Preclinical evaluation of [18F]AlF-FAPI-74 for PET imaging to study cancer-associated fibroblast responses to radiotherapy

Preclinical evaluation of [18F]AlF-FAPI-74 for PET imaging to study cancer-associated fibroblast responses to radiotherapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Preclinical evaluation of [ 18 F]AlF-FAPI-74 for PET imaging to study cancer-associated fibroblast responses to radiotherapy Kristin Lode, Sindhu Kancherla, Yngve Guttormsen, Rodrigo Berzaghi, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7065428/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Cancer-associated fibroblasts (CAFs) are influential elements of the tumor microenvironment with significant roles in tumor progression and therapy resistance. However, if and how CAF-mediated responses to radiotherapy (RT) affects clinical outcomes remains undetermined. Here, we aimed to investigate impact of RT on CAFs using antigen-specific, non-invasive, molecular PET-imaging. The FAP-specific radiotracer [ 18 F]AlF-FAPI-74 was applied to monitor CAF dynamics following external beam RT in two syngeneic subcutaneous murine tumor models (LLC and CT26). Tumors were irradiated using two radiation regimens (1x12 Gy or 2x6 Gy), and PET/MR imaging was performed 7 days post-RT. Additionally, dynamics of FAP + CAFs in tumors was quantified ex vivo using flow cytometry and immunohistochemistry. Results Biodistribution studies of [ 18 F]AlF-FAPI-74 showed radiotracer signal in joint/bone structures and intestines in both mouse strains. Tumor-targeted irradiation led to significant reduction in tumor size. Uptake of [ 18 F]AlF-FAPI-74 in subcutaneous tumors was low but significantly above muscle-background values. Quantification of standardized uptake values (SUV) from static PET-images revealed two-fold increased PET signal in LLC tumors irradiated with 2x6 Gy. Ex vivo analysis confirmed low abundance of FAP + cells in tumors and demonstrated similar RT-induced changes in CAFs across the different models. Conclusions Our findings suggest that CAFs represent a relatively sparse cell population in subcutaneously transplanted tumor models, and that radiotherapy may induce a moderate increase in FAP + cells in LLC tumors. Additionally, we demonstrate that [ 18 F]AlF-FAPI-74 is a reliable biomarker for evaluating levels of FAP + stromal cells in tumors and for addressing potential therapy-induced changes in CAFs. FAP-1 cancer-associated fibroblasts tumor microenvironment radiotherapy ionizing radiation molecular imaging FAPI PET/MRI PET imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Background Among all the stromal cells that reside in the tumor microenvironment (TME), cancer-associated fibroblasts (CAFs) are one of the most abundant and critical components, providing not only physical support for tumor cells, but also playing a key role in promoting or restraining tumorigenesis in a context dependent manner [ 1 ]. The presence of CAFs in the TME is frequently correlated with increased angiogenesis, invasion and metastasis, and thus associated with worse prognosis in a wide variety of solid malignancies [ 2 ]. Besides, CAFs are recognized mediators of immunosuppression in the TME [ 3 , 4 ]. Of note, recent reports highlight the participation of CAFs in therapy resistance [ 5 , 6 ]. In the context of radiotherapy (RT), the ultimate role of CAFs in therapeutic outcomes remain unresolved [ 7 ]. While some studies claim that ionizing radiation have detrimental effects on CAFs by inducing growth arrest and impaired motility [ 8 , 9 ], others argue that exposing fibroblasts to radiation promotes their conversion into a more activated and aggressive phenotype [ 10 ]. Hence, further research is needed to increase our understanding of CAF responses to ionizing radiation, and to elucidate the potential role that CAFs may play in tumor radio-resistance. Given its important role in cancer progression and therapy resistance, the tumor stroma represents an attractive target for delivering diagnostic and therapeutic compounds [ 11 ]. Several approaches have been applied to target CAFs with novel radiolabeled probes based