In vitro and in vivo PET imaging properties of [¹⁸F]KS1, a fluroethoxy ascorbate derivative to track reactive oxygen species in cancer

Objectives: Ascorbate is a water-soluble antioxidant vitamin. Emerging clinical studies have used ascorbate as a cancer chemotherapeutic agent, and to ameliorate chemotherapy-induced side effects in several types of cancers, including glioblastoma, head and neck squamous cell carcinoma (HNSCC), prostate cancer (PCa) and breast cancer. Although hundreds of patients receive ascorbate treatments for advanced cancers, only a few have achieved an anticancer response. Proactively identifying those who will respond to a given treatment will help clinicians provide the right therapeutic alternatives. However, it is unclear how ascorbate works in reactive oxygen species (ROS) environments in tumor cells. We recently reported the design, synthesis, automated radiochemical production of [¹⁸F]KS1, a fluroethoxy-ascorbate derivative and its initial in vitro and in vivo PET imaging in PCa tumor-bearing mice.¹ Herein, we describe the ROS-tracking properties of KS1 at tracer levels and suppression of tumor growth in cancer cells at pharmacologic doses. We also report the PET imaging properties of [¹⁸F]KS1 in a murine model of HNSCC.

Methods: We used DCFDA ROS detection assay kit using the antibody, ab113851 to demonstrate the ROS generation property of KS1. Both SCC-61, a radiation-sensitive HNSCC cell line and PC3, a PCa cell line were incubated with KS1 and ascorbate at 10 µM and 100 µM for 7 h. The same assay was repeated with 0.1 mM and 2 mM of KS1 and ascorbate (n=6). The kit uses tert-butyl hydrogen peroxide, a common ROS inducer as a positive control, and N-acetyl cysteine, a common ROS suppressor as a negative control. ROS intensity was analyzed on a Tecan fluorescent reader and the data was expressed as mean fluorescent values ± SD (n=6). MicroPET imaging and biodistribution studies were performed in SCC-61 and rSCC-61 (radiation-sensitive and radiation-resistant HNSCC respectively) tumor-bearing mice (n=5) for 20 min after 45 min [¹⁸F]KS1 post-injection.

Results: KS1 at 10 µM and 100 µM did not generate any ROS in both SCC-61 and PC3 cell lines. There was no significant change in the intensity between KS1 and ascorbate KS1 intensity at 2 mM increased significantly compared to the intensity at 100 µM. The uptake was close to the positive control TBHP. More importantly, the increased uptake was lowered (~3-fold) with SOD addition. Ascorbate intensity also increased, but not as KS1. With microPET, we observed high [¹⁸F]KS1 uptake in SCC-61 vs. rSCC-61 tumors. From the basic region of interest analysis on mPET scans, ROS-expressing SCC-61 tumors showed ~1.2-fold higher tumor uptake than rSCC-61 tumors, demonstrating high target selectivity. Biodistribution results show excellent tumor to muscle ratio in SCC-61 (10.93) and rSCC-61 tumors (4.11), with ~2.5-fold enrichment in SCC-61 tumors. These biodistribution results were consistent with in vitro cell uptake and microPET/CT imaging results. Additionally, [¹⁸F]KS1’s higher tumor uptake in SCC-61 tumors establishes its validity to distinguish high from low ROS tumor tissue (see supporting info). More importantly, the radioactivity profile is similar to the distribution kinetics in normal mice at 30 min post-injection.

Conclusions: KS1 do not generate ROS in tumor cells at tracer level concentrations (≤100 uM) and tumor-killing properties by inducing ROS generation at pharmacologic doses (≥1.0 mM), and behave like ascorbate. [¹⁸F]KS1 exhibited high tumor uptake in vivo, with superior specificity and selectivity, and favorable pharmacokinetics in several tumor-bearing mice. This would be the first [¹⁸F]-based PET tracer, based on a natural vitamin to image ROS in tumor cells in vivo, with a clear mechanistic profile. Based on our promising data, we hypothesize that ascorbate-based PET ligand strategy will expand the ascorbate scaffold for potential imaging agent(s) to image ROS in vivo. ¹Solingapuram et al; EJNMMI 2019, 9 (1), 43.