pharmaceuticals

Review
New Frontiers in Cancer Imaging and Therapy Based
on Radiolabeled Fibroblast Activation Protein Inhibitors:
A Rational Review and Current Progress
Surachet Imlimthan 1 , Euy Sung Moon 2 , Hendrik Rathke 1 , Ali Afshar-Oromieh 1 , Frank Rösch 2 ,
Axel Rominger 1 and Eleni Gourni 1, *

1 Department of Nuclear Medicine, the Inselspital, Bern University Hospital, University of Bern,
CH-3010 Bern, Switzerland; surachet.imlimthan@extern.insel.ch (S.I.); hendrik.rathke@insel.ch (H.R.);
ali.afshar@insel.ch (A.A.-O.); axel.rominger@insel.ch (A.R.)
2 Department of Chemistry—TRIGA Site, Johannes Gutenberg—University Mainz, 55128 Mainz, Germany;
emoon@students.uni-mainz.de (E.S.M.); frank.roesch@uni-mainz.de (F.R.)
* Correspondence: eleni.gourni@insel.ch

Abstract: 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, immunosup-



pression, 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
Citation: Imlimthan, S.; Moon, E.S.;
diagnostic imaging and treatment. One of the advantages of targeting FAP-expressing CAFs is the
Rathke, H.; Afshar-Oromieh, A.;
absence of FAP expression in quiescent fibroblasts, leading to a controlled targetability of diagnostic
Rösch, F.; Rominger, A.; Gourni, E.
and therapeutic compounds to the malignant tumor stromal area using radiolabeled FAP-based
New Frontiers in Cancer Imaging and
Therapy Based on Radiolabeled
ligands. FAP-based radiopharmaceuticals have been investigated strenuously for the visualization of
Fibroblast Activation Protein malignancies and delivery of theranostic radiopharmaceuticals to the TME. This review provides
Inhibitors: A Rational Review and an overview of the state of the art in TME compositions, particularly CAFs and FAP, and their roles
Current Progress. Pharmaceuticals in cancer biology. Moreover, relevant reports on radiolabeled FAP inhibitors until the year 2021 are
2021, 14, 1023. https://doi.org/ highlighted—as well as the current limitations, challenges, and requirements for those radiolabeled
10.3390/ph14101023 FAP inhibitors in clinical translation.

Academic Editors: Sven Stadlbauer Keywords: fibroblast activation protein; cancer-associated fibroblast; tumor microenvironment;
and Klaus Kopka fibroblast activation protein inhibitor; nuclear imaging; radiotherapy

Received: 3 September 2021

Accepted: 29 September 2021
Published: 5 October 2021
1. Introduction
Publisher’s Note: MDPI stays neutral
Cancer is a heterogeneous disease formed within an extremely complex microenvi-
with regard to jurisdictional claims in
ronment [1]. In fact, a malignant tumor does not only consist of cancerous cells but also a
published maps and institutional affil- vast majority of endogenous host stromal cells (e.g., fibroblasts, and vascular and immune
iations. cells) and extracellular matrix (ECM) components, collectively known as the tumor mi-
croenvironment (TME) [2]. The TME is a unique milieu constituted within the tumor and
can influence tumor development, immune evasion, metastasis, and therapeutic resistance
through complex heterotypic interactions with the cancer cells [3]. The stromal cells in the
Copyright: © 2021 by the authors.
tumor (tumor stroma) are the largest portion of the total tumor mass (over 90%) connected
Licensee MDPI, Basel, Switzerland.
through desmoplastic reaction [4]. Recently, TME components and ECM remodeling have
This article is an open access article
attracted great attention as crucial oncological factors that determine the behavior of cancer
distributed under the terms and cells and disease progression [5]. Among all cells within the TME matrix, fibroblasts are
conditions of the Creative Commons considered to be dominant cells that have a strong association of their biological functions
Attribution (CC BY) license (https:// to all stages of cancer progression and metastasis [6]. Cancer-associated fibroblasts (CAFs),
creativecommons.org/licenses/by/ a type of continuously activated fibroblast, have been implicated to have a strong tumor-
4.0/). modulating effect and are commonly found in most solid tumors—such as breast, prostate,

Pharmaceuticals 2021, 14, 1023. https://doi.org/10.3390/ph14101023 https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 1023 2 of 30

and pancreatic cancers [7,8]. Generally, CAFs account for up to 80% of all fibroblasts in the
TME [9]. The protein molecules secreted by CAFs—such as growth factors, chemokines,
cytokines, and matrix metalloproteinases—play a pivotal role in tumorigenesis through
the stimulation of cell–cell communication and ECM remodeling [10]. In this light, CAFs
have been demonstrated to be a potential biological target for cancer diagnosis and therapy
based on the TME targeting approach [11]. CAFs are identified by the expression of vari-
ous specific biomarkers on their surface, thus providing the opportunity to be used as a
particular site for targeted radio- diagnostic and/or therapeutic applications. Among those,
surface bound biomarkers characterized by neutral pro-tumorigenic or tumor-suppressive
ability are α-smooth muscle actin (α-SMA), fibroblast-specific protein 1 (S100A4 or FSP-1),
platelet-derived growth factor receptors (PDGFRα/β), and fibroblast activation protein
(FAP) [12]. In the field of nuclear medicine in particular, FAP appears to be a promising
target due to its non-expression in normal fibroblasts and the stroma of benign epithelial tu-
mors compared to its significantly high accumulation, mainly on the stromal compartments
of a variety of malignant tumors [13].
Nuclear imaging modalities, including positron emission tomography (PET) and
single-photon emission computed tomography (SPECT), are noninvasive imaging tools
widely used in the clinic. Tumor imaging using PET and SPECT techniques allows real-
time monitoring and determination of target occupancy, pharmacokinetics, biodistribution,
elimination, and treatment responses of established radiopharmaceuticals in vivo [14].
Furthermore, when the imaging agent that identifies the malignant lesions is followed
by the administration of the companion therapeutic agent that treats the same lesions,
the generated pair is integrated into the theranostic platform. The visualization of FAP-
expressing fibroblasts has been investigated with different diagnostic as well as therapeu-
tic radiolabeled agents, which could be grouped into three main categories: antibodies,
FAP inhibitors (FAPIs) of low molecular weight, and peptidomimetic structures [15–20].
Although imaging of FAP showed great promise in both preclinical studies and clinical tri-
als, the therapeutic efficacy of the fibroblast activation protein inhibitor alone is still limited
due to a relatively short retention time of the corresponding radiopharmaceuticals in the
tumor area. Therefore, the current effort has been directed towards the optimization of the
molecular structure of FAPI to exhibit favorable pharmacokinetic performance, resulting
in prolonged retention at the tumor site. Ultimately, this could provide a new convenient
avenue for the development of FAP-based radiopharmaceuticals for the diagnosis and
treatment of cancers predominantly.
In this review, we aim to provide a comprehensive overview of the biological functions
and fates of TME, CAFs, and FAP in cancer prognosis and development. Besides, we further
discuss the recent reports of FAP-based radiopharmaceuticals for nuclear imaging and
targeted radionuclide therapy (endoradiotherapy) in the preclinical level and also provide
a report on the current status with respect to the clinical assessment.

Tumor Microenvironment: A New Arena in Stromal Targeting

In the past decade, tremendous progress in understanding the role of TME partici-
pating in cancer development and growth has been achieved, facilitating the broadening
of tumor targeting and cancer therapeutic approaches [21]. As previously mentioned, the
TME comprises different types of stroma (non-malignant) cells (e.g., various phenotypes
of fibroblasts, immune and inflammatory cells), ECM components, lymph nodes, nerves,
and blood vessels (Figure 1). Within the TME, the interaction between tumor stroma
(non-neoplastic part of the TME) and cancer cells is known to be the key parameter to
direct tumor development and metastasis [22]. The activated stroma undertakes critical
roles in cell invasion, extravasation, migration, angiogenesis, immunosurveillance evasion,
and therapeutic resistance [23].
Pharmaceuticals 2021, 14, 1023 3 of 30

Figure 1. Schematic representation of the tumor microenvironment (TME) depicting various non-malignant and malignant
cells, ECM compositions, and crucial biological processes developed within the TME. CAF: cancer-associated fibroblast;
ECM: extracellular matrix; Treg: regulatory T cell; M1: anti-inflammatory macrophage; M2: tumor-associated macrophage.
The figure is reproduced with permission from [23].

The biological functions of the TME in cancer initiation and progression have been
considered to be prominent for enhanced molecular-based diagnostic and therapeutic
agents [24]. The tumor-associated stromal components in the TME are known to be the
primary support of nutrient supplies for the establishment of metabolic networks with
tumor cell compartments. Typically, the stromal cells secrete growth factors, chemokines,
cytokines, miRNA, and extracellular vesicles in order to interact with cancer cells, the ECM
components, and among themselves, leading to cancer metabolism involved in tumor
development and progression [25]. The upregulation in the expression of oncogenes and
proteins promotes additional oncogenic signaling pathways necessary for cell communica-
tion within tumor stroma, enhancing the proliferation and invasion of cancer cells [5,26,27].
In general, tumor stroma and cancer cells should have at least four critical capabilities
to advance tumor development and progression, including mobility, ECM degradability,
survival in blood circulation, and the ability to adapt and develop in a new tissue envi-
ronment [24]. To acquire those traits, cancer cells employ the transcriptional factors (TFs),
regulatory factors in orchestrating gene expression during the course of cancer develop-
ment [28]. Recent studies demonstrated the potential of TFs to induce TME remodeling
as well as governing the proliferation and migration of cancer cells [28–30]. To this end,
Pharmaceuticals 2021, 14, 1023 4 of 30

the TME transmits the oncogenic signals to activate the TFs. The stromal cells induce
the transcription programs allowing mesenchymal stem cells (MSCs) to invade distant
tissues and create a new TME, after which the cancer cells shut down those transcriptional
processes and reconvert the MSCs into epithelial cells while replicating themselves in the
core of the tumor [24,31]. Therefore, tumorigenesis occurs from the abnormal development
of cells within tissues before turning into a malignancy (Figure 2). Cancer cells secrete
diverse growth factors and degrading proteinases, as well as stimulating the host to release
biomolecules that can degrade the ECM and its component adhesion molecules. The degra-
dation usually occurs at the surface of the tumor cell where there is an outbalance between
degradative enzymes and natural proteinase inhibitors [24,32,33]. These secreted proteins
and enzymes by tumor cells are involved in cell adhesion, cell signaling, cell motility, and
invasion. When the ECM (i.e., collagen) is degraded, activated fibroblasts, inflammatory
cells, and angiogenesis are triggered, thus resulting in the generation of growth factors and
degrading enzymes, which are beneficial for cancer development and progression [24,34].
Taken together, the TME is considered as a target-rich milieu for cancer diagnosis and
therapy as asserted by extensive investigations conducted over the past decade.

Figure 2. Tumorigenesis. (A) Normal epithelium with stromal compartment, including normal epithelial cells, basement
membrane, macrophages, fibroblasts, and general ECM components. (B) Dysplasia; a process during tumorigenesis where
the modified epithelial and stromal cells start excessively propagating and mutating due to cancer invasion. At this stage, the
fibroblasts become activated, and macrophages are decreased. (C) Carcinoma; the ECM is degraded while the angiogenesis
is formed to supply nutrients and oxygen to the tumor area, leading to the continual unregulated proliferation of cancer
cells. The figure is reproduced and adapted with permission from [24].

2. Cancer-Associated Fibroblasts: A Critical Mediator in Cancer Progression

Among all the stromal cells, cancer-associated fibroblasts (CAFs) are dominant popu-
lations in the TME structure. In general, fibroblasts are quiescent and become activated
during wound repair and regeneration, collectively known as myofibroblasts [35]. In the
normal wound healing cycle, myofibroblasts appear in the early phase of granulation tissue
formation, then become the most abundant cell type in the proliferation phase before pro-
gressively disappear in the later stage of the wound healing by apoptotic mechanism [36].
Malignant tumors are conversely recognized as “wounds that do not heal” [37,38], prompt-
ing the activated fibroblasts (CAFs in this case) to become an excellent target for cancer
treatment [39]. Several recent studies have revealed the critical participation of CAFs in
cancer progression as well as modulation of an antitumor immune response [34,40–42].
In tumor stroma, CAFs appear as spindle-shaped cells widely distributed in most con-
nective tissues and responsible for establishing and remodeling the ECM architecture.
Although the exact origin, subtypes, and biology of CAFs are not well defined due to
their heterogeneity and enigmatic cellular components [38,43], increasing investigation of
CAFs in cancer indicates their significance in tumorigenesis, especially in solid tumors [34].
Pharmaceuticals 2021, 14, 1023 5 of 30

CAFs establish a host response to neoplastic transformation and present dynamic pro- and
anti-tumorigenic features during cancer progression. They also exhibit similar functions
as fibrosis-associated fibroblasts, such as epigenetic programing [44]. Due to their hetero-
geneity, CAFs can be recruited and activated through different biological pathways from
various cellular sources (Figure 3), for instance, normal fibroblasts, bone marrow-derived
fibrocytes (BMDFs), mesenchymal stem cells (MSCs), endothelial cells, epithelial cells,
pericyte, smooth muscle cells, and adipocytes [45]. Within the local source (i.e., TME),
cancer cells can induce the activation of those recruited normal resident fibroblasts into
CAFs through the release of miRNA, exosome, and transforming growth factor-β (TGFβ).
Various studies have demonstrated that quiescent pancreatic and hepatic stellate cells
appear to have a myofibroblast-like phenotype upon activation, which means they are
considered as CAFs in pancreatic and liver cancers [46–48].

Figure 3. Potential cellular sources and biological processes involved in CAF formation. CAFs can be generated from
normal resident fibroblasts (by activation), endothelial cells (by EndMT), epithelial cells (by EMT), pericyte/smooth muscle
cell/adipocyte (by transdifferentiation), and circulating fibrocytes (by recruitment). EndMT is known as endothelial-
to-mesenchymal transition, EMT as epithelial-to-mesenchymal transition, SDF1 as stromal-derived factor 1, S100A4 as
fibroblast-specific protein 1, MSC as mesenchymal stem cell, TGFβ as transforming growth factor-β, and α-SMA as α-smooth
muscle actin. The figure is reproduced with permission from [41].

Furthermore, BMDFs and MSCs may participate in the CAF pool triggered within the
tumor stroma through the recruitment process. During the wound healing, BMDFs can
Pharmaceuticals 2021, 14, 1023 6 of 30

migrate to the site of the inflammatory tissue (tumor area in this case) and differentiate
into CAFs, contributing to tumor proliferation [49,50]. On the other hand, MSCs—another
suggested CAF precursor—can undergo differentiation due to the excessive production
of α-SMA responding to the TGFβ secreted by cancer cells for immunosurveillance eva-
sion [51,52]. CAFs can also be differentiated through endothelial and epithelial cells
through endothelial-to-mesenchymal transition (EndMT) and epithelial-to-mesenchymal
transition (EMT), respectively. EndMT and EMT can directly polarize endothelial and
epithelial cells to differentiate into mesenchymal before transforming into CAFs stimulating
by TME secreted factors (e.g., S100A4, growth factors, and cytokines) [53–55]. The least
common procedure in CAF differentiation is transdifferentiation (lineage reprogramming)
where one mature somatic cell is converted into another somatic cell without complete ded-
ifferentiation [56]. The cells that undergo transdifferentiation include adipocytes, smooth
muscle cells, and pericytes [41].
Because of phenotypic heterogeneity in CAFs, the biological markers are diverse but
exhibit specific expression patterns following local TME conditions [34]. CAFs are com-
monly identified based on the expression of various biomarkers, such as α-SMA, S100A4,
platelet-derived growth factor receptors (PDGFRα/β), fibroblast activation protein (FAP),
tenascin C, vimentin, and podoplanin (PDPN). While the expression of these markers is
found on CAFs, several of them also reveal their expression in other cell types (e.g., im-
mune cells, epithelial cells, non-mesenchymal ECM producing cells, lymphatic endothelial
cells, and adipocytes), which also determines the functional heterogeneity of CAF biomark-
ers [34,41,42]. In fact, CAF biomarkers have been intensely explored but still lack precise
identification of specific expression patterns with well-defined features [57]. In particular,
α-SMA, FAP, and PDGFRα/β are highly expressed in CAFs and have been extensively
used in several oncological investigations to determine CAF populations. Among them,
FAP has received tremendous interest as a potential biomarker for CAF identification and
targeting due to its overexpression in most of the cancer types but low to undetectable in
normal fibroblasts presented in the body [11,58–60]. The most relevant CAF biomarkers
and their biological functions are compiled in Table 1.

