Development of 18F-Labeled Resazurin Derivatives for the Detection of Tumor Metabolic Activity Using Cerenkov Imaging

Abstract

This paper describes functional fluorinated bioactivatable molecules to study cancer metabolism using Cerenkov imaging. Resazurin (RA), or Alamar Blue, is a commonly used viability dye and redox sensor. Under reductive conditions or by the action of NADH dehydrogenases, RA is reduced into resorufin (RAred), a highly fluorescent molecule. Cold- and radiolabeled monofluorinated resazurin (MFRA) and difluorinated resazurin (DFRA) were synthesized using electrophilic fluorination. The fluorescence of the reduced probes allowed for the detection of Cerenkov Radiation Energy Transfer (CRET). Cerenkov imaging of MFRAred showed a 4-fold increase in signal at 640 nm relative to MFRA, demonstrating the ability to differentiate between oxidized and reduced species via optical imaging. CRET allows the measurement of signal at longer wavelengths closer to the near infrared (NIR) window, ideal for in vivo imaging. MFRA reduction showed different rates in two breast cancer cell lines: MDA-MB-231, a triple-negative breast cancer, and 4175-Luc+, an aggressive MDA-MB-231 variant, isolated from murine lung metastases. 4175-Luc + cells showed a more rapid reduction of RA and MFRAox than MDA-MB-231 cells. Intratumoral injections of 18F-FDG/MFRA showed a faster reduction of the probe in 4175-Luc + tumors than in MDA-MB-231, suggesting that the metabolic feature observed in the cells is maintained in the tumors. MFRA is a promising probe to determine tumor energy imbalance, reductive environments and assess metastatic potential of tumors. Furthermore, the use of 18F-labeled probes allows for dual modality PET/Cerenkov imaging for probe localization and biodistribution while assessing probe reduction simultaneously.

Phage Biosensor for the Classification of Metastatic Urological Cancers from Urine

“…In turn, the protein concentration of the media has been noticed to have an effect on the change in form [27]. Later, this dye was applied to study the metabolic characteristics of cells [28] and tumor cells [29]. We applied a similar approach to a resazurin reduction test to cancer detection in urine.…”

Molecularly generated light and its biomedical applications

“…[76] Functional Cerenkov agent MFRA, an 18 F-labeled Resazurin that can be reduced into highly fluorescent resorufin, reportedly could be used to determine tumor energy imbalance and reductive environments and assess the metastatic potential of tumors. [77] In addition, the feasibility of Cerenkov luminescence tomography (CLT) has been demonstrated. [78] Given that radio-metal ions, such as 90 Y, 89 Zr, and 68 Ga, yield more photons than 18 F, [71a,79] utilizing such radionuclides holds advantages for imaging and related photodynamic therapy.…”

Theoretical Evaluation of Fluorinated Resazurin Derivatives for In Vivo Applications

“…Living cells metabolise the non-toxic resazurin, and the process can be monitored through the fluorescence emission of resorufin, which is also harmless to living cells [5]. Translating the application of resazurin from in vitro studies to in vivo methods presents certain difficulties, mainly because the optical properties of resazurin and its reduced form, resorufin, fall outside the near-infrared (NIR) imaging window [37,38]. The NIR imaging window, also known as the phototherapeutic window, is defined by a range of wavelengths where light exhibits greater penetration into body tissues [39][40][41].…”

“…Kachur et al [38] reported the synthesis of a new class of compounds, fluorinated resazurin derivatives, aimed at in vivo applications. Arroyo et al [37] tested some of these new compounds for the ex vivo and in vivo detection of tumour metabolic activity using Cerenkov luminescence imaging. Even more ambitious in vivo applications can be envisioned if the direct excitation of resorufin derivatives, inducing its fluorescence emission, is possible in the NIR imaging window.…”

“…Even more ambitious in vivo applications can be envisioned if the direct excitation of resorufin derivatives, inducing its fluorescence emission, is possible in the NIR imaging window. As resorufin is produced through a metabolic mechanism in cells and since tumour cells exhibit enhanced metabolic behaviour compared to normal cells, reprogramming their metabolic fluxes in response to the large demands of NADH and NADPH, highly fluorescent resazurin derivatives that maintain the properties of the redox sensor, would be promising for the in vivo detection of tumours [37,42,43].…”