Over the past decade, the tumor microenvironment (TME) has become a new paradigm of cancer diagnosis and therapy due to its unique biological features, mainly the interconnection between cancer and stromal cells. Within the TME, cancer-associated fibroblasts (CAFs) demonstrate as one of the most critical stromal cells that regulate tumor cell growth, progression, immunosuppression, and metastasis. CAFs are identified by various biomarkers that are expressed on their surfaces, such as fibroblast activation protein (FAP), which could be utilized as a useful target for diagnostic imaging and treatment. One of the advantages of targeting FAP-expressing CAFs is the absence of FAP expression in quiescent fibroblasts, leading to a controlled targetability of diagnostic and therapeutic compounds to the malignant tumor stromal area using radiolabeled FAP-based ligands. FAP-based radiopharmaceuticals have been investigated strenuously for the visualization of malignancies and delivery of theranostic radiopharmaceuticals to the TME. This review provides an overview of the state of the art in TME compositions, particularly CAFs and FAP, and their roles in cancer biology. Moreover, relevant reports on radiolabeled FAP inhibitors until the year 2021 are highlighted—as well as the current limitations, challenges, and requirements for those radiolabeled FAP inhibitors in clinical translation.

Breast Cancer (BC) is the most common cancer worldwide and, despite the advancements made toward early diagnosis and novel treatments, there is an urgent need to reduce its mortality. The Gastrin-Releasing Peptide Receptor (GRPR) is a promising target for the development of theranostic radioligands for luminal BC with positive estrogen receptor (ER) expression, because GRPR is expressed not only in primary lesions but also in lymph nodes and distant metastasis. In the last decades, several GRPR-targeting molecules have been evaluated both at preclinical and clinical level, however, most of the studies have been focused on prostate cancer (PC). Nonetheless, given the relevance of non-invasive diagnosis and potential treatment of BC through Peptide Receptor Radioligand Therapy (PRRT), this review aims at collecting the available preclinical and clinical data on GRPR-targeting radiopeptides for the imaging and therapy of BC, to better understand the current state-of-the-art and identify future perspectives and possible limitations to their clinical translation. In fact, since luminal-like tumors account for approximately 80% of all BC, many BC patients are likely to benefit from the development of GRPR-radiotheranostics.

Radioligand therapy (RLT) targeting prostate specific-membrane antigen (PSMA) is an emerging treatment for metastatic castration-resistant prostate cancer (mCRPC). It administrates ${}^{225}$Ac- or ${}^{177}$Lu-labeled ligands for the targeted killing of tumor cells. Differently from X- or $\gamma$-ray, for the emitted $\alpha$ or $\beta$ particles the ionization of the DNA molecule is less dependent on the tissue oxygenation status. Furthermore, the diffusion range of electrons in a tumor is much larger than the volume typically spanned by hypoxic regions. Therefore, hypoxia is less investigated as an influential factor for PSMA-directed RLT, in particular with $\beta$ emitters. This study proposes an in silico approach to theoretically investigate the influence of tumor hypoxia on the PSMA-directed RLT. Based on mice histology images, the distribution of the radiopharmaceuticals was simulated with an in silico PBPK-based convection–reaction–diffusion model. Three anti-CD31 immunohistochemistry slices were used to simulate the tumor microenvironment. Ten regions of interest with varying hypoxia severity were analyzed. A kernel-based method was developed for dose calculation. The cell survival probability was calculated according to the linear-quadratic model. The statistical analysis performed on all the regions of interest (ROIs) shows more heterogeneous dose distributions obtained with ${}^{225}$Ac compared to ${}^{177}$Lu. The higher homogeneity of ${}^{177}$Lu-PSMA-ligand treatment is due to the larger range covered by the emitted $\beta$ particles. The dose-to-tissue histogram (DTH) metric shows that in poorly vascularized ROIs only 10% of radiobiological hypoxic tissue receives the target dose using ${}^{177}$Lu-PSMA-ligand treatment. This percentage drops down to 5% using ${}^{225}$Ac. In highly vascularized ROIs, the percentage of hypoxic tissue receiving the target dose increases to more than 85% and 65% for the ${}^{177}$Lu and ${}^{225}$Ac-PSMA-ligands, respectively. The in silico study demonstrated that the reduced vascularization of the tumor strongly influences the dose delivered by PSMA-directed RLT, especially in hypoxic regions and consequently the treatment outcome.