Table 1. Typical biomarker expression in CAFs and their biological identities.

Shared
Surface Related Cancer
Biomarker Function Expression with
Expression Model
Other Cells
Cytoskeleton Marker
Normal
Structure Pancreatic fibroblasts
α-SMA Contractility No Liver Smooth muscle
Motility Breast cells
Pericytes
Collagen Normal
induction fibroblasts
S100A4 or FSP-1 No Breast
Fibrosis Macrophages
Motility Epithelial cells
Neurons
Structure Breast
Vimentin No Epithelial cells
Motility Prostate
Endothelial cells
Membrane-bound protein and receptor
Fibrogenesis Stromal
>90% of all
FAP ECM Yes fibroblasts
cancers
remodeling Immune cells
Pharmaceuticals 2021, 14, 1023 7 of 30

Table 1. Cont.

Shared
Surface Related Cancer
Biomarker Function Expression with
Expression Model
Other Cells
Normal
fibroblasts
Tyrosine kinase Cervical Skeletal muscle
PDGFRα/β Yes
activity receptor Colorectal Pericytes
Vascular Smooth
muscle
ECM Component
Breast
Tenascin C Cell adhesion No Malignant Cancer cells
glioma

3. Fibroblast Activation Protein: A Potential Diagnostic and Therapeutic Target across

Stromal Barriers
3.1. Biological Characteristics of FAP
Fibroblast activation protein alpha (FAPα or FAP), also known as prolyl endopeptidase
FAP or seprase, is a type II membrane-bound serine protease (97 kDa subunit) associated
with fibrosis, tissue repair, inflammation, and ECM degradation [57]. Importantly, FAP is
highly expressed in CAFs, a major constituent of tumor stroma, and is upregulated in more
than 90% of human epithelial cancers [61,62]. Due to this reason, FAP is widely recognized
as a crucial biomarker to identify potential CAF-positive tumor stroma. FAP contains
760 amino acids in its structure in which residues 1–4 are in the intracellular domain,
residues 5–25 in the transmembrane domain, and residues 26–760 in the extracellular
domain (Figure 4). Within the extracellular domain, the β-propeller domain comprises the
amino acid residues 54–492 (i.e., substrate selectivity gate) while the residues 26–53 and
493–760 belong to the α/β hydroxylase domain [13,63].

Figure 4. Schematic representations of FAP structural compositions (top panel) and its protein scaffold (bottom panel). In the
protein scaffold, the central pore, seven-bladed β-propeller, single β-propeller, and α/β hydroxylase domains are identified.
“#” represents the position of the amino acid residue in FAP domains. The figure is reproduced with permission from [13].
Pharmaceuticals 2021, 14, 1023 8 of 30

FAP is the homolog of dipeptidyl peptidase IV (DPPIV or CD26), one of the members
of the prolyl peptidase family. DPPIV shares about 50% similarity in amino acid sequence
with FAP, and 70% homology of the catalytic domain [64–66]. FAP contains two types of
enzymatic activity: dipeptidyl peptidase and endopeptidase. Unlike FAP, DPPIV does not
display endopeptidase activity. The endopeptidase allows FAP to mediate the proteolytic
processing of matrix metalloproteinase-cleaved collagen I, leading to the prevention of
morphogenesis, tissue remodeling, and repair [67–69]. Therefore, FAP-specific detection
has been directed towards the endopeptidase activity as well as the development of
novel FAP-targeted inhibitory molecules [13,70]. Through enzymatic and non-enzymatic
activities, FAP demonstrates pro-tumorigenic activity involved in migration, invasion, and
proliferation of stromal fibroblasts, immune, endothelial, and cancer cells, resulting in ECM
degradation, tumor angiogenesis, invasiveness, and immunosurveillance evasion [71–73].
In general, the FAP monomer is considered inactive but can exhibit activity in the
form of homodimers and heterodimers with DPPIV [74]. For the regulation of FAP activity,
dimerization and glycosylation are required. The homodimer (activated FAP) can assem-
ble into a heterodimer by merging FAP and DPPIV, which participates in the fibroblast
migration to the collagenous matrix [75]. FAP can also bind to β-integrins, the important
proteins for cell adhesion, signal transmission, and activation of cellular response. This pro-
cess provides an enhanced cellular localization of FAP in the actin-rich protrusions on
the plasma membrane of malignant cells, which involves the ECM degradation of cancer
invasiveness [76]. Besides, glycosylation is essential for the endopeptidase activity of FAP
for which five potential N-linked glycosylation sites are identified on asparagine residues
in both β-propeller (49, 92, 227, 314) and α/β hydroxylase (679) domains [13,77].

3.2. Relationship between FAP and Immunosuppression in the TME

The complex interactions between stromal and cancer cells in the TME vastly con-
tribute to carcinogenesis and tumor progression. However, the high immune tolerance of
tumors raises complexity and impediment in cancer immunotherapy [65]. The immune
cells within the TME are immunosuppressive cells, such as tumor-associated macrophages
(TAMs), myeloid-derived suppressor cells (MDSCs), natural killer (NK) cells, cytotoxic
CD8 T cells, and regulatory T (Treg) cells [78]. Unlike normal tissues, immune cells in
the TME are significantly low in number and are inactive, which paves the way for can-
cer’s camouflage from the immunosurveillance and the attack of effector cells, leading to
ineffective treatment. Growing evidence has suggested that FAP is one of the immuno-
suppressive components in the TME that induces tumor-promoting inflammation [79].
Feig et al. reported the mediation of immune suppression by chemokine CXCL12 from
FAP-expressing CAFs in pancreatic cancer. The studies showed that the administration of
AMD3100 (CXCL12 inhibitor) induced rapid T-cell accumulation in the region of tumor-
containing cancer cells and performed synergistically with α-programmed cell death 1
ligand 1 (α-PD-L1) to greatly induce the apoptosis of cancer cells. Hence, the CXCL12
protein secreted by FAP-positive cells may direct the immunosuppression in human pan-
creatic ductal adenocarcinoma [80]. Furthermore, a similar approach was investigated
using an oncolytic virus-induced T-cell accumulation. The oncolytic group B, adenovirus
enadenotucirev, was first modified to express a stroma-targeted bispecific T-cell engager
(BiTE) to specifically bind to FAP on CAFs and CD3 epsilon protein on T cells in malignant
ascites and solid prostate cancer biopsies. With the FAP-BiTE encoding virus, tumor-
infiltrating PD-L1 positive T cells were induced to obstruct CAFs. In ascites, this resulted in
depletion of immunosuppressive factors and increased T-cell function and trafficking [81].
Recently, FAP protein expression in 92 colorectal cancers was determined using transcrip-
tomic and immunohistochemical data. The observation showed that FAP expressing genes
were upregulated in both mRNA and protein levels and had a high association with im-
mune cells. Moreover, the abundance of Treg cells within the tumor region was observed, as
well as the depletion of helper T (Th1) cells and NK cells, indicating an immunosuppressive
environment induced by secreted components from FAP positive cells [82]. However, the
Pharmaceuticals 2021, 14, 1023 9 of 30

FAP mechanism on immunosuppression is still in its infancy and needs further investiga-
tion to determine the exact role in the suppression of the antitumor activity of immune
cells in the TME [65,74].

3.3. FAP as a Potential Target in Cancer

As previously described, FAP expression is extremely low to absent in normal tissues
while it is overexpressed in more than 90% of human cancers, such as breast, colorec-
tal, pancreatic, melanoma, myeloma, gastric, brain, and ovarian carcinomas [62,82–87].
Thus, FAP is highly expressed in CAFs within tumor stroma, along with its fast and efficient
internalization, rendering as an attractive biomarker. Although the biological mechanism of
FAP on cancer prognosis is still vague and inconsistent throughout reports in the literature,
the existence of FAP in malignant stroma is determinative as a promising target for cancer
imaging and therapy [11,14]. FAP targeting leads to degradation of the ECM, interfering
in regulatory signaling and subsequently disrupts the supportive biological functions of
stromal CAFs on the tumor growth [63,88]. As a part of the ongoing efforts to develop FAP
targeting agents, several ligands have been reported. They mainly fall into three categories:
antibodies, FAP inhibitors, and peptides.
First, human FAP was originally identified in cultured fibroblasts using the mon-
oclonal antibody (mAb) F19 [89]. Sibrotuzumab/BIBH1, a humanized version of the
F19 antibody as well as other humanized or fully human antibodies against FAP antigen
exhibiting specificity towards the F19 epitope have been reported [90,91]. OS4 is another hu-
manized antibody (CDR-grafted) derived from the F19 antibody [92]. Furthermore, murine
anti-FAP antibodies, including chimeric and humanized versions have been evolved [93].
A second strategy aiming at FAP targeting is based on the inhibition of enzymatic
activity using small molecule inhibitors. Findings, mainly derived from preclinical studies,
suggest that the inhibition of FAP-enzymatic activity induced by low molecular weight
inhibitors has the potential to decrease the invasiveness of malignant cells and further lead
to a considerable reduction of the tumor growth [94]. The design of FAP-specific inhibitors
remains challenging due to the homology of enzymatic substrate domains shared with
other dipeptidyl peptidase members; therefore, a precise characterization of FAP substrate
and inhibitor matching is required for the structural design of novel synthetic FAPIs to
specifically target the endopeptidase activity domain in FAP [74,95]. Val-boroPro (tala-
bostat, PT-100) is a non-selective boronic acid-based inhibitor that targets on both FAP
and DPPIV enzymatic domains. Talabostat has shown promising preclinical results but
demonstrated suboptimal results in the phase II clinical trial in patients with metastatic
colorectal cancer [96]. Moreover, the combination of talabostat with chemotherapeutic
drugs, such as docetaxel and cisplatin, was conducted in non-small cell lung cancer and
metastatic melanoma patients, respectively. However, the results were unsuccessful to
demonstrate significant therapeutic outcomes in phase II clinical trials due to safety and
efficacy reasons [97,98]. On the other hand, d-Ala-boroPro-based FAP inhibitor exhibits
selectivity towards other dipeptidyl peptidase members compared to FAP by a factor of
40 [99]. However, no in vivo pharmacokinetic data for this inhibitor has been reported [100].
Linagliptin, a dual FAP and DPPIV inhibitor, has also shown a worthwhile effect in inhibit-
ing the FAP enzymatic activity [101]. Furthermore, pyroglutamyl(2-cyanopyrrolidine) and
quinolinoylglycyl(2-cyanopyrrolidine) derivatives have demonstrated highly satisfactory
selectivity to FAP over other dipeptidyl peptidase members [99,102].
Thirdly, a FAP binding peptide coupled to the radionuclide chelator DOTA (1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetic acid)—which could serve as a potential radio-
therapeutic tracer named FAP-2286—was evaluated in both preclinical and clinical levels.
FAP-2286 revealed promising data with respect to its potency, selectivity, and efficiency
towards FAP [19,20].
Pharmaceuticals 2021, 14, 1023 10 of 30

4. Development of Radiolabeled-Based FAP Tracers for Tumor Stroma Mediated

Nuclear Imaging and Radionuclide-Based Therapy
FAP targeting using FAP-based radiolabeled tracers allows the delivery of radionu-
clides carrying either imaging photons and/or ionizing particles (α and β+ /β− ) directly to
tumor stroma, resulting in nuclear imaging and/or radionuclide therapy of FAP-positive
tumors. Compared to the flagship PET radiotracer, [18 F]FDG, widely used in the clinic,
FAP-based targeting radiolabeled inhibitors may provide an alternative strategy in nuclear
molecular imaging for detecting tumors with low or heterogeneous glucose metabolism
as well as those located close to highly glycolytic tissues to avoid non-specific uptake
that could cause a high background signal [88]. As stroma occupies a major part in the
tumor volume, FAP-targeted radiotracers may increase the target sensitivity and image
contrast of the disease area compared to the targeting of glucose metabolic pathway in
cancer cells solely due to their lack of brain uptake in contrast to [18 F]FDG. Nevertheless,
the available reports on preclinical and clinical assessments of radiolabeled FAPIs are
still in an early phase, warranting further research and development. Recently, different
radiolabeled FAP-based tracers have been examined for noninvasive nuclear imaging and
targeted radionuclide therapy. In the following section, relevant FAP-targeted radioligands
developed over the past few years are highlighted.

4.1. Radiolabeled FAP-Targeted Antibodies

Because of the consistent presence of FAP on the tumor stroma and the accessibility
of FAP-positive tumor stromal fibroblasts to circulating monoclonal antibodies (mAbs),
several studies have suggested possible diagnostic and therapeutic applications of human-
ized mAb and their constructs with novel immune and nonimmune effector functions.
The murine F19 mAb, which recognizes FAP labeled with 131 I, has been used for that
purpose [89]. Two phase-I quantitative biodistribution studies with [131 I]I-F19 mAb in
patients with hepatic metastasis from primary colorectal cancer and soft tissue sarcoma
have demonstrated the proof of principle of stromal targeting [89,103]. Based on the results
acquired with the use of the murine F19 mAb and aiming at addressing the problems
that appeared due to the immune responses to murine antibodies, a humanized version
of F19—named sibrotuzumab—was preclinically and clinically investigated after radio-
labeling with 131 I. [131 I]I-sibrotuzumab was found to preferentially bind to FAP in vitro
in the same manner as its murine counterpart without affecting the FAP-related enzy-
matic activity. Besides, the results of the first-in-human clinical study demonstrated the
ability of [131 I]I-sibrotuzumab to target stromal FAP and provided evidence that sibro-
tuzumab has a considerable promise in the targeting and therapy of epithelial malignancies.
However, [131 I]I-sibrotuzumab failed to provide a measurable therapeutic activity in phase
I/II clinical trials despite excellent tumor stroma targeting properties. In addition, about
one-third of the sibrotuzumab treated patients developed human-anti-human antibodies
(HAHA) and a reduction in tumor uptake. Therefore, further clinical development of
sibrotuzumab has been discontinued [104]. In an attempt to address the side effects caused
by sibrotuzumab, the successful selection of two human mAbs (ESC11 and ESC14) from a
phage-display library took place. Both ESC11 and ESC14 presented the advantage of being
rapidly internalized by FAP-positive cells. The labeling with the β-emitting 177 Lu of both
exhibited specific accumulation in FAP-positive human melanoma xenografts, providing a
delay in tumor growth in vivo [105].

4.2. Radiolabeled FAP-Based Inhibitors

The first generation of FAP inhibitors was developed based on dipeptide boronic acid
inhibitors (Figure 5). Val-boroPro and D-Ala-boroPro have been proved to be highly
potent against DPPIV and can be combined with the selectivity to DPP8 and DDP9
in vivo, providing a relatively high therapeutic index above 500 in mouse models [106].
However, the biological behavior of these inhibitors has been hampered due to the lack of
their selectivity towards FAP over dipeptidyl peptidases (DPPs) and prolyl oligopeptidase
Pharmaceuticals 2021, 14, 1023 11 of 30

(PREP). In the subsequent work, D-Ala-boroPro was further modified at the amine residue,
forming N-(Pyridine-4-carbonyl)-D-Ala-boroPro (ARI-3099). ARI-3099 demonstrated low
nanomolar potency (IC50 ~36 nM) and high selectivity for FAP (350-fold over PREP), with
negligible potency for DPPs in murine FAP (mFAP) transfected human embryonic kidney
(HEK) 293 cells [100]. Furthermore, the boronic acid-based FAP inhibitor (MIP-1232) was
synthesized and labeled with 125 I [107]. [125 I]I-MIP-1232 showed high accumulation in
FAP-positive SK-Mel-187 melanoma cells and trivial in an NCI-H69 cell line with low
FAP expression. However, the chemical stability and reactivity of boronic acid-based FAP
inhibitors towards FAP enzymes are still ambiguous and not well characterized [108].

