# Potential of TSPO radioligands: Bridging brain tumor diagnostics to the peripheries

F. Avry, C. Rousseau, F. Kraeber-Bodéré, M. Bourgeois, Nicolas Arlicot

► To cite this version:

F. Avry, C. Rousseau, F. Kraeber-Bodéré, M. Bourgeois, Nicolas Arlicot. Potential of TSPO ra-dioligands: Bridging brain tumor diagnostics to the peripheries. Biochimie, 2024, 224, pp.114-119. 10.1016/j.biochi.2024.05.005 . hal-05073064

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# Potential of TSPO radioligands: Bridging brain tumor diagnostics to the peripheries

F. Avry<sup>a</sup>, C. Rousseau<sup>b, c</sup>, F. Kraeber-Bodéré<sup>d</sup>, M. Bourgeois<sup>d, e</sup>, Nicolas Arlicot<sup>a, f, g, *</sup>

<figure><img src="image_2.png" /></figure>

## ARTICLE INFO

**Article history:**

Received 5 January 2024

Received in revised form

2 April 2024

Accepted 4 May 2024

Available online 9 May 2024

Handling Editor: Dr B Friguet

**Keywords:**

TSPO PET imaging

Peripheral cancer

Tumor microenvironment

Preclinical study

## ABSTRACT

Translocator protein (TSPO) is involved in several cellular mechanisms such as steroidogenesis, immunomodulation, cell proliferation and differentiation. Overexpressed in several neurodegenerative diseases and brain cancer, TSPO radioligands have been developed over the last 20 years in positron emission tomography (PET) imaging. Recently, TSPO radioligands have extended beyond their initial application due to their specific binding to activated macrophages, making them a compelling biomarker for deciphering the intricacies of the tumor microenvironment (TME). In this review, we synthesized recent progress from the evaluation of TSPO-specific PET tracers in various peripheral tumor models and highlighted the hurdles and limitations associated with heterogeneous uptake in healthy tissue and tumor regions to achieve the clinical development of such a radiotracer.

© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Contents

<table><tr><td>1.</td><td>Introduction</td><td>115</td></tr><tr><td>2.</td><td>Material and methods</td><td>115</td></tr><tr><td>3.</td><td>Results</td><td>115</td></tr><tr><td>3.1.</td><td>Breast cancer</td><td>115</td></tr><tr><td>3.2.</td><td>Prostate cancer</td><td>115</td></tr><tr><td>3.3.</td><td>Colorectal cancer</td><td>117</td></tr><tr><td>3.4.</td><td>Lung cancer</td><td>117</td></tr><tr><td>3.5.</td><td>Pancreatic cancer</td><td>117</td></tr><tr><td>3.6.</td><td>Head and neck cancer</td><td>117</td></tr><tr><td>4.</td><td>Discussion</td><td>117</td></tr><tr><td>5.</td><td>Conclusion</td><td>118</td></tr><tr><td></td><td>Funding</td><td>118</td></tr><tr><td></td><td>CREdT authorship contribution statement</td><td>118</td></tr><tr><td></td><td>Declaration of competing interest</td><td>118</td></tr><tr><td></td><td>Acknowledgements</td><td>118</td></tr><tr><td></td><td>References</td><td>118</td></tr></table>

<footnote><sup>a</sup> CHRU de Tours, Tours, France</footnote>

<footnote><sup>b</sup> Nantes Université, Univ Angers, INSERM, CNRS, CRCI2NA, F-44000, Nantes, France</footnote>

<footnote><sup>c</sup> ICO, Service de Médecine Nucléaire, F-44800, Saint-Herblain, France</footnote>

<footnote><sup>d</sup> Nantes Université, Univ Angers, CHU Nantes, INSERM, CNRS, CRCI2NA, F-44000, Nantes, France</footnote>

<footnote><sup>e</sup> ARRONAX Cyclotron, F-44800, Saint-Herblain, France</footnote>

<footnote><sup>f</sup> UMR 1253, iBraïN, Université de Tours, INSERM, Tours, France</footnote>

