Heterotopic ossification (HO) is the formation of bone within soft tissue where bone normally does not exist. In general, it is characterized by highly active tissue with high bone turnover and rapid bone formation. It is of an utmost importance to precisely identify and accurately diagnose the maturity of HO as early surgical intervention may result in its recurrence. The objective of this work is the experimental evaluation of HO maturity stage using advanced noninvasive nuclear medicine techniques. The use of PET radiopharmaceuticals may result in a more specific diagnosis between the phases due to their higher sensitivity and better resolution compared to bone scan.
8-week-old Balb/c male mice underwent dual injury procedure, tenotomy and concurrent burn injury on the left side, to induce HO. The progression of HO was monitored by SPECT/CT and PET/CT weekly imaging with 99mTc-MDP, [18F]NaF and [18F]FDG for up to 16 weeks.
There was a statistically significant increase of [18F]FDG uptake from week 1 to 2 and from week 2 to 6 with p values of 0.01 and 0.005; respectively, while there was a statistically significant decrease from week 7 to 14 with a p value of 0.008. There was a statistically significant increase of [18F]NaF uptake from week 2 to 5 and statistically significant decrease between weeks 7 and 14 with p values of 0.016 and 0.003; respectively. As for 99mTc-MDP, the increase in the uptake from week 1 to 2 and from week 2 to 5 were not statistically significant with p values of 0.15 and 0.19; respectively. The decrease of uptake between week 7 and 14 was not statistically significant with a p value of 0.08.
Based on these findings, the use of noninvasive nuclear imaging modalities may assist in distinguishing between the immature and mature phases. The uptake of mainly [18F]FDG may indicate the early inflammatory phase, while the uptake of both [18F]FGD and [18F]NaF may suggest the immature phase, and an uptake of mainly [18F]NaF may indicate the maturity phase of HO.
M2-type tumor-associated macrophages (TAM) residing in the tumor microenvironment (TME) have been linked to tumor invasiveness, metastasis and poor prognosis. M2 TAMs suppress T cell activation, silencing the recognition of the cancer by the immune system. Targeting TAMs in anti-cancer therapy may support the immune system and immune-checkpoint inhibitor therapies to fight the cancer cells. We aimed to develop a PET tracer for the imaging of M2 TAM infiltration of cancer, using activated legumain as the imaging target.
Two P1-mimicking inhibitors with a cyano-warhead were labeled with carbon-11 and evaluated in vitro and in vivo with a CT26 tumor mouse model. Target expression and activity were quantified from RT-qPCR and in vitro substrate conversion, respectively. The co-localization of legumain and TAMs was assessed by fluorescence microscopy. The two tracers were evaluated by PET with subsequent biodistribution analysis with the dissected tissues. Parent-to-total radioactivity in plasma was determined at several time points after i.v. tracer injection, using reverse phase radio-UPLC.
Legumain displayed a target density of 40.7 ± 19.1 pmol per mg total protein in tumor lysate (n = 4) with high substrate conversion and colocalization with M2 macrophages in the tumor periphery. [11C]1 and [11C]2 were synthesized with >95 % radiochemical purity and 12.9–382.2 GBq/μmol molar activity at the end of synthesis. We observed heterogeneous tumor accumulation in in vitro autoradiography and PET for both tracers. However, excess unlabeled 1 or 2 did not compete with tracer accumulation. Both [11C]1 and [11C]2 were rapidly metabolized to a polar radiometabolite in vivo.
The legumain tracers [11C]1 and [11C]2, synthesized with high radiochemical purity and molar activity, accumulate in the legumain-positive CT26 tumor in vivo. However, the lack of competition by excess compound questions their specificity. Both tracers are rapidly metabolized in vivo, requiring structural modifications towards more stable tracers for further investigations.