Figure 5. Different relevant FAPI molecules based on boronic acid warhead. (A) The first generation of boronic acid-based
FAPIs: Val-boroPro, Ala-boroPro, and Glu-boroAla. (B) The later generation of FAP-targeted inhibitors: N-(Pyridine-4-
carbonyl)-D-Ala-boroPro, N-(Pyridine-3-carbonyl)-Val-boroPro, and [125 I]I-MIP-1232.

In 2014, a library of highly potent and selective FAPIs synthesized by utilizing the
core of N-4-quinolinoyl-Gly-(2S)-cyanoPro scaffold as the starting point were vigorously
explored in terms of the structure–activity relationship against dipeptidyl peptidases and
prolyl endopeptidase [109,110]. The first attempt of radiolabeled quinoline-based FAPIs
(i.e., FAPI-01 and FAPI-02) was reported by the Heidelberg research group in Germany [111].
Both FAPIs were synthesized using methods reported earlier by Jansen et al. [109,112].
The basic structure of both compounds consists of a quinoline unit for target selectivity
and retention as well as the Gly-Pro motif containing a nitrile group (CN) for a covalent
bond formation towards the binding pocket of FAP. FAPI-02 was further designed by the
conjugation of the chelator DOTA through a piperazine linker, at position 6 on the aromatic
ring of the quinoline group of FAPI, intended to incorporate suitable diagnostic/therapeutic
radiometals and to improve the pharmacokinetics (Figure 6A). FAPI-01 and FAPI-02 were
radiolabeled with 125 I and 68 Ga/177 Lu, respectively. Although [125 I]I-FAPI-01 exhibited
rapid internalization in both murine FAP-transfected human embryonic kidney (HEK) cells
and human FAP-transfected fibrosarcoma HT-1080 cells in vitro, time-dependent efflux
and enzymatic deiodination of [125 I]I-FAPI-01 eliminated its further preclinical evaluation.
Pharmaceuticals 2021, 14, 1023 12 of 30

In contrast, [68 Ga]Ga-FAPI-02 revealed enhanced binding and uptake in human FAP-
expressing cells in vitro and in vivo compared to [125 I]I-FAPI-01, arising from the enhanced
stability of the radiolabeled compound. [177 Lu]Lu-FAPI-02 biodistribution studies showed
the highest uptake at 2 h after administration in human FAP-transfected HT-1080 tumor-
bearing mice; however, the retention time of [177 Lu]Lu-FAPI-02 in the tumor was relatively
short and might not be enough to achieve a therapeutic response. Therefore, further efforts
of the research group were directed towards the structural modification of FAPI-02 in order
to prolong the tumor retention time and improve lipophilicity. The quinoline-Gly-Pro
FAPI with a carbonitrile warhead, so-called UAMC1110, reported by Jansen et al. [109] was
further functionalized mainly with the chelator DOTA via a variety of linkers (Figure 6B).
UAMC1110 exhibits high affinity to FAP compared to PREP (inhibitory potencies (IC50 ); 3.2
nM and 1.8 µM, respectively). On the basic structure of UAMC1110, there is an additional
difluoro substitution on the pyrrolidine ring in the Gly-Pro motif, which has been shown
to improve target binding affinity [109]. In the first developed series, 13 FAPI derivatives
(FAPI-03 to FAPI-15) were synthesized [113]. Briefly, the molecular structures of the FAPI
derivatives were designed by attaching various linkers to the quinoline moiety of the
UAMC1110 in different positions as well as varying the substitution group (F or H) on
the pyrrolidine ring of the Gly-Pro motif. Among all derivatives, FAPI-04 and FAPI-13
were proved to be the most promising tracers compared to others of the same series,
demonstrating high binding affinity towards the human FAP-transfected HT-1080 cells
with IC50 values of 6.5 nM and 4.5 nM, respectively. The in vivo evaluation of [177 Lu]Lu-
FAPI-04 and [177 Lu]Lu-FAPI-13 was investigated in FAP-transfected HT-1080 xenografts
in comparison with previous data of [177 Lu]Lu-FAPI-02. The uptake in normal tissues
was slightly higher for FAPI-04 than FAPI-02, and even higher for FAPI-13. The tumor
accumulation of FAPI-04 (3.0%ID/g) and FAPI-13 (4.8%ID/g) was improved compared to
FAPI-02 (1.12%ID/g) at 24 h post-injection. However, the tumor-to-blood ratio was more
favorable in FAPI-04 (~28) than FAPI-13 (~22) 24 h post-injection.

Figure 6. Examples of FAPI molecules developed by the Heidelberg group. (A) The first generation of FAPIs (FAPI-01 and
FAPI-02) and subsequent work on FAPI-74 using the same FAP-targeting moiety conjugated NOTA chelator. (B) Relevant
FAPI molecules based on UAMC1110 scaffold, including FAPI-04, FAPI-21, FAPI-34, and FAPI-46.
Pharmaceuticals 2021, 14, 1023 13 of 30

Next, another series containing 15 FAPI derivatives was further developed [110].
In this set of compounds, all FAPI derivatives showed equal or better binding affinity
compared to FAPI-04. The in vivo pharmacokinetic studies by small-animal PET imaging
on FAP-transfected HT-1080 xenografts of the most promising 68 Ga-labeled candidates
of this set (FAPI-21, FAPI-35, FAPI-36, FAPI-46, and FAPI-55), demonstrated rapid tumor
accumulation, low background activity, and predominantly renal elimination. In particular,
FAPI-36 tended to have a prolonged systemic circulation, resulting in an unfavorable
tumor-to-blood ratio and poor imaging contrast. FAPI-21 and FAPI-55 revealed higher
uptake in liver and muscle tissues than FAPI-04 while FAPI-35 demonstrated comparable
tumor-to-blood and tumor-to-liver ratios with an only slight improvement in tumor-to-
muscle ratio. From this series of FAPI tested ligands, FAPI-46 appeared to be the most
promising derivative in the series providing the highest tumor-to-background ratios and
good tumor accumulation.
99m Tc-labeled FAPI tracers for SPECT imaging were also developed from the same

group [114]. These attempts led to the generation of FAPI-19 where the UAMC1110
FAP-targeting moiety was linked to a tricarbonyl chelator suitable for radiolabeling with
99m Tc. Starting from FAPI-19, several FAPI variants with chelators providing the necessary

donor atoms for sufficient coordination of 99m Tc and 188 Re were synthesized, including
FAPI-28, FAPI-29, FAPI-33, FAPI-34, and FAPI-43. All the tested compounds exhibited
high affinity towards FAP (IC50 ranging from 6.4 to 12.7 nM) and a fast internalization
rate in FAP-transfected HT-1080 cells. Their scintigraphy studies presented a variable
pharmacokinetic performance on FAP-transfected HT-1080 xenografts. Due to the high
lipophilicity of the tricarbonyl-99m Tc complex, [99m Tc]Tc-FAPI-19 revealed high liver uptake
and no significant tumor accumulation. As part of the ongoing efforts of the group
to reduce the lipophilicity of [99m Tc]Tc-FAPI-19 while improving the pharmacokinetic
performance, several hydrophilic groups were introduced between the chelator and the
inhibitor. Compared to [99m Tc]Tc-FAPI-19, other derivatives of this series demonstrated
improved tumor uptake and faster clearance from the rest of the body. Overall, [99m Tc]Tc-
FAPI-34 revealed significant tumor uptake with the lowest accumulation in the liver, biliary
gland, and intestine, providing the best in vivo pharmacokinetics among the rest of the
same series.
Another 99m Tc-labeled FAP-targeting ligand (FL-L3) was lately reported by Roy et al.,
which includes the radiometal 99m Tc coordinated via the 99m Tc(V)-oxo moiety while 8-
amino-octanoic acid served as the linker between the metal chelator and the FAP in-
hibitor [115]. [99m Tc]Tc-FL-L3 demonstrated high affinity and specificity for FAP when
the FAP-transfected human embryonic kidney HEK 293 cell line was used. The in vivo
performance of [99m Tc]Tc-FL-L3 on MDA-MB231 breast tumor-bearing mice led to specific
delineation of the experimental tumor and low non-target uptake.
FAPI-74 is another FAP-specific ligand developed by the Heidelberg group aiming
this time at developing a PET tracer suitable for labeling with the two most commonly
used PET nuclides in the clinic (68 Ga and 18 F). Based on the same FAP-targeting moiety
as FAPI-02, FAPI-74 was obtained by exchanging the chelator DOTA with NOTA (1,4,7-
triazacyclononane-1,4,7-triacetic acid) (Figure 6A). The superiority of NOTA over DOTA is
that it can be easily labeled with 68 Ga even at room temperature. Furthermore, NOTA can
also be labeled with 18 F via aluminium fluoride (AlF) chemistry, resulting in a more
practical large-scale production [116]. To the best of our knowledge, although preclinical
data have not been reported yet, the high image contrast and low radiation burden of FAPI-
74 PET/CT in 10 patients with lung cancer and rare cancer entities allow its applicability in
multiple clinical applications [116,117].
The benefits of radiolabeling with 18 F over 68 Ga, such as the higher molar activity of
the final radiolabeled product, the improved physical spatial resolution, and the longer
half-life that allows sufficient time for quality control and transportation, when necessary,
prompt the development of other 18 F-labeled FAPI-based ligands. FAPI-04 was modified
accordingly to achieve radiolabeling with 18 F. The DOTA on FAPI-04 molecule was substi-
Pharmaceuticals 2021, 14, 1023 14 of 30

tuted with an alkyne group, then further radiolabeled with 6-deoxy-6-[18 F]F-fluoroglucosyl
azide through copper-catalyzed click reaction, forming [18 F]F-Glc-FAPI [17]. The in vitro
radiolabel stability of [18 F]F-Glc-FAPI in human serum was maintained above 99% at 55
min of incubation. [18 F]F-Glc-FAPI showed the uptake in the human FAP-transfected
HT-1080 cells but lower affinity to FAP (IC50 = 167 nM) compared to [68 Ga]Ga-FAPI (IC50 =
32 nM). Additionally, the plasma protein binding and lipophilicity of [18 F]F-Glc-FAPI were
higher than [68 Ga]Ga-FAPI. In PET studies, tumor uptake of [18 F]F-Glc-FAPI in human FAP-
transfected HT-1080 tumor-bearing mice was higher (4.6%ID/g) compared to [68 Ga]Ga-
FAPI-04 (2.1%ID/g). However, PET images revealed higher tumor-to-background ratios
for [68 Ga]Ga-FAPI-04 due to the lower plasma protein binding and, consequently, faster
blood clearance.
On the other hand, the Mainz research group utilized an alternative approach to
modify the squaric acid (SA)-based linker bridging between several bifunctional chelators
(DATA5m , DOTA, and DOTAGA) and the UAMC1110 molecule intended to simplify com-
plex synthetic steps of the current chelator-modified FAPIs (Figure 7) [108,118]. The first
generation of precursors, DATA5m .SA.FAPi and DOTA.SA.FAPi molecules, were radio-
labeled with 68 Ga and 177 Lu. Affinity to FAP of the precursors as well as their nat Ga-
and nat Lu-labeled derivatives were excellent, resulting in low nanomolar IC50 values of
0.7–1.4 nM. Additionally, all the tested compounds showed a low affinity for the related pro-
tease PREP (high IC50 values of 1.7–8.7 µM). First proof-of-principle in vivo PET-imaging
animal studies of one of the tested radioligands, [68 Ga]Ga-DOTA.SA.FAPi, in HT29 human
colorectal xenografts indicated promising results with low background signal and high ac-
cumulation in tumor (SUVmean = 0.75), which was higher than [68 Ga]Ga-FAPI-04 (SUVmean
= 0.45) in human FAP-transfected HT-1080 tumors at the same scanning time [108]. How-
ever, the prolongation of the residence time of both DATA5m .SA.FAPi and DOTA.SA.FAPi
in tumor stroma remains a major challenge. Lately, the Mainz group continued to opti-
mize the structure of DOTA.SA.FAPi to enhance tumor uptake and retention time through
the formation of dimeric derivatives of DOTA.SA.FAPi. In this second generation, two
homodimeric derivatives, DOTA.(SA.FAPi)2 and DOTAGA.(SA.FAPi)2 , were developed
through the conjugation of two squaramide ligands with the FAP-targeting moiety [118].
DOTA.(SA.FAPi)2 displayed good radiochemical yield (RCY) when radiolabeled with 68 Ga
in the range of 80–90% in 1 M HEPES buffer (pH 5.5) after 10 min of incubation at 95
◦ C while DOTAGA.(SA.FAPi) revealed an exceptional RCY (>99%) at the same radiola-
2
beling conditions. Moreover, [68 Ga]Ga-DOTAGA.(SA.FAPi)2 demonstrated good in vitro
stabilities in human serum, phosphate-buffered saline (PBS), and isotonic saline solutions
where the percentage of intact radiolabeled compound was kept above 90% over 120 min
of incubation in the corresponding media. DOTAGA.(SA.FAPi)2 was further radiolabeled
with 177 Lu, and the results suggested excellent labeling kinetics in which the RCY was 100%
after 5 min of incubation at 95 ◦ C in 1 M ammonium acetate buffer (pH 5.5). The in vitro
stability of [177 Lu]Lu-DOTAGA.(SA.FAPi)2 proved to be the best in human serum, resulting
in 91% of the radiotracer being intact after 144 h of incubation.
Pharmaceuticals 2021, 14, 1023 15 of 30

Figure 7. FAPI molecules developed by the Mainz research group: DOTA.SA.FAPi, DATA5m .SA.FAPi, DOTA.(SA.FAPi)2 ,
and DOTAGA.(SA.FAPi)2 . ‘R’ represents UAMC1110 scaffold.

Since most of the FAPI molecules currently referred in the literature contain the
chelator DOTA available for radiolabeling with radiometals of oxidation state +3, various
SPECT radionuclides, such as 177 Lu and 111 In have been also used for radiolabeling and the
resulting radiolabeled tracers have been further explored. The first investigation of 177 Lu-
labeled FAPI was carried out with [177 Lu]Lu-FAPI-02 in comparison to [125 I]I-FAPI-01 for
long-term tracking of pharmacokinetic profiles up to 24 h [111]. The efflux kinetic studies
using the FAP-transfected HT-1080 cell line showed that [177 Lu]Lu-FAPI-02 elimination
rate was slower than [125 I]I-FAPI-01, preserving the intracellular accumulated activity of
12% compared to 1.1%, respectively. The biodistribution studies on human FAP-transfected
HT-1080 xenografts showed that the highest uptake of [177 Lu]Lu-FAPI-02 (4.7%ID/g) was
at 2 h, which was gradually decreased to about 1%ID/g at 24 h post-injection.
Although radiolabeled FAP inhibitors proved to be successful in enabling the imaging
of multiple human cancers, the time-dependent clearance from tumors seems to currently
limit their utility as FAP-targeted radiotherapeutics. A trifunctional RPS-309 inhibitor
synthesized based on the UAMC1110 scaffold was reported in the attempt to improve the
therapeutic performance of FAP-based radiotracers [119]. The RPS-309 comprises three
active functional moieties: UAMC1110 for FAP targeting, DOTA chelator for radiolabeling,
and albumin binding group for the plasma binding that enhances longer retention in
plasma. Indeed, [177 Lu]Lu-RPS-309 demonstrated a prolonged circulation time through
the albumin binding group as well as high affinity and retention in liposarcoma SW872
tumor-xenografted mice up to 24 h post-injection. The multifunctional RPS-309 could
be a useful tool for the study of the relationship between FAPI structure and substrate
activity in the future. In addition, an ultra-high-affinity small organic ligand (OncoFAP), a
carboxylic acid moiety, for FAP targeting was very recently reported [120]. The OncoFAP
was chemically modified at position 8 on quinoline moiety of the FAPI molecule and further
functionalization with the chelator DOTAGA, leading to a FAP-based ligand with a high
dissociation constant (Kd ) ranging from 0.68 nM for human FAP to 11.6 nM for murine FAP.
Pharmaceuticals 2021, 14, 1023 16 of 30

In biodistribution studies in animal-bearing SK-RC-52.hFAP tumors, [177 Lu]Lu-DOTAGA-

