**6. Conclusions and Future Perspectives**

In summary, MI plays a vital role in evaluating and managing TCs, especially in accurately finding occult foci that are undetected by traditional ultrasound, CT, and MRI, thereby helping TC patients get the precise therapeutics (Table 1). Transporter-based probes tend to have high sensitivity, and immune-based probes generally have high specificity. So far, there is no single probe to reveal all the lesions both specifically and sensitively. Moreover, although TCs can be classified in a single pathological category, the biomarker expression in TC can vary dramatically. The apparent heterogeneity of TC requires the availability of more than one therapeutic method. By fully elucidating the biological characteristics of TCs and thoroughly exploring the biomarkers enriched in TCs [26,158,159], we believe that we can discover helpful targets for developing diagnostic probes and companion therapeutic agents for different molecular types of TC, not limited to pathological typing and phenotyping.




MI, molecular imaging; SP, speculative; LoE, level of evidence; LL, lesion localization; PE, prognosis evaluation; TT, tumor therapy; RD, radioiodine-131 dosimetry, [131I]NaI dosimetry; TS, tumor staging; IUE, iodine uptake evaluation; PIU, predicting iodine uptake; OS, ongoing study; PRRT, pre-evaluation for receptor radionuclide therapy; TC, thyroid cancer; PTC, papillary thyroid cancer; MTC, medullary thyroid cancer; ATC, anaplastic thyroid cancer; DTC, differentiated thyroid cancer; BsAb, bispecific antibody; Ab, antibody; Fab, antigen-binding fragment.

> With the development of biological techniques and imaging tools, more valuable imaging methods are emerging. Integration of multiple imaging modalities and anatomical features would help physicians diagnose and treat TCs in a timely target-specific manner. Nanoparticles may enable anti-TC drug delivery and multimodality imaging, which may further improve the management of TC. However, the authors are cautious because of the limited clinical evidence in the field of TC. Bispecific MI probes have gained more traction in the past decade. The synthesis of bispecific antibody or antibody fragment tracers has been thoroughly elucidated elsewhere [170]. The importance of bispecific probes is that they enable enhanced affinity and high image quality, providing the ability targeting more occult foci than traditional single-target probes, and inducing more comprehensive application across TC patients with complex biological characteristics.

For antibody probe design, the traditional antibody-based radioactive or fluorescent probes for TCs have simply been prepared by non-selective conjugation on lysine/cysteine residues with or without chelators [171,172]. This approach may reduce target binding affinity, especially at a high conjugate/protein ratio, and in any case, leads to a mixture of products with different numbers of tags per protein molecule [173]. The method may also cause undesirable biodistribution (e.g., high kidney uptake and poor tumor targeting due to in vivo cleavage of the S–S linkage) and pharmacokinetics (fast antibody clearance for modification of interchain disulfide cysteine) [174]. Protein engineering techniques are progressing very rapidly. In this setting, the site-specific and homogeneous introduction of the tags into the targeting moieties would be more advantageous. The emerging techniques mainly include chelator conjunction to the antibody glycan region, enzymeassisted chelator attachment, and incorporating and chelating radioisotope into amino acid sequences [171]. This may help us design molecular imaging agents and companion therapeutic agents with increased possibility for clinical translation.

For the diagnosis of TC, the superiority of MI over conventional anatomical imaging is clear, with advantages of favorable spatial and temporal resolution and functional imaging [175]. The emergence of MI has fundamentally changed the management of TC. For instance, [18F]FDG PET/CT plays its role in optimizing initial therapy, which is mandatory for improving DTC outcome. [18F]FDG PET/CT could be conducted if some foci are radioiodine non-avid before treatment planning. Rosenbaum-Krumme et al. found that the TNM staging and management were changed from standard therapy (surgery plus 131I therapy) to individual therapy (standard therapy plus external beam therapy or targeted therapy etc.) because of the [18F]FDG PET/CT results in 21% of the high-risk DTCs [176]. We suppose that MI in TCs would become a helpful modality for tumor staging, prognosis evaluation, which may lead to immense changes in what treatments the TC patients are given, maximizing the benefits of individual therapy.

For TC therapy, despite the rapid progress in molecular imaging, we firmly believe that the innovation of therapeutic agents should accompany the development of diagnostic agents. For example, radionuclides such as 177Lu, 225Ac 188Re, 67Cu, 47Sc, 166Ho, 90Y, 161Tb, 149Tb, 212Pb, and 213Bi emitting α-particles, β-particles, and Auger electrons can be feasibly chelated with DOTA, NOTA, etc., which is similar to the diagnostic isotopes mentioned in this review, 64Cu or 68Ga chelated with NOTA or DOTA [177]. For theranostic application, diagnostic probes with a chelating agent and a radionuclide suitable for imaging (e.g., NOTA and 64Cu) can be used to map the target and assess the therapeutic potential of the same probe labelled with the same chelating agent and a therapeutic radionuclide (e.g., NOTA and 67Cu) [19]. 149Tb is another theranostic radioisotope that has not been investigated in thyroid cancer research to date. 149Tb can simultaneously emit positrons (β<sup>+</sup> particles), α-particles, and γ-radiation, allowing PET and α particle-based therapy to go on at the same time [177]. The current review focuses on emerging probes for imaging with examples. Further introduction and discussion about the concept of TC therapy or theranostics will be updated and illustrated in an upcoming review. We hope the ever-developing diagnostic and therapeutic probes and theranostic applications can substantially improve the management of TCs, especially the aggressive RR-DTCs, MTCs, and ATCs.

In the last two decades, there has been a general increase in the prevalence of TC. The phenomenon is partially due to environmental factors (e.g., chemical pollution, anthropogenic or natural radiation), but is also due to overdiagnosis by increased screening with more sensitive methods (e.g., high-resolution ultrasound), especially in developed countries (e.g., South Korea and the United States), leading to unnecessary treatment. As mentioned previously, most incident TCs are low-risk DTCs that tend to retain their stability over the years until death due to aging. Therefore, it is unnecessary to treat indolent TCs because of the low cost-effectiveness and the practically unchanged mortality. Something to note is that the ever-developing MI techniques might more sensitively detect TCs, potentially causing overdiagnosis for indolent TC and overtreatment via invasive methods (e.g.,

thyroidectomy or metastasectomy) or noninvasive therapeutics (e.g., chemotherapy agents or multikinase inhibitors). To avoid these pitfalls, future work could focus on discovering prognosis- or progress-related targets or probes, thereby classifying TCs into indolent and active disease. In other words, future MI research should not be limited to the field of finding latent TC lesions.

**Author Contributions:** Conceptualization, Y.J., W.W. and W.C.; Methodology, Y.J., B.L. and W.W.; Writing—original draft preparation, Y.J.; Writing—review and editing, B.L., M.H.Y. and W.W.; Visualization, Y.J.; Supervision, W.W. and W.C.; Project administration, W.W. and W.C.; Funding acquisition, W.W., W.C., J.L. and G.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Key Research and Development Program of China (grant no. 2020YFA0909000), the National Natural Science Foundation of China (grant no. 82001878), the Shanghai Rising-Star Program (Grant No. 20QA1406100), and the University of Wisconsin–Madisons. Y.J. is supported by a postgraduate scholarship from the China Scholarship Council.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** No new data were created or analyzed in this study. Data sharing is not applicable to this article.

**Conflicts of Interest:** Weibo Cai is a scientific advisor, stockholder, and grantee of Focus-X Therapeutics, Inc. All other authors declare no conflict of interest.
