FMISO accumulation in tumor is dependent on glutathione conjugation capacity in addition to hypoxic state

Abstract

Objective 18F-fluoromisonidazole (FMISO), a well-known PET imaging probe for diagnosis of hypoxia, is believed to accumulate in hypoxic cells via covalent binding with macromolecules after reduction of the nitro group. Previously, we showed the majority of 18F-FMISO was incorporated into low-molecular-weight metabolites in hypoxic tumors, and the glutathione conjugate of reduced FMISO (amino-FMISO-GS) distributed in the tumor hypoxic regions as revealed by imaging mass spectrometry (IMS). The present study was conducted to clarify whether FMISO is metabolized to amino-FMISO-GS within tumor cells and how amino-FMISO-GS contributes to FMISO accumulation in hypoxic cells. We also evaluated the relationship between FMISO accumulation and the glutathione conjugation-related factors in the cells.MethodsTumor cells (FaDu, LOVO, and T24) were treated with 18F-FMISO and incubated under normoxic or hypoxic conditions for 4 h. The FMISO metabolites were analyzed with LC–ESI–MS. Several glutathione conjugation-related factors of tumor cells were evaluated in vitro. FaDu tumor-bearing mice were intravenously injected with 18F-FMISO and the tumors were excised at 4 h post-injection. Autoradiography, IMS and histologic studies were performed.ResultsAmino-FMISO-GS was the main contributor to FMISO incorporated in hypoxic FaDu cells in vitro and in vivo. Total FMISO uptake levels and amino-FMISO-GS levels were highest in FaDu, followed by LOVO, and then T24 (total uptake: 0.851 ± 0.009 (FaDu), 0.617 ± 0.021 (LOVO) and 0.167 ± 0.006 (T24) % dose/mg protein; amino-FMISO-GS: 0.502 ± 0.035 (FaDu), 0.158 ± 0.013 (LOVO), and 0.007 ± 0.001 (T24) % dose/mg protein). The glutathione level of FaDu was significantly higher than those of LOVO and T24. The enzyme activity of glutathione-S-transferase catalyzing the glutathione conjugation reaction in FaDu was similar levels to that in LOVO, and was higher than that in T24. Quantitative RT-PCR analysis revealed that the expression levels of efflux transporters of the glutathione conjugate (multidrug resistance-associated protein 1) were lowest in FaDu, followed by LOVO, and then T24.ConclusionsFMISO accumulates in hypoxic cells through reductive metabolism followed by glutathione conjugation. We illustrated the possibility that increased production and decreased excretion of amino-FMISO-GS contribute to FMISO accumulation in tumor cells under hypoxic conditions.Electronic supplementary materialThe online version of this article (doi:10.1007/s12149-017-1189-9) contains supplementary material, which is available to authorized users.

Pharmacokinetic Analysis of Dynamic [18F]FAZA PET Imaging in Pancreatic Cancer Patient

“…A group in Japan studied the mechanism of NI based 18 F-FMISO binding in nude mice by implanting cells from the human FaDu cancer line [14,33,34]. They found that the majority of the tumor radioactivity was from low molecular weight metabolite, glutathione (GST) conjugate of amino-FMISO (amino-FMISO-GH) [14,34,35]. Amino-FMISO-GH is highly hydrophilic and cannot diffuse out of the cell.…”

“…Amino-FMISO-GH is highly hydrophilic and cannot diffuse out of the cell. However, it could efflux out via the adenosine triphosphate (ATP) dependent multi-drug resistant protein (MRP-1) [34,36], which is highly expressed in pancreatic tumor cells [5,[37][38][39] and is responsible for drug resistance. A similar efflux of amino-FAZA-GH could explain the non-trapping of 18 F-FAZA in hypoxic tissue and hence the estimation of non-zero with kinetics modelling.…”

“…Since k4 and distribution volume were comparatively larger for normal than cancerous tissue, it is likely that more amino-FAZA-GH washed out of the normal tissue leading to higher tracer accumulation and contrast between tumor and normal tissue in SUV imaging. As suggested by Masaki et al, NI based tracers may be imaging a complex processes involving nitroreductase, glutathione, and MRP-1 mediated efflux activity [34]. The tracer, 18 F-FAZA could be used to monitor MRP-1 activity and glutathionylation; hence could lead to personalization of treatment protocol by boosting radiation treatment in high hypoxic region and possibly treating high k4 pancreatic cancer with MRP-1 blockers.…”

“…(a) For the F2TC model, distribution volume (DV) and k4 can distinguish the two tissue types with sensitivity of 95% and (b) For the S2TC model, DV achieved a sensitivity of 90%. The solid line in each case is the linear discriminator derived from the Youden index (Cancer 1950; 3(1):[32][33][34][35].…”

Emerging Functional Imaging Biomarkers of Tumour Responses to Radiotherapy

“…18 F-labelled 2-nitroimidazole-based markers have been widely used for PET imaging of hypoxia. In an anoxic environment reduction of the NO 2 moiety of 2-nitroimidazole by nitroreductases produces highly reactive intermediates which bind to many macromolecules and also undergo glutathione conjugation [81]. 18 F-fluoromisonidazole ( 18 F-FMISO) was the first PET tracer for hypoxic imaging to be developed and has subsequently been the most extensively used, with accumulation having been demonstrated in human glioma, head and neck squamous cell carcinomas (HNSCC), breast, lung and renal tumours.…”