OncoFAP showed the localization into tumors at the maximum dose of 1,000 nmol/kg.
Higher than 30%ID/g tumor uptake was achieved within 10 min after administration while
the tumor retention was highly sustained at 6 h post-injection (>20%ID/g).
Recently, the fully automated radiosynthesis of 177 Lu-labeled FAPI-04 and FAPI-
46 radiotracer was established. High radiochemical yields and purities of tested 177 Lu-
labeled FAPI products were achieved while the standard and requirements of the European
Pharmacopoeia were met [121].
FAPI-04 was further radiolabeled with 64 Cu (β+ emitter) and 225 Ac (α emitter) for PET
imaging and radionuclide therapy, respectively [122]. The biodistribution of 64 Cu/68 Ga/
designs 225 Ac-labeled FAPI-04 was conducted in human pancreatic PANC-1 and MIA PaCa-
2 xenografts. [64 Cu]Cu-FAPI-04 showed high accumulation in tumor and most of the
normal tissues compared to [68 Ga]Ga-FAPI-04 in both tumor models. However, high
uptake in the liver of [64 Cu]Cu-FAPI-04 was observed, demonstrating the instability of
[64 Cu]Cu-DOTA complex in vivo. In therapy studies, [225 Ac]Ac-FAPI-04 was found to
significantly suppress tumor growth in the PANC-1 pancreatic cancer xenografts compared
to the control group and no severe radiotoxicity was observed; however, the application
dose of [225 Ac]Ac-FAPI-04 in this study was relatively high compared to [225 Ac]Ac-PSMA-
617 therapy (1.5 MBq/kg compared to 50–200 kBq/kg, respectively) in which further
investigation on safety, hematologic or renal toxicity should be warranted.
The SPECT radionuclide 111 In, has also been used for the generation of radiola-
beled FAP-based inhibitors for SPECT imaging and potential intraoperative applications.
[111 In]In-QCP02, a novel FAP-targeted SPECT imaging agent was synthesized based on the
FAPI-02 analog [123]. The linker between the chelator and the FAP targeting moiety was
modified using a more flexible linear hydrocarbon chain containing a semi-rigid piperazine
group that allows more efficient internalization at FAP binding sites. [111 In]In-QCP02
demonstrated a comparable inhibitory effect for FAP with a Kd value of 16.2 nM and was
proved to be more selective to FAP over DPPIV. The serial SPECT/CT imaging studies in
animals-bearing U87MG (FAP positive) and PC3 (FAP negative) tumors showed that the
uptake of [111 In]In-QCP02 in U87MG tumor was 4-fold higher than the PC3 tumor at 1 h
post-injection. The U87MG tumor could be visualized with [111 In]In-QCP02 for more than
10 h post-injection while the signal from the radiotracer was already undetectable after 3 h
post-injection in the PC3 tumor model.

4.3. Radiolabeled FAP-Targeted Peptides

The targeting of FAP in the tumor stroma using peptide-based radiotracers is still at
its outset. By far, there are only two consecutive reports on peptide-targeted radionuclide
therapy (PTRT) utilizing FAP as a tumor target [19,20]. FAP-2286 is a FAP-targeted pep-
tidomimetic functionalized with a linker bound to DOTA readily for radiolabeling with
68 Ga for PET imaging or 177 Lu for SPECT imaging and radiotherapy. In terms of potency

and selectivity, FAP-2286 peptide revealed high affinity within a low nanomolar concen-
tration in both recombinant human FAP protein (~1.1 nM) and cellular FAP-expressing
WI 38 fibroblast (~2.7 nM) in vitro [19]. Moreover, FAP-2286 was further labeled with
177 Lu and its biological behavior was examined in human FAP-transfected HEK 293 and

patient-derived sarcoma Sarc4809 xenografts. The tumor uptake of [177 Lu]Lu-FAP-2286

in FAP-transfected HEK 293 xenografts was about 14%ID/g at 3 h post-injection and
the retention remained high over 120 days (~9%ID/g). The antitumor activity revealed
that 90% of the animal cohort treated with 60 MBq of [177 Lu]Lu-FAP-2286 were tumor-
free (tumor volume < 10 mm3 ) after 42 days. On the other hand, the biodistribution of
[177 Lu]Lu-FAP-2286 in Sarc4809 xenografts injected at the same radioactivity dose as in
the FAP-transfected HEK xenografted mice showed only 5.9%ID/g tumor uptake after 3 h
post-injection. After 42 days, a significant tumor suppression was observed compared to
the vehicle group; however, the animals were not tumor-free.
Pharmaceuticals 2021, 14, 1023 17 of 30

Here, an overview of the current status of relevant radiolabeled FAP-targeted tracers

is presented (Table 2).

Table 2. Overview of important radiopharmaceutical-based FAP tracers for nuclear imaging and radiotherapy reported in
the literature.

Quality of Radiation
Radionuclide Inhibitor Evaluation Phase Reference
Imaging Radiotherapy
Clinical: patients with lung
FAPI-74 [116]
18 F PET - cancer
Preclinical: fibrosarcoma and
Glc-FAPI-04 [17]
glioblastoma xenografts
FAPI-02 Clinical: various cancers [124]
FAPI-04 Clinical: various cancers
Preclinical: fibrosarcoma
FAPI-20
xenograft
FAPI-21 Clinical: various cancers [110,111,113,
FAPI-22 124]
68 Ga PET -
FAPI-31 Preclinical: fibrosarcoma
FAPI-35 xenograft

FAPI-36
FAPI-37
FAPI-46 Clinical: various cancers
Clinical: patients with lung
FAPI-74
cancer
Preclinical: colorectal
adenocarcinoma
DOTA.SA.FAPi
xenograftClinical: various [108,125–128]
cancer patients
Preclinical: in vitro
modelsClinical: restaging of
DATA5m .SA.FAPi tumor manifestation, liver
tumor and metastases
imaging
DOTA.(SA.FAPi)2 Clinical: patient with thyroid
and pancreatic [118]
DOTAGA.(SA.FAPi)2
neuroendocrine tumors
Preclinical: liposarcoma
RPS-309 [119]
xenograft

111 In Preclinical: glioblastoma

QCP02 SPECT - [123]
xenograft
Clinical: patients with ovarian
FAPI-34 metastasis and pancreatic [114]
99m Tc SPECT -
cancer
Preclinical: breast cancer
FL-L3 [115]
xenograft
225 Ac FAPI-04 - Yes Preclinical: pancreatic cancer
[122]
64 Cu xenograft
FAPI-04 PET Yes
Pharmaceuticals 2021, 14, 1023 18 of 30

Table 2. Cont.

Quality of Radiation
Radionuclide Inhibitor Evaluation Phase Reference
Imaging Radiotherapy
FAPI-02 Preclinical: glioblastoma
[111,129]
xenograft
FAPI-04
177 Lu SPECT Yes Fully automated
FAPI-46 [121]
radiosynthesis unit
Preclinical: liposarcoma
RPS-309 [119]
xenograft
Preclinical: renal carcinoma
OncoFAP [120]
and fibrosarcoma xenografts
Preclinical: HEK-FAP tumor
bearing animalsClinical:
FAP-2286 [19,20]
Patients with diverse
adenocarcinomas
Clinical: Patient with lung
153 Sm FAPI-46 Scintigraphy Yes metastatic, fibrous spindle cell [130]
soft tissue sarcoma
Clinical: metastatic breast
FAPI-04 [113]
cancer patient
90 Y - Yes
Clinical: patient with
metastasized breast and [131]
FAPI-46
colorectal cancers
Clinical: patients with
metastatic soft tissue or bone
[132]
sarcoma, and pancreatic
cancer

5. Clinical Studies of Radiolabeled-Based FAP Inhibitors

As previously described, the clinical study of radiolabeled FAP-based antibodies has
been discontinued due to poor pharmacokinetic performance; meanwhile, to the best
of our knowledge, so far there is one publication reporting on the clinical assessment
of radiolabeled FAP-targeted peptides. The first-in-human report of PTRT of several
adenocarcinomas using the radiolabeled peptide [177 Lu]Lu-FAP-2286 was very recently
published [20]. The theranostic feasibility of [177 Lu]Lu-FAP-2286 was explored in a cohort
of 11 patients evaluating the progressiveness and the metastatic rate of pancreatic, breast,
ovarian, and colorectal adenocarcinomas. The study indicated a favorable safety use
of [177 Lu]Lu-FAP-2286 with manageable severe side effects in few patients. The post-
therapy whole-body scans, including SPECT/CT, showed high tumor accumulation and
prolonged retention time of [177 Lu]Lu-FAP-2286 in all patients at 72 h to 10 days post-
injection. Furthermore, the dosimetric studies demonstrated comparable whole-body and
bone marrow absorbed dose of [177 Lu]Lu-FAP-2286 (0.07 and 0.05 Gy/GBq, respectively)
to [177 Lu]Lu-DOTATATE (0.05 and 0.04 Gy/GBq, respectively) and [177 Lu]Lu-PSMA-617
(0.04 and 0.03 Gy/GBq, respectively). Nevertheless, the limitation of this study remains,
including the small and heterogeneous patient cohort, and the dose escalation due to
a safety concern from the pre-existing red marrow dysfunction from multiple previous
therapies. So far, the reported response rates after PTRT remain improvable, and the
combinational radio-immunotherapy might increase the response rate [131] as FAP and
CAFs are the main drivers of immune evasion [40,133].
Pharmaceuticals 2021, 14, 1023 19 of 30

Below, we give an overview based on the most relevant clinical trials on FAP-targeting
using radiolabeled FAP-based inhibitors, and their performance is discussed mainly in
comparison to [18 F]FDG PET/CT.
The efficacy of [68 Ga]Ga-FAPI-02, the first generation of FAP inhibitors was investi-
gated in comparison with the reference standard [18 F]FDG in patients with breast, lung,
and pancreatic cancer metastases. The [68 Ga]Ga-FAPI-02 showed high specific uptake in
the primary tumors, lymph nodes, and bone metastases with low background activity
while [18 F]FDG accumulated in most of high glucose consumption organs, such as brain,
liver, and spleen. However, the [68 Ga]Ga-FAPI-02 showed a rapid elimination pattern
through the renal clearance and revealed a short tumor retention time, demonstrating
about 75% decrease in tumor uptake from 1 to 3 h after tracer administration [111].
Continuing the clinical assessment of [68 Ga]Ga-FAPI-02, PET/CT scans of [18 F]FDG
and [68 Ga]Ga-FAPI-02 were conducted on six patients with several kinds of cancer (pancre-
atic, esophageal, lung, head and neck, and colorectal cancer) in a side-by-side comparative
study. The observed accumulation of [18 F]FDG and [68 Ga]Ga-FAPI-02 in tumors was com-
parable (average SUVmax : 7.41 for [18 F]FDG and 7.37 for [68 Ga]Ga-FAPI-02; not statistically
significant); however, [68 Ga]Ga-FAPI-02 could not clearly identify primary and metastatic
sites in one patient with iodine-negative thyroid cancer. [68 Ga]Ga-FAPI-02 was found
to be superior compared to [18 F]FDG in terms of background activity, especially in the
brain (SUVmax : 0.32 vs. 11.01), liver (SUVmax : 1.69 vs. 2.77), and oral/pharyngeal mucosa
(SUVmax : 2.57 vs. 4.88), thus leading to higher contrast of PET images for liver metastases
originating from pancreatic and colorectal cancer. It also exhibited a higher ability to
delineate esophageal cancer. In the same cohort of patients, it was observed that three out
of six patients treated with [68 Ga]Ga-FAPI-02 could benefit from low tracer accumulation
in liver and pharyngeal mucosa, leading to high tumor-to-background ratios (Figure 8).

Figure 8. Whole-body PET/CT scans of six selected patients with different tumors entities imaged with [18 F]FDG and
[68 Ga]Ga-FAPI-02 in the range of a 9-day imaging interval. NSCLC: non-small cell lung cancer; Ca: cancer. The figure is
originally published in JNM by Giesel et al. 68 Ga-FAPI PET/CT: Biodistribution and Preliminary Dosimetry Estimate of 2
DOTA-containing FAP-Targeting Agents in Patients with Various Cancers. J Nucl Med. 2019; 60:386–392. © SNMMI [124].
Pharmaceuticals 2021, 14, 1023 20 of 30

The improvement in terms of preclinical pharmacokinetic performance of FAPI-04

compared to FAPI-02, was further clinically assessed [124]. The [68 Ga]Ga-FAPI-04 and
[68 Ga]Ga-FAPI-02 PET/CT scans in two patients with metastasized breast cancer showed
that [68 Ga]Ga-FAPI-04 enhanced tumor retention time by a factor of 3 compared to [68 Ga]Ga-
FAPI-02 at 3 h post-injection. However, both [68 Ga]Ga-FAPI-04 and [68 Ga]Ga-FAPI-02
revealed no significant difference in tumor-to-background ratios at 1 h post-injection.
The promising initial clinical data on FAP-directed targeting imaging paved the way
for clinical trials with a larger cohort of patients to validate the appropriateness of radiola-
beled FAP-based inhibitors as pan-tumor agents. [68 Ga]Ga-FAPI-04 was further evaluated
in 80 patients with 28 different types of tumors (54 primary tumors and 229 metastases)
where a robust [18 F]FDG and other imaging modalities were considered insufficient to ob-
tain the justifiable diagnostic information by physicians [134]. All the enrolled patients were
retrospectively identified with histopathologically proven primary tumors or metastases or
radiologically definite metastases of histologically proven primary tumors. According to
PET/CT scans, the high accumulation of [68 Ga]Ga-FAPI-04 (SUVmax > 12) was presented
in cholangiocarcinoma, sarcoma, esophageal, breast, and lung cancers while low uptake
(SUVmax < 6) was found in renal, thyroid, adenoid cystic, pheochromocytoma, and gastric
cancers, indicating that the known [18 F]FDG limitations in differentiated thyroid and renal
cell carcinoma may not be able to be overcome with [68 Ga]Ga-FAPI. A moderate uptake
(SUVmax = 6–12) was observed in colorectal, head-and-neck, hepatocellular, pancreatic,
ovarian, and prostate cancers. Interestingly, the observed radioactivity in background
tissues (e.g., blood pool and muscle) was relatively low, which resulted in improved tumor-
to-background ratios. Similarly, another clinical study aimed at comparing the diagnostic
efficacy of [18 F]FDG and [68 Ga]Ga-FAPI-04 PET/CT in a small cohort of challenging pa-
tients with primary and metastatic lesions of several origins [135]. The studied cohort
consisted of 75 patients (47 males and 28 females, median age of 61.5 years). All patients
were imaged with [68 Ga]Ga-FAPI-04 while 54 patients with 12 different tumor entities
were selected to have a paired scan with [18 F]FDG in the initial assessment and for the
remaining 21 patients, the pairing scan with [18 F]FDG was performed during the stage of
recurrent imaging. [68 Ga]Ga-FAPI-04 revealed better performance than [18 F]FDG in terms
of detection rate (98.2% vs. 82.1%), especially in hepatocellular, gastric, and pancreatic
cancers, which have well-known limitations with regard to the use of [18 F]FDG PET/CT.
These findings indicate that the use of [68 Ga]Ga-FAPI-04 in PET/CT might be a promis-
ing new approach in the case where [18 F]FDG is limited. Since tumor size has already
been found to influence the delineation of primary tumors by [18 F]FDG due to the partial
volume effect and low tumor metabolic activity, [68 Ga]Ga-FAPI-04 was able to visualize
tumors smaller than 1 cm mainly because of high tumor and low background uptake.
One more important finding from this study was the significantly higher sensitivity for the
detection of lymph node metastases when using [68 Ga]Ga-FAPI-04 compared to [18 F]FDG,
something which happens due to higher uptake of [68 Ga]Ga-FAPI-04. Overall, the study
demonstrated that [68 Ga]Ga-FAPI-04 PET/CT has the potential to outperform [18 F]FDG
PET/CT in identifying liver metastases, peritoneal carcinomatosis, brain tumors, and target
sites for many types of metastatic cancers. However, the [68 Ga]Ga-FAPI-04 performance
was not more tumor-specific than [18 F]FDG because [68 Ga]Ga-FAPI-04 PET was found to
yield more false-positive findings.
The two most common types of primary hepatic malignancies, hepatocellular car-
cinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), are currently identified by
MRI and CT or ultrasound imaging techniques and/or biopsy. Although MRI is superior
compared to CT/ultrasound in detecting intrahepatic lesions, its use remains limited for
the detection of lesions smaller than 2 cm. In addition to this limitation, the molecular
characterization of those malignancies may contribute to their therapeutic management.
Given the high FAP expression on HCC and ICC in combination with the great promise of
[68 Ga]Ga-FAPI-04 in clinical settings, a pilot clinical study in 17 patients who underwent
surgery or biopsy revealed the diagnostic ability of [68 Ga]Ga-FAPI-04 for detecting and
Pharmaceuticals 2021, 14, 1023 21 of 30