<footnote><sup>g</sup> INSERM CIC 1415, Université de Tours, INSERM, Tours, France</footnote>

<footnote>* Corresponding author. CHRU de Tours, Tours, France.
E-mail address: nicolas.arlicot@univ-tours.fr (N. Arlicot).</footnote>
# 1. Introduction

Positron emission tomography (PET) imaging is currently developed as non-invasive imaging method for the early diagnosis, staging and monitoring of cancer. TSPO radioligands that have shown specific binding to activated microglia/macrophages, have also shown relevance in tumor imaging with interesting signal to noise ratios (S/N) and for deciphering the tumor microenvironment (TME) through specifically Tumor-Associated Macrophage (TAM) [1]. TSPO is a 18 kDa mitochondrial protein initially assessed in neurodegenerative disease, neuroinflammation [2] and brain cancer. Considering the several limitations of magnetic resonance (MR) and computed tomography (CT) methods [3], added to the low physiological biomarker expression in this area [4], PET TSPO appeared to be of interest. Further to the first trials with [11C] PK11195 [5], two other generations of PET TSPO radiotracers followed, enhancing binding sensitivity and selectivity and overcoming the genetic limit linked to the rs6971 polymorphism [2] (A147T).

As TSPO is involved in immunomodulation, cell proliferation and differentiation [6], several cancer types present an increased expression of this biomarker such as brain, head and neck, lungs, colorectal, liver prostate, endometrial and breast cancer [7–9]. This upregulation has been related to tumor aggressiveness and/or poor prognosis [7,10,11].

In their review, Filippi et al. reported the clinical evidence of TSPO radioligands in defining glioma grade and associated TME, with the possibility for the patients to be rapidly referred for targeted therapy [12]. While some teams begin to assess other brain tumors, such as in IDH-wild type glioblastoma [10] or schwannoma [13], the clinical application of TSPO radioligands to peripheral tumors has not yet been set.

Up to now, numerous PET radioligands targeting TSPO have been developed. Reviews by Zhang et al. and Singh et al. summarized their structures and highlighted the challenges encountered in the TSPO radiotracer use [2,14]. In the present review, we explore the recent progress of TSPO molecular imaging in non-primo brain tumors, by comparing the radioligands' characteristics and highlighting the obstacles that remain to be overcome in order to achieve the clinical development of such a radiotracer.

# 2. Material and methods

The PubMed and Google Scholar database was screened using pre-defined search dates (January 1995–October 2023). The search terms used were as follows: "PET imaging" AND "TSPO" AND "cancer OR tumor". We screened *in vivo/ex vivo* preclinical and/or *ex vivo* clinical results not performed in brain tumor cell lines. Eleven original papers fulfilled these criteria and were thus included in the scope of the present review. Cell line, animal model and PET radioligands used are presented. The main conditions and results of these PET imaging preclinical studies assessing TSPO radiotracers in peripheral tumor xenograft models are summarized in Table 1.

# 3. Results

## 3.1. Breast cancer

In 2011, Zheng et al. paved the way of peripheral potential of TSPO radiotracer in breast tumor imaging with [18F]DPA-714 in nude NMRI and FVB/NRj mice [15]. Comparing TSPO Western-blot (WB) and [18F]DPA-714 *ex vivo* uptake in MDA-MB-231, PyMT, MCF-7 and HBCx-12 B tumor cells bearing, they demonstrated that TSPO expression was distributed between the tumor and the