on antibodies, peptides and small molecule inhibitors in different cancer types [ 12 ]. Currently, some of the most practiced strategies are represented by radiotracers targeting fibroblast activation protein (FAP) [ 13 ]. FAP is a membrane bound proline-specific serine protease with dipeptidyl peptidase and endopeptidase activities [ 14 ], known to degrade denatured type-I collagen, alpha-2 antiplasmin and FGF21 in vivo [ 15 ]. High FAP expression is associated with pathologic remodeling of the extracellular matrix, a process that is inherent to the development of solid malignancies [ 16 ]. Reactive stromal fibroblasts, i.e. CAFs in solid tumors, are featured by abundant surface expression of FAP, and their presence is frequently associated with bad prognosis, whereas low or no expression is observed on normal fibroblasts in healthy tissues in humans [ 14 ]. However, FAP can also be expressed on stromal fibroblasts during non-malignant processes, such as tissue remodeling, fibrosis, wound healing and inflammation [ 17 ]. Development of the selective FAP inhibitor UAMC-1110 has led to promising radiolabeled FAP inhibitors (FAPIs) that have been tested in different tumor entities [ 18 , 19 ]. Quinoline-based FAP inhibitors specifically bind to the enzymatic domain of FAP prior to cellular internalization. Different methods for conjugation of quinoline-based FAP ligands with chelators suitable for radiolabeling have been developed [ 18 , 20 ]. In this study, we are using a FAPI-74 variant that includes a NOTA chelator. Other FAPI based radiotracers have been successfully used as tumor-specific imaging biomarkers in preclinical and clinical models [ 21 – 23 ]. Here, we investigate the impact of radiotherapy on CAFs in vivo , using PET/MR imaging of a FAP-targeting radiotracer in two different preclinical tumor models. Results indicate low but significant tumor uptake of FAPI-74, and some background PET-signal in joints. Fractionated medium-dose radiotherapy is inducing two-fold enhanced tracer-uptake, visible as hyperintense tumor-specific PET signals. 2 Materials and methods Cell cultures Murine cell lines of Lewis lung carcinoma (LLC) expressing luciferase (LL/2-Luc2) and colon carcinoma (CT26) were purchased from ATCC (Virginia, USA; Cat # CRL-1642-LUC2 and # CRL-2638). LLC cells were cultured in RPMI high glucose (Sigma Life Science; Cat #D5796) supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin and 2 µg/mL blasticidine, whereas CT26 cells were cultured in RPMI-1640 (Sigma Life Science; Cat # R8758) supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. In vivo models Female C57BL/6J and BALB/cJ mice (age 6–8 weeks, 21.1 ± 4.5 g, Charles River Sulzfeld, Germany), were acclimatized in the local animal facility for minimum five days prior to experimentation. Animals had access to water and standard chow (Scanbur, BK, Norway) ad libitum . All procedures and experiments involving animals were conducted according to regulations by the Federation of European Laboratory Animal Science Association (FELASA) and the Norwegian law FOR-2017-04-05-451 and approved by the Norwegian Food and Safety Authority (Project FOTS ID 18956 and 25795). The LL/2-Luc (luciferase-expressing Lewis lung adenocarcinoma-LLC), which is syngeneic to C56Bl6J mice, and the CT26 (colon adenocarcinoma), which is syngeneic to Balb/c mice were used in this study. Here, the LL/2-luc cell line is referred to as LLC (Lewis Lung Carcinoma) for simplification. Prior to inoculation, all cell lines were tested and proven pathogen free by Idexx Bioanalytics (Mice Comprehensive test). For inoculation, cells were prepared in sterile RPMI and Geltrex™ (Gibco, Cat # A1413201) at 1:1 ratio. For tumor-cell inoculation, 100 µL cell suspension (5x10 5 cells) were injected subcutaneously into the right hind flank of mice under anesthesia. During growth, tumors were measured at least three times per week using a digital caliper, and volumes calculated using the modified ellipsoidal formula ( \(\:V=\frac{1}{2}(length\times\:{width}^{2})\) ). Animal tumor irradiation Animals were subjected to image-guided radiotherapy in a dedicated