Background: FAP radiopharmaceuticals show promise for cancer diagnosis; however, their limited tumor residency hinders treatment. This study compared two FAPi derivatives, DOTA.SA.FAPi and DOTAGA.(SA.FAPi)₂, labeled with gallium-68 and lutetium-177, aiming to determine an optimum combination for creating theranostic pairs. Methods: The radiotracers were studied for lipophilicity, binding to human serum proteins, and binding to human cancer-associated fibroblasts (CAFs) in vitro, including saturation and internalization/externalization studies. PET/SPECT/CT and biodistribution studies were conducted in PC3 and U87MG xenografts for [⁶⁸Ga]Ga-DOTA.SA.FAPi and [⁶⁸Ga]Ga-DOTAGA.(SA.FAPi)₂. [¹⁷⁷Lu]Lu-DOTA.SA.FAPi and [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂, were evaluated in PC3 xenografts. Biodistribution studies of [⁶⁸Ga]Ga-DOTA.SA.FAPi were performed in healthy male and female mice. Results: All radiotracers exhibited strong binding to FAP. Their internalization rate was fast while only [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂ was retained longer in CAFs. [⁶⁸Ga]Ga-DOTAGA.(SA.FAPi)₂ and [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂ displayed elevated lipophilicity and affinity for human serum proteins compared to [⁶⁸Ga]Ga-DOTA.SA.FAPi and [¹⁷⁷Lu]Lu-DOTA.SA.FAPi. In vivo studies revealed slower washout of [⁶⁸Ga]Ga-DOTAGA.(SA.FAPi)₂ within 3 h compared to [⁶⁸Ga]Ga-DOTA.SA.FAPi. The tumor-to-tissue ratios of [⁶⁸Ga]Ga-DOTAGA.(SA.FAPi)₂ versus [⁶⁸Ga]Ga-DOTA.SA.FAPi did not exhibit any significant differences. [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂ maintained a significant tumor uptake even after 96 h p.i. compared to [¹⁷⁷Lu]Lu-DOTA.SA.FAPi. Conclusions: Dimeric compounds hold promise for therapy, while monomers are better suited for diagnostics. Finding the right combination is essential for effective disease management.

Purpose: This study explores the use of fibroblast activation protein inhibitors (FAPI) targeting radiopharmaceuticals as a new approach for pan-cancer treatment, focusing on key factors affecting their effectiveness. We hypothesized that adjusting the administered radiotracer dose one could enhance the tumor-to-background ratios. Methods: In a dose-escalation study with PC3 xenografts, all radiotracers were administered at doses between 10 and 1500 pmol, followed by biodistribution and PET/CT imaging. Their selectivity towards FAP, PREP, and DDP4, along with their stability in vivo, was assessed by biodistribution and metabolite analysis, respectively. Organ FAP expression was quantified using qPCR, and circulating FAP (sFAP) levels were measured in mouse and human blood samples via ELISA. Proof-of-principle human studies were also conducted. Results: Increasing the dose from 10 to 600 pmol significantly reduced blood uptake and enhanced tumor uptake, optimizing their in vivo performance. All radiotracers showed peak efficacy at 350–600 pmol, with altered pharmacokinetics beyond 600 pmol. Biodistribution studies validated the in vivo selectivity of all radiotracers towards FAP, even in the presence of PREP and DPP4 inhibitors, while they demonstrated remarkable stability in vivo. FAP expression was confirmed in various organs, with sFAP quantified in both healthy mice and humans. Human studies with [⁶⁸Ga]Ga-DOTA.SA.FAPI revealed reduced off-target uptake (e.g., pancreas, salivary glands, heart), aligning with the preclinical findings. Conclusion: The study highlights the crucial need for precise FAPI-radiotracer dosing, optimizing PET imaging, reducing radiation exposure, and enhancing treatment by accounting for FAP biology and sFAP’s influence on pharmacokinetics.