stroma/tumor micro-environment (TME). Autoradiographic imaging of this second generation TSPO radiotracer showed a visually higher uptake in tumor than muscle. Co-administration of 100-fold excess of cold PK11195 or DPA-714 showed an important and a significantly displaceable radiotracer uptake in the spleen, lungs and liver tissue in addition to the tumor. High uptake in peripheral organs was also supported *in vivo* by Vasdev et al., which assessed [18F]FEPPA tumor uptake in the high, expressing TSPO MDA-MB-231 cells bearing CD1 nu/nu mice [16]. At 90 min p.i., tumor uptake was significantly lower than surrounding muscle (T/M = 0.4) and display a poor specificity with cold ligand addition. *Ex vivo* uptake biodistribution assessed with a γ-counter exhibited a blood, spleen, heart, lung, kidneys, muscles, intestine radioligand uptake 3 to more than 30 times higher than MDA-MB-231 excised cells (tumor = 0.7 vs. muscle = 4.5 %ID/g). Pre-administration of an excess of cold FEPPA does not decrease significantly (1.5 %ID/g vs. 0.7 %ID/g) tumor uptake. On the contrary, several TSPO-expressing organs displayed a significant binding displacement with cold FEPPA co-administration.

Later, Zheng et al. assessed again their experiment protocol with [18F]FEPPA in the same breast tumors compared to dextran sulfate sodium bowel induced-inflammation and turpentine induced-inflammation model in Wistar rats, nude NMRI and FVB/NRj mice [17]. They confirmed the low radiotracer tumor uptake (0.9 % ID/g), T/M ratio (1.2) and tumor to other organ ratio in human and murine breast xenografted tumors. While breast xenografted tumors [18F]FEPPA uptake was not assessed in rats, the 2 peripheral inflammation models displayed *in vivo* a markedly higher lesion-to-control ratio in rats than in mice (mean SUV value: 9.1 vs. 1.1), although mice tumor uptake is higher in mice than in rats (0.85 % ID/g vs 0.23 %ID/g). Biodistribution investigations highlighted a significant lower uptake in rat muscle, organs and brain compared to mice. An *ex vivo* radio-HPLC analysis showed a faster [18F]FEPPA metabolism in rats than in mice (97 % vs. 85 % of intact [18F]FEPPA 60 min p.i.) and an overall muscle-to plasma ratio significantly higher in mice than in rats (2.5 vs. 10.2).

In their comparative study, Morais de Souza et al. assessed [11C](R)-PK11195 and [18F]FDG uptake in 4T1 murine tumor bearing Balb/C mice to visualize the Tumor-Associated Macrophages (TAM) in the TME [18]. At 60 min post injection, [11C](R)-PK11195 showed similar T/M ratio to [18F]FDG at 3 days (2.8 vs. 2.3), 1 week (2.3 vs. 3.2) and 2 weeks (1.7 vs 3.9) after tumor cells inoculation. However, [11C](R)-PK11195 SUV value significantly decreased 2 weeks after inoculation (3 days: 0.3; 1 week: 0.4; 2 weeks: 0.17), contrasted to [18F]FDG (3 days: 0.64; 1 week: 0.59; 2 weeks: 0.6). Interestingly, *ex vivo* study reported a [11C](R)-PK11195 center-to-edge tumor uptake gradient accentuated at week 2, attesting metabolic and TAM activity are predominant in the tumor border. Immunofluorescence images performed on the same tumor sampled support this correlation with TSPO expression gradient.