characterizing hepatic nodules in patients with suspected carcinoma. The same study
also indicated that patients with less advanced lesions may be more suitable for FAP-
targeted imaging compared to those with advanced lesions [136]. Nevertheless, a larger
number of patients, as well as a side-by-side comparison with [18 F]FDG, are essential for
the confirmation of these findings.
Continuing the clinical assessment of the radiolabeled FAP-based inhibitors, FAPI-21
and FAPI-46—the other two outstanding FAPI derivatives that developed in an attempt to
improve FAPI-04 performance—indeed, showed superior tumor accumulation and tumor-
to-background ratios over FAPI-04 [110]. Although the preclinical data suggested that
FAPI-21 provided more specific tumor uptake than FAPI-46, FAPI-21 exhibited high uptake
in normal organs in clinical studies of 8 patients with colorectal, ovarian, oropharyngeal,
and pancreatic cancers, resulting in lower tumor-to-background ratios compared to FAPI-46.
The radiation dosimetry of [68 Ga]Ga-FAPI-46 was initially evaluated in six cancer patients
with cholangiocarcinoma, pancreatic, breast, oropharynx, head-and-neck, and gastric
cancers [137]. The mean absorbed radiation dose was determined at three time points after
the injection of the radiotracer (10 min, 1 h, and 3 h). After three serial scans, the average
effective whole-body absorbed dose of [68 Ga]Ga-FAPI-46 was about 1.56 mSv with a
200 MBq injected dose, which was lower than other robust 68 Ga-labeled radiotracers used in
the current clinic (e.g., [68 Ga]Ga-DOTATATE and [68 Ga]Ga-PSMA-11). The biodistribution
study further revealed high tumor-to-background ratios, which were increasing over
time, these data suggest superior diagnostic performance and favorable pharmacokinetics.
Additionally, a clinical trial comparing the diagnostic performance of [68 Ga]Ga-FAPI-46
and [68 Ga]Ga-FAPI-04, and further evaluation of their clinical roles, was performed in
22 patients with lower gastrointestinal tract (LGT) tumors [138]. The radiotracer uptake
was quantified by SUVmax and SUVmean values. The studies showed that both tracers
were able to detect and restage both primary tumors and metastases arising from the LGT,
suggesting that these properties may open new possibilities in guiding radiation therapy of
LGT tumors. Given the fact that no fasting in patients before the examination is required in
combination with the short retention time of the tracers in malignant lesions, FAP targeted
imaging agents might be an alternative option for the successful management of patients
with LGT and their personalized therapeutic treatment approach.
FAP imaging using [68 Ga]Ga-FAPI PET/CT was further investigated in multiple sarco-
mas [139], gynecological tumors [140], and pancreatic ductal adenocarcinomas (PDAC) [141].
These preliminary clinical data also suggest that FAPI ligands may have the potential to
highly contribute to the diagnosis and potential therapy of those types of cancer; however,
larger comprehensive studies are required for confirmation.
As 18 F for various reasons logistically easier to handle, a study was conducted to
investigate the biodistribution, radiation dosimetry, and tumor delineation of [18 F]F-FAPI-
74 and [68 Ga]Ga-FAPI-74 in patients with lung cancer [116]. A high image contrast (SUVmax
> 10) was observed in tumors, lymph nodes, and metastases after 1 h post-injection.
The dosimetric studies in patients showed the normalized effective dose of 1.4 mSv/100
MBq for [18 F]F-FAPI-74 and 1.6 mSv/100 MBq for [68 Ga]Ga-FAPI-74, which were lower
than [18 F]FDG (2 mSv/100 MBq). Although [18 F]FDG PET/CT is considered as the standard
radiotracer for staging and target volume delineation in lung cancers, the preliminary
experience with 10 patients was not yet sufficient to determine the sensitivity, specificity,
and accuracy of [18 F]F-FAPI-74 PET/CT. Like [18 F]FDG, [18 F]F-FAPI-74 PET/CT was able
to identify additional distant metastases compared with a diagnostic CT scan. In a case
report from the same group, it was shown that [68 Ga]Ga-FAPI-04 PET/CT delineated brain
metastases originated from primary lung cancer [142]. Based on the so far generated data,
the FAPI-74 radioligand appears to be a versatile PET radiotracer with the potential for
multiple clinical applications.
Besides, 99m Tc was applied for FAPI-34 radiolabeling and [99m Tc]Tc-FAPI-34 was
used for diagnostic scintigraphy and SPECT imaging for the follow-up of [90 Y]Y-FAPI-46
radiotherapy in patients with ovarian and pancreatic cancers [114]. [99m Tc]Tc-FAPI-34
Pharmaceuticals 2021, 14, 1023 22 of 30

demonstrated high contrast in SPECT/CT images obtained by rapid tumor uptake and fast
clearance from the body. The authors also suggested that FAPI-34 could be labeled with
188 Re (high-energy β- emitter) for radionuclide therapy in future studies. By far, studies

on the therapeutic applications of FAPI radiotracers are limited due to the fast washout
from the tumors. Although the effort to overcome this obstacle is ongoing, this seems to be
one of the biggest challenges on the development of radiotherapeutic FAP-based inhibitors.
The first FAP targeted radiotherapeutic application was reported in 2018 [113] when FAPI-
04 was labeled with β- emitter 90 Y. [90 Y]Y-FAPI-04 exhibited high accumulation in the
metastatic site in patients with advanced breast cancer; however, the target occupancy
of [90 Y]Y-FAPI-04 was relatively short (<3 h), eliminating its applicability to achieve the
therapeutic response within this time frame.
[68 Ga]Ga-DATA5m .SA.FAPi, [68 Ga]Ga-DOTA.SA.FAPi, and [68 Ga]Ga-DOTAGA.
(SA.FAPi)2 developed by the Mainz research team were recently investigated for the first
time in clinical PET/CT studies. [68 Ga]Ga-DATA5m .SA.FAPi was used to test the restaging
of the potential tumor manifestation in a patient with thyroid carcinoma and pulmonary
metastasis after treatment with surgery and eight cycles of 131 I radiotherapy [125]. The de-
cision for the FAP imaging was made because the tumor marker (Tg) was rising and 131 I
whole-body scans were negative. [68 Ga]Ga-DATA5m .SA.FAPi was used instead of [68 Ga]Ga-
DOTA.SA.FAPi for this study due to its slightly better IC50 value and a facile preparation
based on an instant kit-type protocol. PET/CT scans did not show increased uptake in
the neck area and pulmonary lesions but in focal nodular hyperplasia (a benign liver le-
sion). The observed enhanced tracer uptake demonstrated that [68 Ga]Ga-DATA5m .SA.FAPi
PET/CT may be a useful tool to characterize a variety of not only malignant but also
benign tumors. Based on this finding, it is important to verify if other benign liver tumors,
such as adenomas or hemangiomas, could be detected by this tracer. Moreover, the cor-
relation between [68 Ga]Ga-DATA5m .SA.FAPi PET/CT and Ki-67 as the marker of tumor
proliferation, grading, and tumor aggressiveness was investigated in 13 patients with liver
metastases of neuroendocrine tumors (NET) [128]. Patients with histologically proven NET
from liver biopsy or primary tumor underwent [68 Ga]Ga-DATA5m .SA.FAPi, [18 F]FDG, and
[68 Ga]Ga-DOTATOC PET/CT scans. In general, 12/13 patients were categorized as posi-
tive uptake of [68 Ga]Ga-DATA5m .SA.FAPi and [68 Ga]Ga-DOTA-TOC in liver metastases
while [18 F]FDG showed the positive uptake only in eight patients. In terms of SUVmax
and Ki-67 correlation, FDGSUVmax revealed positive correlation coefficient with Ki-67
(rho = 0.543, p < 0.05) while DOTATOCSUVmax was in a negative range (rho = −0.618,
p < 0.05). There was no significant correlation between FAPiSUVmax and Ki-67 (rho =
0.382, p > 0.05). However, [68 Ga]Ga-DATA5m .SA.FAPi-positive tumor fraction (FAPiTF )
demonstrated a strong positive correlation with Ki-67 (rho = 0.770, p < 0.01) followed by
FDGTF (rho = 0.524, p < 0.05) while DOTATOCTF showed a strong negative correlation
(rho = −0.828, p < 0.01). Besides, the correlation of PET-positive tumor volume with Ki-67
was also determined. [68 Ga]Ga-DATA5m .SA.FAPi-positive tumor volume (FAPiVOL ) exhib-
ited a moderate correlation coefficient (rho = 0.510, p < 0.05), but no significant correlation
was observed with FDGVOL and DOTATOCVOL (p > 0.05). For dedifferentiation, 2/13
patients had a Ki-67 of above 55% (FAPiVOL :DOTATOCVOL ratio > 10) while 11/13 patients
had a Ki-67 of max. 40% (max. FAPiVOL :DOTATOCVOL ratio = 6.4). Although the results
showed that FAPiVOL :DOTATOCVOL ratio might enable the prediction of Ki-67 with a
high correlation coefficient in patients with NET metastasis in the liver, further study is
required with a higher patient population for the assessment of suspicious dedifferentiation
and prognosis.
Furthermore, the biodistribution, pharmacokinetics, and dosimetry of [68 Ga]Ga-
DOTA.SA.FAPi, were determined in 54 patients with various cancers while a head-to-
head comparison with [18 F]FDG PET/CT scans was also performed [126]. [68 Ga]Ga-
DOTA.SA.FAPi revealed high target-to-background ratios in various types of cancers while
the diagnostic accuracy of [68 Ga]Ga-DOTA.SA.FAPi was comparable to [18 F]FDG PET/CT.
The results from this study with respect to the selectivity of [68 Ga]Ga-DOTA.SA.FAPi
Pharmaceuticals 2021, 14, 1023 23 of 30

in various cancers may also contribute to the improvement of the already existing data
on FAP molecular imaging. Next, [68 Ga]Ga-DOTA.SA.FAPi PET/CT-guided [177 Lu]Lu-
DOTA.SA.FAPi radiotherapy was lately reported by the same group [127]. The clini-
cal trial of [68 Ga]Ga-DOTA.SA.FAPi was conducted in an end-stage breast cancer pa-
tient, who had atypical cellular cords and tubules in the fibrotic stroma as shown in the
histopathologic examination. [68 Ga]Ga-DOTA.SA.FAPi provided similar intense match-
ing lesion uptake as observed in [18 F]FDG PET/CT scan, after which the radionuclide
was switched from 68 Ga to 177 Lu for radiotherapy. [177 Lu]Lu-DOTA.SA.FAPi showed a
similar biodistribution pattern as [68 Ga]Ga-DOTA.SA.FAPi where the intense tracer ac-
cumulation was found in all lesions. However, this cohort of the study was conducted
in only one patient while a larger cohort is required to confirm the uptake pattern of the
tracer. The latest clinical report on the new class of FAPI-radiopharmaceuticals utilizing the
squaric acid (SA) motif as the linker between the FAP inhibitor and the bifunctional chela-
tor concerning the homodimeric FAP-based inhibitor, DOTAGA.(SA.FAPI)2 was also re-
ported [118]. Six patients were recruited for this study who underwent [18 F]FDG, [68 Ga]Ga-
DOTA.SA.FAPi and [68 Ga]Ga-DOTAGA.(SA.FAPi)2 PET/CT scans, respectively [118].
[68 Ga]Ga-DOTAGA.(SA.FAPi)2 showed an increase in tumor uptake at 1 h post-injection
compared to [68 Ga]Ga-DOTA.SA.FAPi, which was further increased after 3 h post-injection.
Thus, the use of dimeric structure DOTAGA.(SA.FAPi)2 could be a useful tool to enhance
tumor accumulation and retention and may represent an advanced step towards radionu-
clide FAP targeted therapy. However, [68 Ga]Ga-DOTAGA.(SA.FAPi)2 is accompanied by
high, delayed, and heterogeneous blood pool uptake across the patients, thus attributing
to a risk of increased radiation dose to the non-target organs. All these findings warrant
further investigations in a larger patient population.
Although the clinical studies are currently in the early stage and prospective studies
are ongoing, it is evident that FAP is a highly promising target for cancer imaging and
therapy. All the so far acquired PET images utilizing several FAP-based radiolabeled
inhibitors document a high tumor-to-background ratio in different tumor subtypes, render-
ing FAP-based radiotracers as potential pan-tumor imaging agents. FAPI-directed PET/CT
seems to play a crucial role in some cancers for which the robust [18 F]FDG directed PET/CT
is known to have limited applications. The FAPI PET/CT provides complementary di-
agnostic, phenotypic, and biomarker information compared to [18 F]FDG PET/CT. Since
FAP-based inhibitors can also be labeled with therapeutic radionuclides while this is not
the case for FDG, this allows research groups to shift their attention to the direction of the
theranostic approach where the diagnostic radiotracers will serve as predictive biomarkers
for therapeutic responses to FAP-targeted treatments.

6. Conclusions
In summary, the critical role of the TME has been recently considered as an integral
part of the initiation and progression of tumorigenesis. Monitoring the biological changes
in the tumor microenvironment would provide essential information to identify related
oncological targets for cancer prevention and treatment. CAFs are of particular interest due
to the variety in subtype and phenotype of expressing biomarkers. Among all markers,
FAP has been considered as an attractive target that involves in the mediation of immuno-
suppression in the TME. The introduction of radiolabeled FAP-based inhibitors has led to a
new class of radiopharmaceuticals used for visualizing FAP presented in the tumor stroma.
Over the past few years, several research groups have demonstrated the potential of FAPI
radiotracers in cancer diagnosis and therapy through extensive studies at both preclinical
and clinical levels. Based on current reports, FAP imaging with 68 Ga-labeled FAPI PET/CT
appears to be a promising strategy for the visualization of several cancers. However, limita-
tions remain due to the suboptimal specificity of those FAPI molecules to cancer-associated
FAP substrate, rapid clearance from the systemic circulation, and short retention time in the
tumor. These constraints can hamper the long-term tracking of radiotracers and obstruct
the therapeutic efficacy in targeted radionuclide therapy. Therefore, the current effort has
Pharmaceuticals 2021, 14, 1023 24 of 30

been directed towards the structural optimization of FAPI molecules to improve the overall
pharmacokinetic properties in vivo. Moreover, the effectiveness of FAP-targeted radio-
therapy is ambiguous due to a small number of validated studies while the consolidation
with other therapeutic approaches (e.g., immunotherapy and combinatorial molecular
radiotherapy) may provide better treatment outcomes.

