Toward in vivo proof of binding of 18 F-labeled inhibitor [ 18 F]TRACK to peripheral tropomyosin receptor kinases.

Background: Tropomyosin receptor kinases (TrkA, TrkB, TrkC) are a family of tyrosine kinases primarily expressed in neuronal cells of the brain. Identification of oncogenic alterations in Trk expression as a driver in multiple tumor types has increased interest in their role in human cancers. Recently, first- and second-generation ¹¹ C and ¹⁸ F-labeled Trk inhibitors, e.g., [ ¹⁸ F]TRACK, have been developed. The goal of the present study was to analyze the direct interaction of [ ¹⁸ F]TRACK with peripheral Trk receptors in vivo to prove its specificity for use as a functional imaging probe.
Methods: In vitro uptake and competition experiments were carried out using the colorectal cancer cell line KM12. Dynamic PET experiments were performed with [ ¹⁸ F]TRACK, either alone or in the presence of amitriptyline, an activator of Trk, entrectinib, a Trk inhibitor, or unlabeled reference compound TRACK in KM12 tumor-bearing athymic nude mice as well as B6129SF2/J and corresponding B6;129S2-Ntrk2 ^tm1Bbd /J mice. Western blot and immunohistochemistry experiments were done with KM12 tumors, brown adipose tissue (BAT), and brain tissue samples.
Results: Uptake of [ ¹⁸ F]TRACK was increasing over time reaching 208 ± 72% radioactivity per mg protein (n = 6/2) after 60 min incubation time. Entrectinib and TRACK competitively blocked [ ¹⁸ F]TRACK uptake in vitro (IC ₅₀ 30.9 ± 3.6 and 29.4 ± 9.4 nM; both n = 6/2). [ ¹⁸ F]TRACK showed uptake into KM12 tumors (SUV _mean,60 min 0.43 ± 0.03; n = 6). Tumor-to-muscle ratio reached 0.9 (60 min) and 1.2 (120 min). In TrkB expressing BAT, [ ¹⁸ F]TRACK uptake reached SUV _mean,60 min 1.32 ± 0.08 (n = 7). Activation of Trk through amitriptyline resulted in a significant radioactivity increase of 21% in KM12 tumor (SUV _mean,60 min from 0.53 ± 0.01 to 0.43 ± 0.03; n = 6; p < 0.05) and of 21% in BAT (SUV _mean,60 min from 1.32 ± 0.08; n = 5 to 1.59 ± 0.07; n = 6; p < 0.05) respectively. Immunohistochemistry showed TrkB > TrkA expression on BAT fat cells, but TrkA > TrkB in whole brain. WB analysis showed sevenfold higher TrkB expression in BAT versus KM12 tumor tissue.
Conclusion: The present data show that radiotracer [ ¹⁸ F]TRACK can target peripheral Trk receptors in human KM12 colon cancer as well as brown adipose tissue as confirmed through in vitro and in vivo blocking experiments. Higher TrkB versus TrkA protein expression was detected in brown adipose tissue of mice confirming a peripheral functional role of brain-derived neurotrophic factor in adipose tissue.
(© 2022. The Author(s).)