## 3.2. Prostate cancer

[18F]VUIIS1008, a parent ligand of [18F]DPA-714 with higher TSPO affinity was assessed in a *in situ* spontaneous development of prostate cancer in 4–6 months old Pten/Trp53 conditional mouse model (Ptenpc −/−; Trp53pc −/−) [19]. Following MR tumor size imaging and tumor proliferation, dynamic PET/CT was acquired during 60 min p.i. A constant T/M ratio of 10 was exhibited between 5 and 60 min p.i. (tumor uptake: 7.1 %ID/mL vs. muscle: 0.7 %ID/mL). Immunohistochemically analysis confirmed the high [18F]VUIIS1008 uptake tumor cells presented TSPO expression. Reasonable liver uptake was observed considering the preponderant [18F]VUIIS1008 liver clearance.
<table><caption>**Table 1**
Ex vivo/in vivo TSPO radioligands studies in peripheral assessment.</caption><thead><tr><th>Tumor organ model</th><th>Radiotracer</th><th>In vivo model</th><th>Cell line</th><th>Imaging modalities</th><th>results</th><th>reference</th></tr></thead><tbody><tr><td rowspan="4">Breast</td><td>[18F]DPA-714</td><td>Nude NMRI and FVB/NRj mice</td><td>Human: MCF-7<br>MDA-MB-231<br>HBCx-12 B<br>Mouse: PyMT</td><td>30 min static acquisition 30 p.i.</td><td>Autoradiography study showed a T/M ratio >1 and an important uptake and specificity in the spleen, tumor, kidney, liver and kidneys</td><td>[15]</td></tr><tr><td>[18F]FEPPA</td><td>CD1 nu/nu mice</td><td>MDA-MB-231</td><td>10 min static acquisition 90 p.i.</td><td>T/M ratio = 0.4 and poor a poor in vivo specificity<br>Higher uptake in heart, muscle, lungs, kidneys and spleen than tumor ex vivo</td><td>[16]</td></tr><tr><td>[18F]DPA-714</td><td>NMRI, C57BL/6 and FVB/NRj mice</td><td>Human: MCF-7<br>MDA-MB-231<br>HBCx-12 B<br>Mouse: PyMT</td><td>30 min static acquisition 30 min p.i.</td><td>T/M ratio = 1.2 in vivo and an important uptake and specificity in the spleen, tumor, kidney, liver and kidneys<br>Significantly lower peripheral, muscle and brain radiotracer uptake in rats than mice</td><td>[17]</td></tr><tr><td>[11C](R)-PK11195</td><td>Balb/C mice</td><td>Mouse: 4T1</td><td>30 min static acquisition 60 min p.i.</td><td>[11C](R)-PK11195 and [18F]FDG similar T/M ratios (≈2–3)<br>[11C](R)-PK11195 uptake decrease 2 weeks post cells inoculation unlike a constant [18F]FDG uptake value<br>Increased [11C](R)-PK11195 tumor edge uptake with time<br>Decreased [11C](R)-PK11195 tumor uptake in immunodeficient mice</td><td>[18]</td></tr><tr><td rowspan="2">Prostate</td><td>[18F] VUIIS1008</td><td>Pten/Trp53<sup>−/−</sup> conditional mouse</td><td>/</td><td>Static acquisition during 60 min p.i.</td><td>T/M ratio = 10 with a significantly higher uptake in tumor than muscle.<br>Low general uptake in other organs.<br>Tumor uptake steady state reached about 25 min p.i.</td><td>[19]</td></tr><tr><td>[18F] PBR316</td><td>Sprague-Dawley rats and Balb/c nu/nu mice</td><td>PC-3MLuc-C6</td><td>Static acquisition from 0 to 240 min p.i.</td><td>Enhanced T/M ratio from 1.3 30 min p.i. to 2.5 3 h p.i. trend towards increased uptake in the tumor with time, unlike in other organs<br>Pre-injection of low PK11195 dose increased tumor uptake in rats and mice</td><td>[20]</td></tr><tr><td>colorectal</td><td>[18F]FEPPA</td><td>/</td><td>HT-29 Patient tumor sample</td><td>autoradiography</td><td>Tumor to healthy peripheral tissue ratio at least of 3 HT-29 cells and tumor displayed important specificity</td><td>[21]</td></tr><tr><td rowspan="2">lung</td><td>[18F]PBR06</td><td>FVB/N and athymic nude mice</td><td>A549 cells Turpentine oil</td><td>Biodistribution: 120 min p.i.<br>In vivo: during 90 min p.i.</td><td>Ex vivo T/M was lower than inflamed to muscle ratio (2.33 vs 1.45)<br>In vivo TACs study exhibit low and constant T/M ratio unlike high inflamed-to-muscle ratio<br>Major uptake in the liver, stomach and lungs</td><td>[22]</td></tr><tr><td>Pancreas</td><td>[11C]PBR28</td><td>CD11b-DTR mice</td><td>PDAC 4668 cells</td><td>Autoradiography 20 min p.i.<br>In vivo: Dynamic acquisition during 60 min p.i.</td><td>Autoradiography T/M ratio = 2.4<br>Significantly higher radiotracer uptake in immunocompetent than macrophage depleted mice<br>Tumor is visible in vivo despite important uptake in lung, spleen and kidneys</td><td>[23]</td></tr><tr><td>[18F]V-1008</td><td></td><td>In situ KTC and KSC / mice</td><td>/</td><td>Dynamic acquisition during 60 min p.i.</td><td>Significantly higher radiotracer uptake in premalignant tumor cells than control (tumor to normal ratio = 1.4)<br>No [18F]V-1008 uptake association with tumor progression<br>Blocking study was not performed with the cold radiotracer</td><td>[24]</td></tr><tr><td>Head and neck</td><td>[18F]F-DPA</td><td>Athymic Nude Foxn1<sup>nu</sup> mice</td><td>hypopharyngeal tumor FaDu and tongue squamous carcinomas Cal33 cells</td><td>Static 20 min acquisition 20 p.i.</td><td>Significantly higher radiotracer uptake in RT treated than non-RT treated FaDu cells bearing mice unlike [18F]FDG in vivo</td><td>[25]</td></tr><tr><td></td><td></td><td></td><td></td><td>Static acquisition during 60 min p.i.</td><td>Cold PK11195 pretreated no RT mice showed higher [18F]F-DPA uptake than no pretreated no RT mice<br>Tumor uptake showed a similar trend ex vivo after PK11195 pretreatment</td><td></td></tr></tbody></table>

