concentrations can induce living cells’ hypoxia in different degrees effectively and suggest that intracellular level of reducing substances and reductase (such as cytochrome P450 reductase and NTR27,28), may elevate under hypoxia stimulation from a side. Furthermore, HBL was confirmed to be capable for monitoring hypoxia at cellular level with a series of dynamic changes. According to the results of cytotoxicity test accomplished by SRB cytotoxicity assay, the hypoxia bioluminescent probe HBL is low-toxic and fine-biocompatible (Figure S5). Meanwhile, the cytotoxicity of cobalt chloride was also tested (Figure S5). Exhibiting low cytotoxicity at high concentrations, a large excess amount of cobalt ion may decrease the activi-



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ty of enzymes in cells, bringing down the bioluminescence intensity in turn.

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manner along with the increasing concentration of probe. In brief, these results further confirmed that HBL is capable of hypoxia detection at the cellular level. To some extent, the results also coincided with and experimentally proved the oxygen-dependent manner of CYP450 enzymes6.

Figure 4. Bioluminescence measurement in ES-2-luc cells under hypoxic condition (1% pO2). (A) Bioluminescence imaging in hypoxic living cells with diverse concentrations (0, 5, 10, 20, 40, 60, 70, 80, 90, 100 µM) of HBL; (B) quantification of bioluminescence intensity of HBL for each condition.

Figure 3. Hypoxia assay in ES-2-luc cells using HBL. (A) Bioluminescence imaging in hypoxic living cells induced by diverse concentrations (0, 25, 50, 100, 200, 400, 600, 800, 1000, 2000 µM) of cobalt chloride; (B) quantification of bioluminescence intensity of HBL for each condition; (C) variation of relative bioluminescence intensity in 45 min. In order to validate the capability of HBL for hypoxic microenvironment detection, ES-2-luc cells were incubated under hypoxic condition (1% pO2) for 6 h. Compared with the control group (cells incubated in normoxic conditions), an increasing bioluminescence intensity was observed in hypoxic cells (Figure 4). Meanwhile, the total photon emission exhibited a concentration-dependent

Finally, having confirmed that HBL can provide a method for monitoring oscillations of hypoxia with significant selectivity and sensitivity in living cells, we interrogated the capability of HBL for hypoxia visualization in human tumor xenograft models. ES-2-luc cells were subcutaneously grown on the axilla of BALB/c nude mice. We attempted to inject the bioluminescent probe in tumors with different diameters (less than 1.0 cm and more than 1.2 cm) on the nude mice, respectively. Thereafter, the subsequent bioluminescence intensity was kinetically recorded with living imaging system. As shown in Figure 5, after intratumoral injection of HBL, distinctly enhancing emission signal from tumor regions in nude mice were observed, reaching peak values immediately. The enhanced bioluminescence intensity from the whole tumors lasted for about 5 min. Within 1 min after HBL injected, the average flux value of the “big” group (over 1.2 cmdiameter tumors) was 3-fold higher than that of the “small” group (less-than 1.0 cm-diameter tumors). The bioluminescence intensity of the “big” group declined significantly over time, while the “small” group decreased slightly. The emission signals of the two groups trended


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to be uniform about 30 min after HBL injection. Conclusion In summary, by introducing an azo group, which can be reduced by CYP450 reductase, into amino luciferin, we designed and prepared a Hypoxia BioLuminescent probe (HBL). The investigation results obviously demonstrated that the bioluminescent probe is able to monitor hypoxia visually both in cellulo and in vivo. Moreover, not only did the experimental data establish a clear correlation between dynamic hypoxia degrees and tumors with different size, but also implied that it is capable of HBL offer-

ing a valuable approach for dynamic hypoxia monitoring in tumor development in living animals. Therefore, exhibiting fine selectivity and sensitivity as well as other beneficial characteristics, HBL presents a powerful tool in hypoxia bioimaging and provides a feasible approach for visualizing hypoxia via CYP450 enzymes in the development of tumors. As a result, by using such a promising toolkit, a valuable opportunity was supplied to understand hypoxia and its role in tumor processes. It is believable that the novel hypoxia bioluminescent probe will expand the hypoxia imaging toolkit as well as applications of bioluminescence technology.

Figure 5. Bioluminescence imaging of living ES-2-fluc-GFP ovarian tumor xenografts on BALB/c nude mice. (A) Timedependent bioluminescence imaging of tumors with different diameters (“big”: more than 1.2 cm, “small”: less than 1.0 cm) (B) Fluctuation of total flux from the tumoral areas of the nude mice of triplicates (n=3 mice) at 0−30 min. (C) Quantification of bioluminescence intensity from the tumoral areas of the nude mice of triplicates (n=3). Corresponding Author

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Supporting Information Full experimental procedure, NMR, MS, fluorescence, and cell imaging data. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENTS

AUTHOR INFORMATION



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The present work was supported by grants from the National Program on Key Basic Research Project of China (No. 2013CB734000), the National Natural Science Foundation of China (No. 81673393), the Taishan Scholar Program at Shandong Province, the Qilu Scholar Program at Shandong University and the Major Project of Science and Technology of Shandong Province (No. 2015ZDJS04001). Notes The authors declare no competing financial interest.

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