Author Contributions: Conceptualization S.I. and E.G.; writing—original draft preparation S.I. and
E.G.; writing—review and editing S.I., E.S.M., H.R., A.A.-O., F.R., A.R., and E.G. All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Roma-Rodrigues, C.; Mendes, R.; Baptista, P.; Fernandes, A. Targeting Tumor Microenvironment for Cancer Therapy. Int. J. Mol.
Sci. 2019, 20, 840. [CrossRef]
2. Whiteside, T.L. The tumor microenvironment and its role in promoting tumor growth. Oncogene 2008, 27, 5904–5912. [CrossRef]
3. Baghban, R.; Roshangar, L.; Jahanban-Esfahlan, R.; Seidi, K.; Ebrahimi-Kalan, A.; Jaymand, M.; Kolahian, S.; Javaheri, T.; Zare, P.
Tumor microenvironment complexity and therapeutic implications at a glance. Cell Commun. Signal. 2020, 18, 59. [CrossRef]
4. Lindner, T.; Loktev, A.; Giesel, F.; Kratochwil, C.; Altmann, A.; Haberkorn, U. Targeting of activated fibroblasts for imaging and
therapy. EJNMMI Radiopharm. Chem. 2019, 4, 16. [CrossRef] [PubMed]
5. Hanahan, D.; Weinberg, R.A. Hallmarks of Cancer: The Next Generation. Cell 2011, 144, 646–674. [CrossRef]
6. Öhlund, D.; Elyada, E.; Tuveson, D. Fibroblast heterogeneity in the cancer wound. J. Exp. Med. 2014, 211, 1503–1523. [CrossRef]
[PubMed]
7. Smith, N.R.; Baker, D.; Farren, M.; Pommier, A.; Swann, R.; Wang, X.; Mistry, S.; McDaid, K.; Kendrew, J.; Womack, C.; et al.
Tumor Stromal Architecture Can Define the Intrinsic Tumor Response to VEGF-Targeted Therapy. Clin. Cancer Res. 2013, 19,
6943–6956. [CrossRef] [PubMed]
8. Neesse, A.; Michl, P.; Frese, K.K.; Feig, C.; Cook, N.; Jacobetz, M.A.; Lolkema, M.P.; Buchholz, M.; Olive, K.P.; Gress, T.M.; et al.
Stromal biology and therapy in pancreatic cancer. Gut 2011, 60, 861–868. [CrossRef]
9. Xing, F.; Saidou, J.; Watabe, K. Cancer associated fibroblasts (CAFs) in tumor microenvironment. Front. Biosci. Landmark Ed. 2010,
15, 166–179. [CrossRef] [PubMed]
10. Álvarez-Teijeiro, S.; García-Inclán, C.; Villaronga, M.; Casado, P.; Hermida-Prado, F.; Granda-Díaz, R.; Rodrigo, J.; Calvo, F.;
del-Río-Ibisate, N.; Gandarillas, A.; et al. Factors Secreted by Cancer-Associated Fibroblasts that Sustain Cancer Stem Properties
in Head and Neck Squamous Carcinoma Cells as Potential Therapeutic Targets. Cancers 2018, 10, 334. [CrossRef]
11. Koustoulidou, S.; Hoorens, M.W.H.; Dalm, S.U.; Mahajan, S.; Debets, R.; Seimbille, Y.; de Jong, M. Cancer-Associated Fibroblasts
as Players in Cancer Development and Progression and Their Role in Targeted Radionuclide Imaging and Therapy. Cancers 2021,
13, 1100. [CrossRef]
12. Han, C.; Liu, T.; Yin, R. Biomarkers for cancer-associated fibroblasts. Biomark. Res. 2020, 8, 64. [CrossRef]
13. Fitzgerald, A.A.; Weiner, L.M. The role of fibroblast activation protein in health and malignancy. Cancer Metastasis Rev. 2020, 39,
783–803. [CrossRef]
14. Garcia, E.V. Physical attributes, limitations, and future potential for PET and SPECT. J. Nucl. Cardiol. 2012, 19, 19–29. [CrossRef]
[PubMed]
15. Laverman, P.; van der Geest, T.; Terry, S.Y.A.; Gerrits, D.; Walgreen, B.; Helsen, M.M.; Nayak, T.K.; Freimoser-Grundschober, A.;
Waldhauer, I.; Hosse, R.J.; et al. Immuno-PET and Immuno-SPECT of Rheumatoid Arthritis with Radiolabeled Anti-Fibroblast
Activation Protein Antibody Correlates with Severity of Arthritis. J. Nucl. Med. 2015, 56, 778–783. [CrossRef] [PubMed]
16. Van der Geest, T.; Laverman, P.; Gerrits, D.; Walgreen, B.; Helsen, M.M.; Klein, C.; Nayak, T.K.; Storm, G.; Metselaar, J.M.;
Koenders, M.I.; et al. Liposomal Treatment of Experimental Arthritis Can Be Monitored Noninvasively with a Radiolabeled
Anti–Fibroblast Activation Protein Antibody. J. Nucl. Med. 2017, 58, 151–155. [CrossRef]
17. Toms, J.; Kogler, J.; Maschauer, S.; Daniel, C.; Schmidkonz, C.; Kuwert, T.; Prante, O. Targeting Fibroblast Activation Protein:
Radiosynthesis and Preclinical Evaluation of an 18F-Labeled FAP Inhibitor. J. Nucl. Med. 2020, 61, 1806–1813. [CrossRef]
[PubMed]
18. Huang, S.; Fang, R.; Xu, J.; Qiu, S.; Zhang, H.; Du, J.; Cai, S. Evaluation of the tumor targeting of a FAPα-based doxorubicin
prodrug. J. Drug Target. 2011, 19, 487–496. [CrossRef] [PubMed]
Pharmaceuticals 2021, 14, 1023 25 of 30

19. Zboralski, D.; Osterkamp, F.; Simmons, A.D.; Bredenbeck, A.; Schumann, A.; Paschke, M.; Beindorff, N.; Mohan, A.-M.;
Nguyen, M.; Xiao, J.; et al. 571P Preclinical evaluation of FAP-2286, a peptide-targeted radionuclide therapy (PTRT) to fibroblast
activation protein alpha (FAP). Ann. Oncol. 2020, 31, S488. [CrossRef]
20. Baum, R.P.; Schuchardt, C.; Singh, A.; Chantadisai, M.; Robiller, F.C.; Zhang, J.; Mueller, D.; Eismant, A.; Almaguel, F.;
Zboralski, D.; et al. Feasibility, Biodistribution and Preliminary Dosimetry in Peptide-Targeted Radionuclide Therapy (PTRT)
of Diverse Adenocarcinomas using 177Lu-FAP-2286: First-in-Human Results. J. Nucl. Med. Off. Publ. Soc. Nucl. Med. 2021.
[CrossRef]
21. Jin, M.-Z.; Jin, W.-L. The updated landscape of tumor microenvironment and drug repurposing. Signal Transduct. Target. Ther.
2020, 5, 166. [CrossRef] [PubMed]
22. McMillin, D.W.; Negri, J.M.; Mitsiades, C.S. The role of tumour-stromal interactions in modifying drug response: Challenges and
opportunities. Nat. Rev. Drug Discov. 2013, 12, 217–228. [CrossRef] [PubMed]
23. Rodrigues, J.; Heinrich, M.A.; Teixeira, L.M.; Prakash, J. 3D In Vitro Model (R)evolution: Unveiling Tumor–Stroma Interactions.
Trends Cancer 2021, 7, 249–264. [CrossRef] [PubMed]
24. Mbeunkui, F.; Johann, D.J. Cancer and the tumor microenvironment: A review of an essential relationship. Cancer Chemother.
Pharmacol. 2009, 63, 571–582. [CrossRef]
25. Ramamonjisoa, N.; Ackerstaff, E. Characterization of the Tumor Microenvironment and Tumor–Stroma Interaction by Non-
invasive Preclinical Imaging. Front. Oncol. 2017, 7, 3. [CrossRef]
26. Liotta, L.A.; Kohn, E.C. The microenvironment of the tumour-host interface. Nature 2001, 411, 375–379. [CrossRef]
27. Kalluri, R.; Zeisberg, M. Fibroblasts in cancer. Nat. Rev. Cancer 2006, 6, 392–401. [CrossRef]
28. Liu, H.; Ni, S.; Wang, H.; Zhang, Q.; Weng, W. Charactering tumor microenvironment reveals stromal-related transcription factors
promote tumor carcinogenesis in gastric cancer. Cancer Med. 2020, 9, 5247–5257. [CrossRef]
29. Tania, M.; Khan, M.A.; Fu, J. Epithelial to mesenchymal transition inducing transcription factors and metastatic cancer. Tumour
Biol. J. Int. Soc. Oncodev. Biol. Med. 2014, 35, 7335–7342. [CrossRef] [PubMed]
30. Johnston, S.J.; Carroll, J.S. Transcription factors and chromatin proteins as therapeutic targets in cancer. Biochim. Biophys. Acta
2015, 1855, 183–192. [CrossRef]
31. Vishnoi, K.; Viswakarma, N.; Rana, A.; Rana, B. Transcription Factors in Cancer Development and Therapy. Cancers 2020, 12,
2296. [CrossRef] [PubMed]
32. Handsley, M.M.; Edwards, D.R. Metalloproteinases and their inhibitors in tumor angiogenesis. Int. J. Cancer 2005, 115, 849–860.
[CrossRef] [PubMed]
33. Görögh, T.; Beier, U.H.; Bäumken, J.; Meyer, J.E.; Hoffmann, M.; Gottschlich, S.; Maune, S. Metalloproteinases and their inhibitors:
Influence on tumor invasiveness and metastasis formation in head and neck squamous cell carcinomas. Head Neck 2006, 28, 31–39.
[CrossRef] [PubMed]
34. Liu, T.; Han, C.; Wang, S.; Fang, P.; Ma, Z.; Xu, L.; Yin, R. Cancer-associated fibroblasts: An emerging target of anti-cancer
immunotherapy. J. Hematol. Oncol. 2019, 12, 86. [CrossRef] [PubMed]
35. Kalluri, R. The biology and function of fibroblasts in cancer. Nat. Rev. Cancer 2016, 16, 582–598. [CrossRef] [PubMed]
36. Li, B.; Wang, J.H.-C. Fibroblasts and myofibroblasts in wound healing: Force generation and measurement. J. Tissue Viability 2011,
20, 108–120. [CrossRef]
37. Dvorak, H.F. Tumors: Wounds that do not heal. Similarities between tumor stroma generation and wound healing. N. Engl. J.
Med. 1986, 315, 1650–1659.
38. Byun, J.S.; Gardner, K. Wounds that will not heal: Pervasive cellular reprogramming in cancer. Am. J. Pathol. 2013, 182, 1055–1064.
[CrossRef] [PubMed]
39. Kobayashi, H.; Enomoto, A.; Woods, S.L.; Burt, A.D.; Takahashi, M.; Worthley, D.L. Cancer-associated fibroblasts in gastrointestinal
cancer. Nat. Rev. Gastroenterol. Hepatol. 2019, 16, 282–295. [CrossRef]
40. Ziani, L.; Chouaib, S.; Thiery, J. Alteration of the Antitumor Immune Response by Cancer-Associated Fibroblasts. Front. Immunol.
2018, 9, 414. [CrossRef]
41. Chen, X.; Song, E. Turning foes to friends: Targeting cancer-associated fibroblasts. Nat. Rev. Drug Discov. 2019, 18, 99–115.
[CrossRef] [PubMed]
42. Pape, J.; Magdeldin, T.; Stamati, K.; Nyga, A.; Loizidou, M.; Emberton, M.; Cheema, U. Cancer-associated fibroblasts mediate
cancer progression and remodel the tumouroid stroma. Br. J. Cancer 2020, 123, 1178–1190. [CrossRef] [PubMed]
43. Sahai, E.; Astsaturov, I.; Cukierman, E.; DeNardo, D.G.; Egeblad, M.; Evans, R.M.; Fearon, D.; Greten, F.R.; Hingorani, S.R.;
Hunter, T.; et al. A framework for advancing our understanding of cancer-associated fibroblasts. Nat. Rev. Cancer 2020, 20,
174–186. [CrossRef]
44. Mishra, R.; Haldar, S.; Suchanti, S.; Bhowmick, N.A. Epigenetic changes in fibroblasts drive cancer metabolism and differentiation.
Endocr. Relat. Cancer 2019, 26, R673–R688. [CrossRef] [PubMed]
45. Öhlund, D.; Handly-Santana, A.; Biffi, G.; Elyada, E.; Almeida, A.S.; Ponz-Sarvise, M.; Corbo, V.; Oni, T.E.; Hearn, S.A.;
Lee, E.J.; et al. Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer. J. Exp. Med. 2017, 214,
579–596. [CrossRef]
46. Omary, M.B.; Lugea, A.; Lowe, A.W.; Pandol, S.J. The pancreatic stellate cell: A star on the rise in pancreatic diseases. J. Clin.
Invest. 2007, 117, 50–59. [CrossRef]
Pharmaceuticals 2021, 14, 1023 26 of 30

47. Yin, C.; Evason, K.J.; Asahina, K.; Stainier, D.Y.R. Hepatic stellate cells in liver development, regeneration, and cancer. J. Clin.
Invest. 2013, 123, 1902–1910. [CrossRef]
48. Pereira, B.A.; Vennin, C.; Papanicolaou, M.; Chambers, C.R.; Herrmann, D.; Morton, J.P.; Cox, T.R.; Timpson, P. CAF Subpopula-
tions: A New Reservoir of Stromal Targets in Pancreatic Cancer. Trends Cancer 2019, 5, 724–741. [CrossRef]
49. Terai, S.; Fushida, S.; Tsukada, T.; Kinoshita, J.; Oyama, K.; Okamoto, K.; Makino, I.; Tajima, H.; Ninomiya, I.; Fujimura, T.; et al.
Bone marrow derived “fibrocytes” contribute to tumor proliferation and fibrosis in gastric cancer. Gastric Cancer 2015, 18, 306–313.
[CrossRef]
50. LeBleu, V.S.; Neilson, E.G. Origin and functional heterogeneity of fibroblasts. FASEB J. 2020, 34, 3519–3536. [CrossRef]
51. Mishra, P.J.; Mishra, P.J.; Humeniuk, R.; Medina, D.J.; Alexe, G.; Mesirov, J.P.; Ganesan, S.; Glod, J.W.; Banerjee, D. Carcinoma-
Associated Fibroblast–Like Differentiation of Human Mesenchymal Stem Cells. Cancer Res. 2008, 68, 4331–4339. [CrossRef]
[PubMed]
52. Kurashige, M.; Kohara, M.; Ohshima, K.; Tahara, S.; Hori, Y.; Nojima, S.; Wada, N.; Ikeda, J.; Miyamura, K.; Ito, M.; et al. Origin of
cancer-associated fibroblasts and tumor-associated macrophages in humans after sex-mismatched bone marrow transplantation.
Commun. Biol. 2018, 1, 131. [CrossRef] [PubMed]
53. Wang, X.; Zhang, W.; Sun, X.; Lin, Y.; Chen, W. Cancer-associated fibroblasts induce epithelial-mesenchymal transition through
secreted cytokines in endometrial cancer cells. Oncol. Lett. 2018, 15, 5694–5702. [CrossRef]
54. Yeon, J.H.; Jeong, H.E.; Seo, H.; Cho, S.; Kim, K.; Na, D.; Chung, S.; Park, J.; Choi, N.; Kang, J.Y. Cancer-derived exosomes trigger
endothelial to mesenchymal transition followed by the induction of cancer-associated fibroblasts. Acta Biomater. 2018, 76, 146–153.
[CrossRef] [PubMed]
55. Zhuang, J.; Lu, Q.; Shen, B.; Huang, X.; Shen, L.; Zheng, X.; Huang, R.; Yan, J.; Guo, H. TGFβ1 secreted by cancer-associated
fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT. Sci. Rep. 2015, 5, 11924.
[CrossRef]
56. Jopling, C.; Boue, S.; Izpisua Belmonte, J.C. Dedifferentiation, transdifferentiation and reprogramming: Three routes to regenera-
tion. Nat. Rev. Mol. Cell Biol. 2011, 12, 79–89. [CrossRef]
57. Nurmik, M.; Ullmann, P.; Rodriguez, F.; Haan, S.; Letellier, E. In search of definitions: Cancer-associated fibroblasts and their
markers. Int. J. Cancer 2020, 146, 895–905. [CrossRef]
58. Brennen, W.N.; Isaacs, J.T.; Denmeade, S.R. Rationale behind targeting fibroblast activation protein-expressing carcinoma-
associated fibroblasts as a novel chemotherapeutic strategy. Mol. Cancer Ther. 2012, 11, 257–266. [CrossRef] [PubMed]
59. Busek, P.; Mateu, R.; Zubal, M.; Kotackova, L.; Sedo, A. Targeting fibroblast activation protein in cancer—Prospects and caveats.
Front. Biosci. Landmark Ed. 2018, 23, 1933–1968.
60. Fabre, M.; Ferrer, C.; Domínguez-Hormaetxe, S.; Bockorny, B.; Murias, L.; Seifert, O.; Eisler, S.A.; Kontermann, R.E.; Pfizenmaier,
K.; Lee, S.Y.; et al. OMTX705, a Novel FAP-Targeting ADC Demonstrates Activity in Chemotherapy and Pembrolizumab-Resistant
Solid Tumor Models. Clin. Cancer Res. 2020, 26, 3420–3430. [CrossRef]
61. Wikberg, M.L.; Edin, S.; Lundberg, I.V.; Van Guelpen, B.; Dahlin, A.M.; Rutegård, J.; Stenling, R.; Oberg, A.; Palmqvist, R. High
intratumoral expression of fibroblast activation protein (FAP) in colon cancer is associated with poorer patient prognosis. Tumour
Biol. J. Int. Soc. Oncodev. Biol. Med. 2013, 34, 1013–1020. [CrossRef] [PubMed]
62. Huber, M.A.; Kraut, N.; Park, J.E.; Schubert, R.D.; Rettig, W.J.; Peter, R.U.; Garin-Chesa, P. Fibroblast activation protein: Differential
expression and serine protease activity in reactive stromal fibroblasts of melanocytic skin tumors. J. Invest. Dermatol. 2003, 120,
182–188. [CrossRef] [PubMed]
63. Aertgeerts, K.; Levin, I.; Shi, L.; Snell, G.P.; Jennings, A.; Prasad, G.S.; Zhang, Y.; Kraus, M.L.; Salakian, S.; Sridhar, V.; et al.
Structural and Kinetic Analysis of the Substrate Specificity of Human Fibroblast Activation Protein α. J. Biol. Chem. 2005, 280,
19441–19444. [CrossRef]
64. Lee, K.N.; Jackson, K.W.; Christiansen, V.J.; Lee, C.S.; Chun, J.-G.; McKee, P.A. Antiplasmin-cleaving enzyme is a soluble form of
fibroblast activation protein. Blood 2006, 107, 1397–1404. [CrossRef] [PubMed]
65. Jiang, G.-M.; Xu, W.; Du, J.; Zhang, K.-S.; Zhang, Q.-G.; Wang, X.-W.; Liu, Z.-G.; Liu, S.-Q.; Xie, W.-Y.; Liu, H.-F.; et al. The ap-
plication of the fibroblast activation protein α-targeted immunotherapy strategy. Oncotarget 2016, 7, 33472–33482. [CrossRef]
[PubMed]
66. Juillerat-Jeanneret, L.; Tafelmeyer, P.; Golshayan, D. Fibroblast activation protein-α in fibrogenic disorders and cancer: More than
a prolyl-specific peptidase? Expert Opin. Ther. Targets 2017, 21, 977–991. [CrossRef]
67. Park, J.E.; Lenter, M.C.; Zimmermann, R.N.; Garin-Chesa, P.; Old, L.J.; Rettig, W.J. Fibroblast Activation Protein, a Dual Specificity
Serine Protease Expressed in Reactive Human Tumor Stromal Fibroblasts. J. Biol. Chem. 1999, 274, 36505–36512. [CrossRef]
68. Fan, M.-H.; Zhu, Q.; Li, H.-H.; Ra, H.-J.; Majumdar, S.; Gulick, D.L.; Jerome, J.A.; Madsen, D.H.; Christofidou-Solomidou, M.;
Speicher, D.W.; et al. Fibroblast Activation Protein (FAP) Accelerates Collagen Degradation and Clearance from Lungs in Mice. J.
Biol. Chem. 2016, 291, 8070–8089. [CrossRef]
69. Jabłońska-Trypuć, A.; Matejczyk, M.; Rosochacki, S. Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM)
enzymes in collagen degradation, as a target for anticancer drugs. J. Enzyme Inhib. Med. Chem. 2016, 31, 177–183. [CrossRef]
70. Huang, C.-H.; Suen, C.-S.; Lin, C.-T.; Chien, C.-H.; Lee, H.-Y.; Chung, K.-M.; Tsai, T.-Y.; Jiaang, W.-T.; Hwang, M.-J.; Chen, X.
Cleavage-site specificity of prolyl endopeptidase FAP investigated with a full-length protein substrate. J. Biochem. 2011, 149,
685–692. [CrossRef]
Pharmaceuticals 2021, 14, 1023 27 of 30