Mattner et al. successfully synthesized [18F]PBR316, a third generation TSPO radioligands derived from PBR111 and PBR102 with 20 ± 5 % radiochemical yield and a 10-times higher binding selectivity than its parents [20]. Time-activity study performed in healthy SD rats indicated [18F]PBR316 quickly diffuse in tissues resulting in low blood radioactivity load and a decreasing uptake kinetic in all organs expected bone marrow and adrenals. Uptake was important in lungs, heart, kidneys, adrenals and bone marrow. [18F]PBR316 and metabolites did not seem to cross the BBB considering the weak %ID/g in the brain uptake almost 4 h p.i. Interestingly, pre-administration of some competitive binders such as PK11195 or Ro 5-4864 increased adrenals and plasma uptake in rats while uptake decreased in other organs. In PC-3MLuc-C6 tumor bearing Balb/c nu/nu mice showed in vivo and ex vivo an increased [18F]PBR316 uptake with time, with a maximum of 1.8 %ID/g

reached about 4 h p.i. In vivo and ex vivo uptake kinetics of measured organs are similar than rats, with a decreased of [18F] PBR316 %ID/g overtime. Thus, T/M ratio increased from 1.3 (30min p.i.) to 2.5 (3 h p.i.), with an interesting enhanced 1.8 %ID/g to 3.8 % ID/g uptake value. Displacement study performed with low PK11195 dose (1 mg/kg) 30 min p.i. displayed ex vivo a significantly enhanced [18F]PBR316 tumor uptake (about 1.5 %ID/g to 3%ID/g) and a significant [18F]PBR316 decrease uptake in several organs excepted in blood and muscle. Similar trend was reported on in vivo imaging. Authors explained this paradox considering (i) the lower TSPO binding affinity of PK11195 vs. PBR316 (ii) the insufficient dose of i.v. cold ligand injected regarding the TSPO receptor density in these tissues (iii) cold PBR316 i.v. injection significantly decrease [18F]PBR316 uptake in all organs, although tumor uptake specificity was not assessed with PBR316.
### 3.3. Colorectal cancer

Up to now, only one paper assessed TSPO radiotracer *ex vivo* in human colorectal cancer. After the incubation with automated radiosynthesis of [¹⁸F]FEPPA, autoradiography of patient colorectal cancer tumor exhibited at least 3 times higher uptake compared to adjacent healthy mucosa collected slices [21]. Tumor samples TSPO expression was confirmed via WB and was similar to the known high TSPO expression unlike in healthy colon lysate. In high expression TSPO expression HT-29 cell line, [¹⁸F]FEPPA binding equilibrium was reached after 20 min and cold FEPPA injection displaced the binding intensity by around 50 %. Co-incubated with cold FEPPA or PBR28, [¹⁸F]FEPPA patient tumor *ex vivo* samples uptake decreased by 80 % demonstrating an interesting specificity.