small-animal irradiator (Precision X-ray, North Branford) when tumors had reached 5–6 mm in diameter (8–10 days upon cell inoculation). Animals were anesthetized prior to CT imaging and RT-delivery by continuous isoflurane gas (0.5 L/min oxygen with 4% isoflurane) in induction chambers. Anesthesia was maintained throughout the full procedure by continuous isoflurane gas via nose cone (0.4 L/min oxygen with 2.0% isoflurane). Anatomical CT images were acquired for each tumor and imported into the SmART-plan advanced treatment planning system (Precision X-ray, North Branfor) for tumor delineation, pretreatment dose-calculations and plan evaluations, as previously described [ 24 ]. Next, RT was delivered to tumors by the treatment plan, using two opposing photon beams, with maximum energy 225 kV, dose-rate 3.1 Gy/min, and collimator-size Ø=10 mm. Radiation-regimens included one single-high dose of 12 Gy (1x12 Gy), or two intermediate doses of 6 Gy (2x6 Gy) delivered 24 hrs apart. Radiosynthesis of [ 18 F]AlF-FAPI-74 Chemicals for radiotracer production were purchased from VWR (Oslo, Norway), unless otherwise stated. Fluoride ( 18 F-) batches (~ 10 GBq) were locally produced via the nuclear reaction 18O(p,n) 18 F in the “PETtrace 860” medical cyclotron (GE Healthcare, Uppsala, Sweden), using a proton beam (16.5 MeV) to bombard a Niobium target prefilled with [ 18 O]H 2 O (Rotem Industries, Israel). Radiosynthesis of [ 18 F]AlF-FAPI-74 was performed following the procedure by Dahl [ 25 ] and Giesel [ 21 ] et al, with some modifications. Briefly, the [ 18 F]AlF moiety was inserted to the FAPI-74-NOTA precursor (SOFIE Biosciences, VA, US) in a Tracerlab FX2N automatic fixed-tube module as follow: 10 GBq of [18F-] ion were trapped on an anion exchange cartridge (Waters Accel Plus QMA Light cartridge) (Waters, Oslo, Norway) preconditioned with NaOAc (5 mL, 0.5 M, pH 3.9) and rinsed with diH2O. The fluoride [18F-] ion was then eluted with a 3:4 (v/v) mixture of NaOAc (0.5M, pH 3.9) and DMSO. The solution was further incubated in the secondary reactor vessel with the precursor FAPI-74 (60 µg in 20 µL diH2O9), together with AlCl3 (10mM)) and sodium ascorbate (44.16 mM in 0.5 M NaOAc, pH 3.9)). This mixture was allowed to react (95°C, 15 min), then cooled down (to 20°C) and diluted with sodium ascorbate in 0.5M phosphate buffer, pH 8.19. Final purification was performed by solid-phase extraction in an Oasis HLB Plus Light cartridge (Waters, Oslo, Norway). Final product was eluted with ethanol and formulated in sodium ascorbate (50.8 mM, 7 mL) spiked with phosphate buffer to a final pH of 7.4. Simplified quality control of the formulated product was performed by high-pressure liquid chromatography (HPLC) coupled with photodiode array (PDA) detection set to detect at λ = 254 nm (Agilent Technologies, Waldbronn, Germany) and a FlowRam radioflow (RAD) detector (Lablogic, Sheffield, UK) in series. Analysis of the formulated product [ 18 F]AlF-FAPI-74, the non-radioactive standard [ 19 F]AlF-FAPI-74 and the precursor FAPI-74 were performed on an X-Bridge BEH C18 (4.6X150 mm, 3.5 µm) (Waters, Oslo, Norway) analytical column by gradient elution with a mobile phase (0.1% NH3 and 95% acetonitrile). during 30 min, using a Flowrate of 1 mL/min. In vitro and in vivo [ 18 F]AlF-FAPI-74 metabolic stability tests The in vitro metabolic stability of [ 18 F]FAPI-74 was first studied in mouse plasma obtained by full-blood centrifugation (2500 rpm, 5 min, 4°C). Next, plasma was incubated with ~ 10 MBq of [ 18 F]AlF-FAPI-74 in a shaking water bath (37°C) with circulation, and samples (100 µL) collected 5, 15, 30, 45, 60 and 120 min after adding the radiotracer, followed by inclusion of 100 µL ice-cold acetonitrile per sample to deproteinize the plasma. Upon a reaction-time for indicated timepoints, samples were subjected to vortex mixing followed by ultrasound bath ( 5 min) and final centrifugation (4°C, 10 min, 14500 rpm). A volume of 100 uL from the clear supernatants were analyzed by HPLC, following the procedure described above. For checking the in vivo stability of [ 18 F]AlF-FAPI-74, sample preparations were carried out in a