71. Zi, F.; He, J.; He, D.; Li, Y.; Yang, L.; Cai, Z. Fibroblast activation protein α in tumor microenvironment: Recent progression and
implications (review). Mol. Med. Rep. 2015, 11, 3203–3211. [CrossRef]
72. Jacob, M.; Chang, L.; Puré, E. Fibroblast activation protein in remodeling tissues. Curr. Mol. Med. 2012, 12, 1220–1243. [CrossRef]
73. Hamson, E.J.; Keane, F.M.; Tholen, S.; Schilling, O.; Gorrell, M.D. Understanding fibroblast activation protein (FAP): Substrates,
activities, expression and targeting for cancer therapy. Proteom. Clin. Appl. 2014, 8, 454–463. [CrossRef]
74. Puré, E.; Blomberg, R. Pro-tumorigenic roles of fibroblast activation protein in cancer: Back to the basics. Oncogene 2018, 37,
4343–4357. [CrossRef] [PubMed]
75. Ghersi, G.; Dong, H.; Goldstein, L.A.; Yeh, Y.; Hakkinen, L.; Larjava, H.S.; Chen, W.-T. Regulation of fibroblast migration on
collagenous matrix by a cell surface peptidase complex. J. Biol. Chem. 2002, 277, 29231–29241. [CrossRef]
76. Mueller, S.C.; Ghersi, G.; Akiyama, S.K.; Sang, Q.X.; Howard, L.; Pineiro-Sanchez, M.; Nakahara, H.; Yeh, Y.; Chen, W.T. A novel
protease-docking function of integrin at invadopodia. J. Biol. Chem. 1999, 274, 24947–24952. [CrossRef]
77. Sun, S.; Albright, C.F.; Fish, B.H.; George, H.J.; Selling, B.H.; Hollis, G.F.; Wynn, R. Expression, purification, and kinetic
characterization of full-length human fibroblast activation protein. Protein Expr. Purif. 2002, 24, 274–281. [CrossRef] [PubMed]
78. Duan, Q.; Zhang, H.; Zheng, J.; Zhang, L. Turning Cold into Hot: Firing up the Tumor Microenvironment. Trends Cancer 2020, 6,
605–618. [CrossRef] [PubMed]
79. Chen, L.; Qiu, X.; Wang, X.; He, J. FAP positive fibroblasts induce immune checkpoint blockade resistance in colorectal cancer via
promoting immunosuppression. Biochem. Biophys. Res. Commun. 2017, 487, 8–14. [CrossRef]
80. Feig, C.; Jones, J.O.; Kraman, M.; Wells, R.J.B.; Deonarine, A.; Chan, D.S.; Connell, C.M.; Roberts, E.W.; Zhao, Q.; Caballero, O.L.;
et al. Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in
pancreatic cancer. Proc. Natl. Acad. Sci. USA 2013, 110, 20212–20217. [CrossRef] [PubMed]
81. Freedman, J.D.; Duffy, M.R.; Lei-Rossmann, J.; Muntzer, A.; Scott, E.M.; Hagel, J.; Campo, L.; Bryant, R.J.; Verrill, C.;
Lambert, A.; et al. An Oncolytic Virus Expressing a T-cell Engager Simultaneously Targets Cancer and Immunosuppressive
Stromal Cells. Cancer Res. 2018, 78, 6852–6865. [CrossRef]
82. Coto-Llerena, M.; Ercan, C.; Kancherla, V.; Taha-Mehlitz, S.; Eppenberger-Castori, S.; Soysal, S.D.; Ng, C.K.Y.; Bolli, M.; von Flüe,
M.; Nicolas, G.P.; et al. High Expression of FAP in Colorectal Cancer Is Associated With Angiogenesis and Immunoregulation
Processes. Front. Oncol. 2020, 10, 979. [CrossRef]
83. Huang, Y.; Simms, A.E.; Mazur, A.; Wang, S.; León, N.R.; Jones, B.; Aziz, N.; Kelly, T. Fibroblast activation protein-α promotes
tumor growth and invasion of breast cancer cells through non-enzymatic functions. Clin. Exp. Metastasis 2011, 28, 567–579.
[CrossRef]
84. Li, M.; Cheng, X.; Rong, R.; Gao, Y.; Tang, X.; Chen, Y. High expression of fibroblast activation protein (FAP) predicts poor
outcome in high-grade serous ovarian cancer. BMC Cancer 2020, 20, 1032. [CrossRef]
85. Lee, H.-O.; Mullins, S.R.; Franco-Barraza, J.; Valianou, M.; Cukierman, E.; Cheng, J.D. FAP-overexpressing fibroblasts produce
an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells. BMC Cancer 2011, 11, 245.
[CrossRef] [PubMed]
86. Ge, Y.; Zhan, F.; Barlogie, B.; Epstein, J.; Shaughnessy, J.; Yaccoby, S. Fibroblast activation protein (FAP) is upregulated in
myelomatous bone and supports myeloma cell survival. Br. J. Haematol. 2006, 133, 83–92. [CrossRef]
87. Wang, R.-F.; Zhang, L.-H.; Shan, L.-H.; Sun, W.-G.; Chai, C.-C.; Wu, H.-M.; Ibla, J.C.; Wang, L.-F.; Liu, J.-R. Effects of the fibroblast
activation protein on the invasion and migration of gastric cancer. Exp. Mol. Pathol. 2013, 95, 350–356. [CrossRef] [PubMed]
88. Calais, J. FAP: The Next Billion Dollar Nuclear Theranostics Target? J. Nucl. Med. 2020, 61, 163–165. [CrossRef]
89. Welt, S.; Divgi, C.R.; Scott, A.M.; Garin-Chesa, P.; Finn, R.D.; Graham, M.; Carswell, E.A.; Cohen, A.; Larson, S.M.; Old, L.J.
Antibody targeting in metastatic colon cancer: A phase I study of monoclonal antibody F19 against a cell-surface protein of
reactive tumor stromal fibroblasts. J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 1994, 12, 1193–1203. [CrossRef]
90. Schmidt, A.; Müller, D.; Mersmann, M.; Wüest, T.; Gerlach, E.; Garin-Chesa, P.; Rettig, W.J.; Pfizenmaier, K.; Moosmayer, D.
Generation of human high-affinity antibodies specific for the fibroblast activation protein by guided selection. Eur. J. Biochem.
2001, 268, 1730–1738. [CrossRef] [PubMed]
91. Mersmann, M.; Schmidt, A.; Rippmann, J.F.; Wüest, T.; Brocks, B.; Rettig, W.J.; Garin-Chesa, P.; Pfizenmaier, K.; Moosmayer, D.
Human antibody derivatives against the fibroblast activation protein for tumor stroma targeting of carcinomas. Int. J. Cancer
2001, 92, 240–248. [CrossRef]
92. Wüest, T.; Moosmayer, D.; Pfizenmaier, K. Construction of a bispecific single chain antibody for recruitment of cytotoxic T cells to
the tumour stroma associated antigen fibroblast activation protein. J. Biotechnol. 2001, 92, 159–168. [CrossRef]
93. Ostermann, E.; Garin-Chesa, P.; Heider, K.H.; Kalat, M.; Lamche, H.; Puri, C.; Kerjaschki, D.; Rettig, W.J.; Adolf, G.R. Effective
immunoconjugate therapy in cancer models targeting a serine protease of tumor fibroblasts. Clin. Cancer Res. Off. J. Am. Assoc.
Cancer Res. 2008, 14, 4584–4592. [CrossRef]
94. Wäster, P.; Rosdahl, I.; Gilmore, B.F.; Seifert, O. Ultraviolet exposure of melanoma cells induces fibroblast activation protein-α in
fibroblasts: Implications for melanoma invasion. Int. J. Oncol. 2011, 39, 193–202. [PubMed]
95. Altmann, A.; Haberkorn, U.; Siveke, J. The Latest Developments in Imaging of Fibroblast Activation Protein. J. Nucl. Med. 2021,
62, 160–167. [CrossRef]
Pharmaceuticals 2021, 14, 1023 28 of 30

