MCT1 is a critical biological target for glycolysis of tumor. Several MCT1-targeting radiotracers have been developed in recent years [21, 23–27]. However, one series of MCT1-targeting probe [18F]FACH is still not evaluated in tumor PET imaging [23–27]; another 11C-labeld coumarin analog probe was restricted for further application with its low radioactive tumor accumulation and rapid clearance [21]. Herein, we designed and synthesized a novel MCTs targeting PET probe for tumor-bearing mice imaging and evaluated it in vitro and in vivo.
Based on the structure of MCTs inhibitor α-cyano-4-hydroxycinnamic acid (CHC) [29], α-cyano-4-hydroxyethyl cinnamate was directly chosen as the lead compound in this study, and the precursor compound α-cyano-4-(2-p-toluenesulfonyl ethoxy)-ethyl cinnamate (compound 2) was obtained through a one-step chemical reaction [27]. Intriguingly, we discovered our radiotracer could be obtained in one-step procedure using the precursor compound 2 under 110℃ for 15 min with the chemical yield of 52.08 ± 6.74% (n = 7, decay corrected), indicating the hydrolysis of ester group in the nucleophilic substitution process. Additionally, the elevation of reaction temperature and time can effectively increase the yield of the target tracer by HPLC analysis. Although the report of one-step radiosynthesis of [18F]FACH using CHC analog without the protection of carboxylic acid group have been published [25], to the aim of simplifying radiolabeling steps and increasing the yield, their precursor need to be prepared for unprotection in the chemical synthesis period. Compared with that, our synthesis of [18F]FEtO-CHC demonstrates the benefits of streamlining chemical and radiolabeling processes, while maintaining a high yield in radiochemical production. However, the phenomenon of hydrolysis during the nucleophilic substitution procedure lacks relevant literature reports, necessitating further exploration of its specific mechanism.
The stability studies conducted in vivo and in vitro revealed a certain level of degradation in the urine of SD rats with a 60 min post-intravenous administration of [18F]FEtO-CHC, which is a common occurrence in many other fluorine-labeled organic compounds due to the inherent instability of the C-F bond [30]. Furthermore, the lipophilicity measurement for [18F]FEtO-CHC obtained a logD7.4 value of 0.29 ± 0.03 (n = 3), demonstrating its restricted liposolubility. By calculating logD7.4, we can give a valuable estimate for the absorption and distribution of drugs within the body, while also identifying the capacity of drug transportation across the blood-brain barrier (BBB) [31], providing us with guidance for subsequent cell experiments and tumor imaging.
Given the 4T1 breast cancer cell line has been used in the study of AZD3965 and AR-C155858 inhibitors and is potently inhibited by CHC [32–33] and the slight overexpression of MCT1 in BxPC3 pancreatic cancer cell line [34], we applied 4T1 and BxPC3 cell lines for our evaluation. The cellular uptake analysis revealed that both BxPC3 and 4T1 cell lines exhibited a similar peak uptake at 15 min, following which the uptake in 4T1 cells continued to increase while BxPC3 cells showed a gradual decrease, matched with the distinct expression levels of MCT1 protein in these two kinds of cell lines from western blot results. Significant uptake inhibition was observed both in vitro and in vivo when using CHC as a competitive MCT1 inhibitor, confirming the specific targeting capability of [18F]FEtO-CHC towards MCT1. The cellular efflux experiment demonstrated a high and similar efflux rate in BxPC3 and 4T1 cells, suggesting its short retention time in tumors. The biodistribution results were consistent with the 2h-dynamic Micro-PET/CT imaging findings in a mouse tumor-bearing model as well. The liver and kidneys exhibited predominant metabolic activity for [18F]FEtO-CHC and significant radioactive accumulation was observed in bladder, confirming the metabolic organs of [18F]FEtO-CHC in both liver and kidneys. In vivo dynamic Micro-PET imaging demonstrated that the tumor-to-muscle (T/M) values in both BxPC3 and 4T1 tumor-bearing mice reached approximately 1.5 at 40 min, after which the T/M gradually decreased for BxPC3 but continued to rise slowly for 4T1, consistent with the findings from the in vitro uptake experiment. Immunohistochemical analysis further confirmed the higher expression of MCTs in both BxPC3 and 4T1 tumor tissue compared to that observed in muscle. These results collectively indicate that probe uptake, both in vitro and in vivo, is associated with MCT1 expression levels. Although the uptake of our radiotracer is relatively low in tumor due to its rapid clearance, we overcome the high plasma binding rate of 11C-labeld coumarin analog probe and enhanced the retention capacity in tumors within 2h [21]. Furthermore, it is the first time to give a comprehensive evaluation for novel MCTs targeting probes in vitro tumor cell lines and in vivo tumor-bearing mice. To enhance the radioactive accumulation in MCT1-positive tumors, we will optimize its structural design from improving the retention in u and slowing the rate of clearance from blood. Meanwhile, we will focus on further exploration of its potential applications in diagnosing and treating specific tumors.
The prospects for tumor therapy targeting MCTs have been promising in recent years. AZD3965 are in advanced clinical trial, demonstrating excellent efficacy against lymphoma [4, 19]. The probe targeting MCT not only enables the early non-invasive diagnosis of tumors, but also holds the promise of screening MCT-positive tumors and monitoring the therapeutic efficacy of MCT- targeting therapy. In addition, owing to the study of monocarboxylate transports and glycolysis mechanism is still in progress, exploring the mechanism in diversities of diseases for MCT-dependent tumors and non-tumors such as glioblastoma, Alzheimer's disease and multiple sclerosis are promising [35], which may provide an effective technical tool to explore the transport mechanism of monocarboxylic acid.