### 3.4. Lung cancer

Zhang et al. injected the 2nd generation [¹⁸F]PBR06 or [¹⁸F]FDG to A549 cells to FVB/N and athymic nude mice to differentiate turpentine-induced-muscle inflammation from lung tumor [22]. After immunofluorescence TSPO expression validation, gamma-counter 2 h p.i. displayed a significantly higher uptake in inflamed region than tumor (4.8 %ID/g vs. 1.55 %ID/g) and significantly higher inflamed to muscle ratio than T/M (2.33 vs 1.45). MicroPET/CT revealed important [¹⁸F]PBR06 mice liver, spleen, kidney, stomach intestine, pancreas and especially lungs uptake. In vivo time activity curves displayed an interesting [¹⁸F]PBR06 inflamed to normal muscle ratio, whereas T/M ratios were similar over the 90 min acquisition. Interestingly, inflamed muscle imaging with [¹⁸F]PBR06 indicated an enhanced uptake at the edge of the inflammatory zone unlike [¹⁸F]FDG. [¹⁸F]FDG seems to exhibit similar uptake in both cases.

### 3.5. Pancreatic cancer

[¹¹C]PBR28, a second TSPO radiotracer was assessed in a murine pancreatic ductal adenocarcinoma (PDAC) 4668 cells grafted CD11b-DTR mice model which possess a diphtheria toxin receptor inducing macrophage death [23]. Western Blot analysis confirmed that 4668 PDAC cells did not express in vitro TSPO receptors unlike peri-tumoral infiltrating macrophage revealed by immunoblot and IHC. At 20 min p.i., autoradiographic study displayed high kidney and PDAC tumor [¹¹C]PBR28 (ranging from 1.6 to 3.4 %ID/g), with an interesting 2.4 mean T/M ratio. Pre-treated mice with diphtheria toxin (i.e. macrophage depleted mice) presented significantly lower *ex vivo* tumor [¹¹C]PBR28 uptake, supporting specific radioligand binding to infiltrating macrophages. Enhanced [¹¹C]PBR28 uptake of the edge of the grafted PDAC was confirmed by autoradiography of whole-mount cross-sections of the mice. *In vivo*, tumor was visible 1 h p.i. although a clearly higher lung, kidneys and spleen radiotracer uptake.

Through a dual modality *in situ* approach, Cohen et al. attempted to image premalignant and malignant pancreatic tumor [24]. PET/MR imaging was performed with the 2nd TSPO radiotracer [¹⁸F]V-1008 injected in 2 genetically engineered pancreatic advanced grade tumor KTC and KSC mouse model known to frequently develop PDAC in 20 weeks. Compared to control group, 7–8 weeks age-matched premalignant-IHC confirmed KSC mice showed a significant higher *ex vivo* [¹⁸F]V-1008 uptake (2.52 %ID/g vs. 3.59% ID/g) 1 h p.i. In vivo, 11–12 weeks KTC mice did not exhibit higher tumor [¹⁸F]V-1008 uptake than younger mice (8.8 %ID/g vs 8.4 %ID/g), despite IHC and optical dye imaging confirmed higher TSPO expression. [¹⁸F]V-1008 uptake is important in mice lungs, spleen and kidneys in *ex vivo* imaging.