96. Narra, K.; Mullins, S.R.; Lee, H.-O.; Strzemkowski-Brun, B.; Magalong, K.; Christiansen, V.J.; McKee, P.A.; Egleston, B.;
Cohen, S.J.; Weiner, L.M.; et al. Phase II trial of single agent Val-boroPro (talabostat) inhibiting fibroblast activation protein in
patients with metastatic colorectal cancer. Cancer Biol. Ther. 2007, 6, 1691–1699. [CrossRef]
97. Eager, R.M.; Cunningham, C.C.; Senzer, N.N.; Stephenson, J.; Anthony, S.P.; O’Day, S.J.; Frenette, G.; Pavlick, A.C.; Jones, B.;
Uprichard, M.; et al. Phase II assessment of talabostat and cisplatin in second-line stage IV melanoma. BMC Cancer 2009, 9, 263.
[CrossRef]
98. Eager, R.M.; Cunningham, C.C.; Senzer, N.; Richards, D.A.; Raju, R.N.; Jones, B.; Uprichard, M.; Nemunaitis, J. Phase II trial of
talabostat and docetaxel in advanced non-small cell lung cancer. Clin. Oncol. 2009, 21, 464–472. [CrossRef]
99. Jansen, K.; Heirbaut, L.; Cheng, J.D.; Joossens, J.; Ryabtsova, O.; Cos, P.; Maes, L.; Lambeir, A.-M.; De Meester, I.;
Augustyns, K.; et al. Selective Inhibitors of Fibroblast Activation Protein (FAP) with a (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine
Scaffold. ACS Med. Chem. Lett. 2013, 4, 491–496. [CrossRef]
100. Poplawski, S.E.; Lai, J.H.; Li, Y.; Jin, Z.; Liu, Y.; Wu, W.; Wu, Y.; Zhou, Y.; Sudmeier, J.L.; Sanford, D.G.; et al. Identification of
selective and potent inhibitors of fibroblast activation protein and prolyl oligopeptidase. J. Med. Chem. 2013, 56, 3467–3477.
[CrossRef] [PubMed]
101. Thomas, L.; Eckhardt, M.; Langkopf, E.; Tadayyon, M.; Himmelsbach, F.; Mark, M. (R)-8-(3-amino-piperidin-1-yl)-7-but-2-ynyl-3-
methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione (BI 1356), a novel xanthine-based dipeptidyl peptidase
4 inhibitor, has a superior potency and longer duration of action compared with other dipeptidyl peptidase-4 inhibitors. J.
Pharmacol. Exp. Ther. 2008, 325, 175–182.
102. Tsai, T.-Y.; Yeh, T.-K.; Chen, X.; Hsu, T.; Jao, Y.-C.; Huang, C.-H.; Song, J.-S.; Huang, Y.-C.; Chien, C.-H.; Chiu, J.-H.; et al.
Substituted 4-carboxymethylpyroglutamic acid diamides as potent and selective inhibitors of fibroblast activation protein. J. Med.
Chem. 2010, 53, 6572–6583. [CrossRef] [PubMed]
103. Tanswell, P.; Garin-Chesa, P.; Rettig, W.J.; Welt, S.; Divgi, C.R.; Casper, E.S.; Finn, R.D.; Larson, S.M.; Old, L.J.; Scott, A.M.
Population pharmacokinetics of antifibroblast activation protein monoclonal antibody F19 in cancer patients. Br. J. Clin.
Pharmacol. 2001, 51, 177–180. [CrossRef] [PubMed]
104. Scott, A.M.; Wiseman, G.; Welt, S.; Adjei, A.; Lee, F.-T.; Hopkins, W.; Divgi, C.R.; Hanson, L.H.; Mitchell, P.; Gansen, D.N.; et al.
A Phase I dose-escalation study of sibrotuzumab in patients with advanced or metastatic fibroblast activation protein-positive
cancer. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 2003, 9, 1639–1647.
105. Fischer, E.; Chaitanya, K.; Wüest, T.; Wadle, A.; Scott, A.M.; van den Broek, M.; Schibli, R.; Bauer, S.; Renner, C. Radioimmunother-
apy of fibroblast activation protein positive tumors by rapidly internalizing antibodies. Clin. Cancer Res. Off. J. Am. Assoc. Cancer
Res. 2012, 18, 6208–6218. [CrossRef] [PubMed]
106. Connolly, B.A.; Sanford, D.G.; Chiluwal, A.K.; Healey, S.E.; Peters, D.E.; Dimare, M.T.; Wu, W.; Liu, Y.; Maw, H.; Zhou, Y.; et al.
Dipeptide boronic acid inhibitors of dipeptidyl peptidase IV: Determinants of potency and in vivo efficacy and safety. J. Med.
Chem. 2008, 51, 6005–6013. [CrossRef] [PubMed]
107. Meletta, R.; Müller Herde, A.; Chiotellis, A.; Isa, M.; Rancic, Z.; Borel, N.; Ametamey, S.; Krämer, S.; Schibli, R. Evaluation of the
Radiolabeled Boronic Acid-Based FAP Inhibitor MIP-1232 for Atherosclerotic Plaque Imaging. Molecules 2015, 20, 2081–2099.
[CrossRef]
108. Moon, E.S.; Elvas, F.; Vliegen, G.; De Lombaerde, S.; Vangestel, C.; De Bruycker, S.; Bracke, A.; Eppard, E.; Greifenstein, L.;
Klasen, B.; et al. Targeting fibroblast activation protein (FAP): Next generation PET radiotracers using squaramide coupled
bifunctional DOTA and DATA5m chelators. EJNMMI Radiopharm. Chem. 2020, 5, 19. [CrossRef]
109. Jansen, K.; Heirbaut, L.; Verkerk, R.; Cheng, J.D.; Joossens, J.; Cos, P.; Maes, L.; Lambeir, A.-M.; De Meester, I.;
Augustyns, K.; et al. Extended structure-activity relationship and pharmacokinetic investigation of (4-quinolinoyl)glycyl-2-
cyanopyrrolidine inhibitors of fibroblast activation protein (FAP). J. Med. Chem. 2014, 57, 3053–3074. [CrossRef]
110. Loktev, A.; Lindner, T.; Burger, E.-M.; Altmann, A.; Giesel, F.; Kratochwil, C.; Debus, J.; Marmé, F.; Jäger, D.; Mier, W.; et al.
Development of Fibroblast Activation Protein-Targeted Radiotracers with Improved Tumor Retention. J. Nucl. Med. Off. Publ. Soc.
Nucl. Med. 2019, 60, 1421–1429. [CrossRef] [PubMed]
111. Loktev, A.; Lindner, T.; Mier, W.; Debus, J.; Altmann, A.; Jäger, D.; Giesel, F.; Kratochwil, C.; Barthe, P.; Roumestand, C.; et al.
A Tumor-Imaging Method Targeting Cancer-Associated Fibroblasts. J. Nucl. Med. 2018, 59, 1423–1429. [CrossRef] [PubMed]
112. Jansen, K.; De Winter, H.; Heirbaut, L.; Cheng, J.D.; Joossens, J.; Lambeir, A.-M.; De Meester, I.; Augustyns, K.; Van der Veken,
P. Selective inhibitors of fibroblast activation protein (FAP) with a xanthine scaffold. Med. Chem. Commun. 2014, 5, 1700–1707.
[CrossRef]
113. Lindner, T.; Loktev, A.; Altmann, A.; Giesel, F.; Kratochwil, C.; Debus, J.; Jäger, D.; Mier, W.; Haberkorn, U. Development of
Quinoline-Based Theranostic Ligands for the Targeting of Fibroblast Activation Protein. J. Nucl. Med. 2018, 59, 1415–1422.
[CrossRef] [PubMed]
114. Lindner, T.; Altmann, A.; Krämer, S.; Kleist, C.; Loktev, A.; Kratochwil, C.; Giesel, F.; Mier, W.; Marme, F.; Debus, J.; et al. Design
and Development of 99mTc-Labeled FAPI Tracers for SPECT Imaging and 188Re Therapy. J. Nucl. Med. Off. Publ. Soc. Nucl. Med.
2020, 61, 1507–1513. [CrossRef] [PubMed]
115. Roy, J.; Hettiarachchi, S.U.; Kaake, M.; Mukkamala, R.; Low, P.S. Design and validation of fibroblast activation protein alpha
targeted imaging and therapeutic agents. Theranostics 2020, 10, 5778–5789. [CrossRef]
Pharmaceuticals 2021, 14, 1023 29 of 30

116. Giesel, F.L.; Adeberg, S.; Syed, M.; Lindner, T.; Jiménez-Franco, L.D.; Mavriopoulou, E.; Staudinger, F.; Tonndorf-Martini, E.;
Regnery, S.; Rieken, S.; et al. FAPI-74 PET/CT Using Either 18F-AlF or Cold-Kit 68Ga Labeling: Biodistribution, Radiation
Dosimetry, and Tumor Delineation in Lung Cancer Patients. J. Nucl. Med. 2021, 62, 201–207. [CrossRef] [PubMed]
117. Dendl, K.; Finck, R.; Giesel, F.L.; Kratochwil, C.; Lindner, T.; Mier, W.; Cardinale, J.; Kesch, C.; Röhrich, M.; Rathke, H.; et al. FAP
imaging in rare cancer entities-first clinical experience in a broad spectrum of malignancies. Eur. J. Nucl. Med. Mol. Imaging 2021.
[CrossRef]
118. Moon, E.S.; Ballal, S.; Yadav, M.P.; Bal, C.; Rymenant, Y.V.; Stephan, S.; Bracke, A.; der Veken, P.V.; Meester, I.D.; Roesch, F.
Homodimeric Fibroblast Activation Protein (FAP) Targeting Radiotheranostics to Improve Tumor Uptake and Retention Time.
Pharmaceuticals 2021. [CrossRef]
119. Kelly, J.M.; Jeitner, T.M.; Ponnala, S.; Williams, C.; Nikolopoulou, A.; DiMagno, S.G.; Babich, J.W. A Trifunctional Theranostic
Ligand Targeting Fibroblast Activation Protein-α (FAPα). Mol. Imaging Biol. 2021, 23, 686–696. [CrossRef] [PubMed]
120. Millul, J.; Bassi, G.; Mock, J.; Elsayed, A.; Pellegrino, C.; Zana, A.; Dakhel Plaza, S.; Nadal, L.; Gloger, A.; Schmidt, E.; et al.
An ultra-high-affinity small organic ligand of fibroblast activation protein for tumor-targeting applications. Proc. Natl. Acad. Sci.
USA 2021, 118, e2101852118. [CrossRef]
121. Eryilmaz, K.; Kilbas, B. Fully-automated synthesis of 177Lu labelled FAPI derivatives on the module modular lab-Eazy. EJNMMI
Radiopharm. Chem. 2021, 6, 16. [CrossRef] [PubMed]
122. Watabe, T.; Liu, Y.; Kaneda-Nakashima, K.; Shirakami, Y.; Lindner, T.; Ooe, K.; Toyoshima, A.; Nagata, K.; Shimosegawa, E.;
Haberkorn, U.; et al. Theranostics Targeting Fibroblast Activation Protein in the Tumor Stroma: 64Cu- and 225Ac-Labeled
FAPI-04 in Pancreatic Cancer Xenograft Mouse Models. J. Nucl. Med. Off. Publ. Soc. Nucl. Med. 2020, 61, 563–569. [CrossRef]
[PubMed]
123. Slania, S.L.; Das, D.; Lisok, A.; Du, Y.; Jiang, Z.; Mease, R.C.; Rowe, S.P.; Nimmagadda, S.; Yang, X.; Pomper, M.G. Imaging
of Fibroblast Activation Protein in Cancer Xenografts Using Novel (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine-Based Small
Molecules. J. Med. Chem. 2021, 64, 4059–4070. [CrossRef] [PubMed]
124. Giesel, F.L.; Kratochwil, C.; Lindner, T.; Marschalek, M.M.; Loktev, A.; Lehnert, W.; Debus, J.; Jäger, D.; Flechsig, P.;
Altmann, A.; et al. 68Ga-FAPI PET/CT: Biodistribution and Preliminary Dosimetry Estimate of 2 DOTA-Containing
FAP-Targeting Agents in Patients with Various Cancers. J. Nucl. Med. 2019, 60, 386–392. [CrossRef]
125. Kreppel, B.; Gärtner, F.C.; Marinova, M.; Attenberger, U.; Meisenheimer, M.; Toma, M.; Kristiansen, G.; Feldmann, G.; Moon, E.S.;
Roesch, F.; et al. [68Ga]Ga-DATA5m.SA.FAPi PET/CT: Specific Tracer-uptake in Focal Nodular Hyperplasia and potential Role in
Liver Tumor Imaging. Nukl. Nucl. Med. 2020, 59, 387–389. [CrossRef]
126. Ballal, S.; Yadav, M.P.; Moon, E.S.; Kramer, V.S.; Roesch, F.; Kumari, S.; Tripathi, M.; ArunRaj, S.T.; Sarswat, S.; Bal, C. Biodistribu-
tion, pharmacokinetics, dosimetry of [68Ga]Ga-DOTA.SA.FAPi, and the head-to-head comparison with [18F]F-FDG PET/CT in
patients with various cancers. Eur. J. Nucl. Med. Mol. Imaging 2021, 48, 1915–1931. [CrossRef]
127. Ballal, S.; Yadav, M.P.; Kramer, V.; Moon, E.S.; Roesch, F.; Tripathi, M.; Mallick, S.; ArunRaj, S.T.; Bal, C. A theranostic approach of
[68Ga]Ga-DOTA.SA.FAPi PET/CT-guided [177Lu]Lu-DOTA.SA.FAPi radionuclide therapy in an end-stage breast cancer patient:
New frontier in targeted radionuclide therapy. Eur. J. Nucl. Med. Mol. Imaging 2021, 48, 942–944. [CrossRef]
128. Kreppel, B.; Gonzalez-Carmona, M.A.; Feldmann, G.; Küppers, J.; Moon, E.S.; Marinova, M.; Bundschuh, R.A.; Kristiansen, G.;
Essler, M.; Roesch, F.; et al. Fibroblast activation protein inhibitor (FAPi) positive tumour fraction on PET/CT correlates with
Ki-67 in liver metastases of neuroendocrine tumours. Nuklearmedizin 2021, 60, 344–354. [CrossRef]
129. Röhrich, M.; Loktev, A.; Wefers, A.K.; Altmann, A.; Paech, D.; Adeberg, S.; Windisch, P.; Hielscher, T.; Flechsig, P.;
Floca, R.; et al. IDH-wildtype glioblastomas and grade III/IV IDH-mutant gliomas show elevated tracer uptake in fibroblast
activation protein-specific PET/CT. Eur. J. Nucl. Med. Mol. Imaging 2019, 46, 2569–2580. [CrossRef]
130. Kratochwil, C.; Giesel, F.L.; Rathke, H.; Fink, R.; Dendl, K.; Debus, J.; Mier, W.; Jäger, D.; Lindner, T.; Haberkorn, U.
[153Sm]Samarium-labeled FAPI-46 radioligand therapy in a patient with lung metastases of a sarcoma. Eur. J. Nucl. Med.
Mol. Imaging 2021, 48, 3011–3013. [CrossRef]
131. Rathke, H.; Fuxius, S.; Giesel, F.L.; Lindner, T.; Debus, J.; Haberkorn, U.; Kratochwil, C. Two Tumors, One Target: Preliminary
Experience With 90Y-FAPI Therapy in a Patient With Metastasized Breast and Colorectal Cancer. Clin. Nucl. Med. 2021, 46,
842–844. [CrossRef]
132. Ferdinandus, J.; Fragoso Costa, P.; Kessler, L.; Weber, M.; Hirmas, N.; Kostbade, K.; Bauer, S.; Schuler, M.; Ahrens, M.;
Schildhaus, H.-U.; et al. Initial clinical experience with 90Y-FAPI-46 radioligand therapy for advanced stage solid tumors: A case
series of nine patients. J. Nucl. Med. Off. Publ. Soc. Nucl. Med. 2021, jnumed.121.262468. [CrossRef]
133. Fearon, D.T. The carcinoma-associated fibroblast expressing fibroblast activation protein and escape from immune surveillance.
Cancer Immunol. Res. 2014, 2, 187–193. [CrossRef]
134. Kratochwil, C.; Flechsig, P.; Lindner, T.; Abderrahim, L.; Altmann, A.; Mier, W.; Adeberg, S.; Rathke, H.; Röhrich, M.;
Winter, H.; et al. 68Ga-FAPI PET/CT: Tracer Uptake in 28 Different Kinds of Cancer. J. Nucl. Med. Off. Publ. Soc. Nucl.
Med. 2019, 60, 801–805. [CrossRef]
135. Chen, H.; Pang, Y.; Wu, J.; Zhao, L.; Hao, B.; Wu, J.; Wei, J.; Wu, S.; Zhao, L.; Luo, Z.; et al. Comparison of [68Ga]Ga-DOTA-FAPI-04
and [18F] FDG PET/CT for the diagnosis of primary and metastatic lesions in patients with various types of cancer. Eur. J. Nucl.
Med. Mol. Imaging 2020, 47, 1820–1832. [CrossRef] [PubMed]
Pharmaceuticals 2021, 14, 1023 30 of 30

136. Shi, X.; Xing, H.; Yang, X.; Li, F.; Yao, S.; Zhang, H.; Zhao, H.; Hacker, M.; Huo, L.; Li, X. Fibroblast imaging of hepatic carcinoma
with 68Ga-FAPI-04 PET/CT: A pilot study in patients with suspected hepatic nodules. Eur. J. Nucl. Med. Mol. Imaging 2021, 48,
196–203. [CrossRef] [PubMed]
137. Meyer, C.; Dahlbom, M.; Lindner, T.; Vauclin, S.; Mona, C.; Slavik, R.; Czernin, J.; Haberkorn, U.; Calais, J. Radiation Dosimetry
and Biodistribution of 68Ga-FAPI-46 PET Imaging in Cancer Patients. J. Nucl. Med. Off. Publ. Soc. Nucl. Med. 2020, 61, 1171–1177.
[CrossRef]
138. Koerber, S.A.; Staudinger, F.; Kratochwil, C.; Adeberg, S.; Haefner, M.F.; Ungerechts, G.; Rathke, H.; Winter, E.; Lindner, T.;
Syed, M.; et al. The Role of 68Ga-FAPI PET/CT for Patients with Malignancies of the Lower Gastrointestinal Tract: First Clinical
Experience. J. Nucl. Med. 2020, 61, 1331–1336. [CrossRef] [PubMed]
139. Koerber, S.A.; Finck, R.; Dendl, K.; Uhl, M.; Lindner, T.; Kratochwil, C.; Röhrich, M.; Rathke, H.; Ungerechts, G.; Adeberg, S.; et al.
Novel FAP ligands enable improved imaging contrast in sarcoma patients due to FAPI-PET/CT. Eur. J. Nucl. Med. Mol. Imaging
2021, 48, 3918–3924. [CrossRef] [PubMed]
140. Dendl, K.; Koerber, S.A.; Finck, R.; Mokoala, K.M.G.; Staudinger, F.; Schillings, L.; Heger, U.; Röhrich, M.; Kratochwil, C.;
Sathekge, M.; et al. 68Ga-FAPI-PET/CT in patients with various gynecological malignancies. Eur. J. Nucl. Med. Mol. Imaging 2021,
48, 4089–4100. [CrossRef]
141. Röhrich, M.; Naumann, P.; Giesel, F.L.; Choyke, P.L.; Staudinger, F.; Wefers, A.; Liew, D.P.; Kratochwil, C.; Rathke, H.;
Liermann, J.; et al. Impact of 68Ga-FAPI PET/CT Imaging on the Therapeutic Management of Primary and Recurrent Pancreatic
Ductal Adenocarcinomas. J. Nucl. Med. Off. Publ. Soc. Nucl. Med. 2021, 62, 779–786.
142. Giesel, F.L.; Heussel, C.P.; Lindner, T.; Röhrich, M.; Rathke, H.; Kauczor, H.-U.; Debus, J.; Haberkorn, U.; Kratochwil, C. FAPI-
PET/CT improves staging in a lung cancer patient with cerebral metastasis. Eur. J. Nucl. Med. Mol. Imaging 2019, 46, 1754–1755.
[CrossRef] [PubMed]