similar manner as for the in vitro stability study, except that animals (n = 3 per timepoint per strain) were injected i.v. with 100 ± 20 MBq [ 18 F]AlF-FAPI-74 (in 50 µL) through a tail-vein catheter, blood collected by cardiac puncture (1 mL) at different time points (15, 30 and 60 min) post-injection, with further sample processing as described for the in vitro stability test. In vivo imaging procedures of whole-body PET and MRI Animals were anesthetized with isoflurane (4% induction in O 2 ) and subjected to simultaneous whole-body PET and MRI (MR Solutions 7.0T PET/MR, Guildford, UK) one day prior to first dose of RT (baseline pre-RT FAPI values), and one-week post-RT. For PET/MR imaging,7 MBq (± 3 MBq) in 100 µL [ 18 F]AlF-FAPI-74 were injected via retro-orbital or tail-vein under anesthesia (Isoflurane, 0.4 L/min, 2% in O 2 ). Radioligand biodistribution was compared between the two routes of administration showing near identical biodistribution patterns (not shown). This observation has also been demonstrated by others [ 26 ]. Following tracer-injection, animals were returned to their cages and remained awake until imaging, 40 min post-injection. For imaging, animals were placed in a prone position in a multi-mouse bed (Mouse hotel, MINERVE, Esternay, France) holding two mice. To acquire static images, PET scans were performed for 20 min in all animals. The list-mode data were rebinned into 24x5s, 8x60s, 10x300s time frames and reconstructed into 3D datasets using OSEM (two iterations, 32 subsets) and DICOM data exported for further analysis. PET data analyses In vivo organ biodistribution was assessed and quantified on PET/MR images. In each animal, 3D organs were carefully delineated on T1-weighted MR images followed by PET image co-registration. The muscle VOI was defined around the whole thigh muscle of the mouse hindleg excluding the bone (hypointense/dark in MRI). Average activities (in kBq/mL) were extracted from all organ volume-of-interest (VOIs) and converted into standardized uptake values (SUV) or percentage injected dose per mL (%ID/mL), using the decay-value corrected for total injected dose. This value is equivalent to percentage injected dose per gram (%ID/g) and calculated for in vivo organ biodistribution [ 17 ]. Tumor volumes were segmented using PMOD (v4.3, PMOD Technologies LLC, Switzerland) based on T1 weighed MRI, and SUV or %ID/g for the VOIs were obtained. For tumors, regions with higher accumulation of [ 18 F]AlF-FAPI-74 were defined on the PET data by applying a threshold of 75% of the maximum VOI value to obtain the tumor-biological-active-volume (TBV75). BTV75 was defined by retaining the tumor sub-volume comprising the top 25% of PET signal intensities, i.e., all voxels within the tumor VOI with uptake ≥ 75% of the maximum SUV. This region was used to highlight areas of highest FAPI tracer accumulation and to evaluate radiotherapy-induced changes in biologically active tumor regions. The VOI of muscle in the contralateral leg from the tumor was used to obtain SUV values of the physiological perfusion of [ 18 F]AlF-FAPI-74, to calculate tumor-to-background ratio. Quantitative data are presented as fold-change uptake in tumor relative to contralateral muscle. Ex vivo tumor analyses of CAFs For immunohistochemistry evaluations, tumors were excised 7d post-RT, fixed in paraformaldehyde (4% in saline buffer) immediately after resection and embedded in paraffin blocks. The Discovery Ultra Research instrument (Roche 05987750001) was used for automated preparation and immunohistochemical staining of tumor tissue sections (4 µm thick). Anti-mouse αSMA antibody (D4K9N, Cell Signaling) for identifying and quantifying levels of CAFs in tumors was used at 1:100 dilution for staining. The antibody was validated for IHC-P (formalin fixed and paraffin-embedded murine tissue) by the supplier. Optimization of dilutions, incubation times, antigen retrieval and temperatures were performed in-house (Supplementary figure ). Staining and antibody specificity was verified by an internal tissue control containing several normal and cancer tissues. Negative controls were conducted