### 3.6. Head and neck cancer

A comparative study was conducted in 2021 to assess the interest of [¹⁸F]F-DPA, a derivative of [¹⁸F]DPA-714 with 4-fold higher affinity for TSPO, in measuring radiotherapy tumor induced inflammation vs. [¹⁸F]FDG [25]. After the inoculation of the human hypopharyngeal tumor FaDu cells in Foxn1<sup>nu</sup> nude mice, PET imaging with both radiotracers were achieved 1 or 2 weeks after the delivery of 5 Gy on 2 consecutive days to the tumor. Following whole body TSPO expression confirmation, FaDu cell bearing mice exhibited 20 min p.i. a significantly higher [¹⁸F]F-DPA uptake than *in vivo* control group 1 and 2 weeks after the radiotherapy (RT) exposure (1.5 %ID/mL vs. 0.74 %ID/mL and 1.1 %ID/mL vs. 0.64 %ID/mL respectively). In contrast, [¹⁸F]FDG uptake did not highlight a significant change 1-week post-RT (1.9 %ID/mL vs. 1.9 %ID/mL) but rather an uptake reduction 2 weeks post RT (0.9 %ID/mL vs. 1.8 %ID/mL). The team duplicated this experiment protocol with the tongue squamous carcinomas cell line Cal33 and the tumor enhanced uptake reached a value slightly under the significance (p = 0.063). The pre-injection of 1 mg of cold PK11195 in mice with non-RT tumor interestingly highlighted a comparable uptake than RT mice during a 60 min p.i. timeframe acquisition (about 1.5 %ID/mL). Likewise, pretreated no-RT mice showed higher radiotracer uptake than non-pretreated no-RT mice.

The *ex vivo* study confirmed that RT increased [¹⁸F]F-DPA tumor uptake in FaDu and Cal33 cell lines two weeks post-mortem radiotracers injection (3.3 %ID/mL vs. 1.2 %ID/mL), with a tumor-to-blood ratio significantly higher than control groups (8 vs. 4)²⁵. Radiotracer uptake is important in adrenals, kidneys, lung, heart, spleen brown fats and thymus. After RT, a proportion of migratory monocytes/macrophage significantly increased as opposed to a decreased number of M1 and M2 macrophages. Interestingly, PK11195 pre-administration in no-RT mice significantly increased [¹⁸F]F-DPA levels in blood (plasma + erythrocytes) and muscle, with a similar increased FaDu tumor uptake than in the *in vivo* model (p = 0.07). The authors explained that PK11195 may relocate [¹⁸F]F-DPA by a competitive mechanism.

## 4. Discussion

This short review focused on the pre-clinical evaluation of PET TSPO radioligands in peripheral tumors models. These eleven studies about various peripheral *in vivo/ex vivo* tumor models display heterogeneous results. Zheng et al. and Vasdev et al. concluded to a limited benefit of TSPO radiotracers in their breast tumor mouse model considering the lack of specificity and the low tumor uptake. On the other hand, pancreas, colorectal and prostate cancer mice models reported interesting uptakes and T/M or tumor to normal ratios value from 1.3 up to 10.

Although radiotracers included here present inherent variations in affinity (Ki), logP value, selectivity and specificity for TSPO and tissues [14], it cannot explain such discrepancies. In their bio-distribution study, Zheng et al. reported that *in vivo* metabolism and elimination of [¹⁸F]DPA-714 in healthy Wistar rats are clearly faster than in mice model [17]. Thus, 60 min p.i., at least mice kidney, liver and spleen radiotracer uptake are still increasing. Tantawy et al. presented a similar increasing [¹⁸F]VUIIS1008 muscle uptake trend from 0 to 60 min p.i. in their prostate tumor model [19]. They supposed that weak mice tumor contrast highlighted through the distinct rat and mice T/M values (13.9 vs. 1.2) is partly resulting from significant non-specific radiotracer accumulation in healthy tissues [17]. In this sense, the bio-distribution study realized up to 4 h p.i. by Mattner et al. exhibited in mice and/or rats specific tissue presenting delayed kinetics, such as adrenals, bone marrow and prostate tumor unlike in other organs showing constant
decreasing uptake [20]. Hence, T/M ratio and tumor-to-other organ were improved with time over 1 h. Enhancing TSPO imaging contrast and tumor selectivity is even more important as several peripheral tissues presented high TSPO radiotracer uptake constitutively [16–18,20,22,24,26]. This delayed kinetics could partly explain the failure of some displacement studies.

While brain is ideally suited for the use of TSPO radiotracers considering its anatomical position in the skull and basically low TSPO expression in healthy tissue, peripheral organs uptake seems to limit the use of these radiotracers for tumors related to these organs. To illustrate, Zheng et al. emphasized in 9 L xenografted glioma cells bearing mice and rats interesting selectivity with low healthy brain tissue uptake in intrathecally (i.c.) injected tumors whereas subcutaneously (s.c.) injected cells displayed massive uptake in digestive organs [17]. This obvious limitation of TSPO radioligands in peripheral use was not discussed given that only 1 article explores in situ tumor. Thus, delayed imaging could be considered to refine contrast especially in tumors located in high constitutive TSPO uptake organs or in tumors with low radiotracer overall uptake. This lack of contrast can also be explained by a lack of radiotracer specificity. In this sense, while some displacement studies have failed to display a significant uptake reduction, 3 articles interestingly showed an increased uptake of tumor uptake with co-administration of cold TSPO tracer, improving contrast and the T/M ratio [16,20,25]. According to Mattner et al., it is conceivable that an excess of cold tracer increases the radiotracer blood pool, leading to enhanced non-specific binding, this type of binding being considered irreversible. Thus, if the overall uptake increase in tumor, it may reflect a low level of specific targeting and a large number of non-specific targets.

Furthermore, Zheng et al. exhibited variable tumor and peritumoral TSPO expression considering the cell line grafted. In fact, overall TSPO radiotracer uptake is composed of an interplay of tumor and peritumor cells uptake, such as pericyte, progenitor, immune and endothelial cells [27]. Given the cumulative signal from TSPO, quantifying the TSPO radiotracer can be tricky as uptake is influenced by TSPO expression in the tumor, which can fluctuate over time. Interestingly, it has recently been demonstrated in glioma tumor model that tumor aggressiveness is primarily driven by TSPO expression of M2 TAMs [28], which seems to be similar in PDAC model [23]. On the other hand, TSPO tumor overexpression shows high variability and its role in the mechanism of cancer is still unclearly understood [29]. Its expression appears to decrease with undifferentiated cancer cells [7]. Autoradiography performed by Morais de Souza et al. highlighted decreasing tumor uptake over time while the direct peritumoral environment seems to be enhanced [18]. Same trends were showed in PDAC model [23] and confirmed in vivo in lung [22] and breast [16] tumor. Hence, T/M ratio could be improved by defining the ROI by the peritumoral crown to avoid the highly variable center tumor uptake. This hypothesis must be considered with caution, as the immune and TAM infiltration of a grafted orthotopic tumor may not be as physiological as that of a spontaneous tumor, which could lead to a contrived uptake gradient. However, preliminary results from the pilot study of Rousseau et al. in triple negative breast cancer display enhanced uptake at the tumor periphery in some patients [30] (data unavailable).

## 5. Conclusion

The field of PET imaging in cancer has witnessed significant progress with the exploration of various molecular targets and physiological pathways such as hypermetabolism, apoptosis, neangiogenesis, and amino acid metabolism. While TSPO-targeted PET imaging has demonstrated its interest in several neurological

disorders and brain cancers, its application in peripheral tumors suffers from limitations due to the highly heterogeneous physiological expression of TSPO in peripheral tissues. However, it may appear to offer an additional layer of understanding in the pathophysiological processes, especially in the microtumoral environment.

## Funding

None to declare.

## CRediT authorship contribution statement

**F. Avry:** Data curation, Writing – original draft, Writing – review & editing.
**C. Rousseau:** Formal analysis, Validation, Visualization.
**F. Kraeber-Bodéré:** Funding acquisition, Visualization.
**M. Bourgeois:** Formal analysis, Validation, Visualization.
**Nicolas Arlicot:** Conceptualization, Methodology, Project administration, Supervision, Validation, Funding acquisition.

## Declaration of competing interest

The authors declare that they have no competing interests.

## Acknowledgements

This work has been supported in part by grants from the French National Agency for Research "France 2030 investment plan" Labex IRON (ANR-11-LABX-18-01) and from INCa-DGOS-INSERM/ITMO Cancer_18011 (SIRIC ILIAD).

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