by omitting the primary antibody. Positive staining on specimens were quantified digitally using the QuPath software (Version v.0.5.1, tool: Positive Cell Detection). In addition, presence of FAP + cells within irradiated tumors was compared to non-irradiated tumors by flow cytometry. For this purpose, tumors were collected 7 days post-radiotherapy, minced and enzymatically digested. The resulting cell suspensions were stained with viability dye and murine anti-FAP antibody (Bioss Antibodies, Cat# bs 5758-A680). Data was obtained by flow cytometry from cells gated according to their scatter properties (FSC-A vs SSC-A) and doublet exclusion (FSC-A vs FSC-H). Cell debris were excluded from the analyses based on scatter signals. Data acquired by flow cytometry were analyzed by FlowJo software (Version v.10.10; TreeStar, OR, USA). Statistical analyses All values are expressed as the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism (GraphPad Software Inc., La Jolla, CA). Comparison of data with three or more experimental groups were conducted using one-way ANOVA followed by Dunnett post hoc corrections for multiple comparisons. Comparison of data with two groups were conducted using unpaired two-tailed student’s t-test. Level of significance was defined as p ≤ 0.05. 3 Results [ 18 F]AlF-FAPI-74 radiosynthesis & in vivo/in vitro stability tests FAPI-74 was efficiently radiolabeled by [ 18 F]AlF insertion onto the NOTA chelator (Fig. 1 a), which resulted in a radiochemical yield of 14.8 ± 2.4% (n = 8) and > 99% purity. The radiolabeled compound eluted in a single HPLC peak in the radio-analysis at a retention time of 12 min (Fig. 1 b) and demonstrated high molar activities (213 ± 44 GBq/µmol). Evaluation of metabolic stability of [ 18 F]AlF-FAPI-74 in vitro , in mouse plasma, showed full stability up to 120 min n (Fig. 1 c). In vivo stability of the radiotracer was assessed in healthy female BALB/cJ and C57BL/6J mice and showed full stability up to 60 min (Fig. 1 d&e, respectively), allowing flexibility in the timing of image acquisition, as the radiotracer remains unchanged over a longer period. The in vitro and in vivo stability tests revealed that the radiolabeled compound, upon exposure to blood plasma, still eluted in one single peak upon a retention time of 12 min. Dynamic PET scans & organ biodistribution in healthy animals Organ biodistribution in vivo was assessed by dynamic PET/MR image analysis and quantitative average tracer uptake expressed in kBq/mL for each animal. The VOI data was subsequently used to calculate percentage injected dose pr mL (%ID/mL) using the decay corrected total injected dose. Interestingly, organ uptake (Fig. 2 A) in C57BL/6J mice was calculated to be slightly higher in brain, spleen, kidneys, urimary bladder and muscle than in the albino BALB/cJ mice, based on PET dynamic data (Fig S1 ). Radiotracer-uptake in urinary bladder demonstrated the largest difference between these two strains, but with large standard deviations between animals (coefficient of variance 129.7% or 138.4%, BALB/cJ or C57BL/6J (Fig S2 ). Furthermore, the radiotracer showed quick clearance from the organs through the kidneys, resulting in major uptake in the urinary bladder (Fig. 2 a & b). [ 18 F]AlF-FAPI-74 uptake in tumor lesions To assess tumor-associated [ 18 F]AlF-FAPI-74 uptake, tumor volumes were carefully delineated in a series of MR images, and co-registered with the corresponding PET images (Fig. 3 a & d). The injected dose was decay corrected to the starting time for PET scanning, and the SUV (Fig. 3 b-c & e-f), the SUV max/mean (Fig S3 &S4) and the %ID/ml (Fig S5 ) was calculated. To compensate for physiological tissue perfusion, a VOI in the contralateral leg muscle was delineated and used as reference region in each animal, calculating the ratio tumor-to-muscle. Furthermore, tumor-to-blood ratios were calculated for both models to observe the blood clearance and the tumor-to-lung ratio for the LLC model (Fig. 3 c & d). In general, tumor uptake was low and heterogenous in both the LLC and the CT26 models, but with values higher than muscle background the CT26 tumor model (Fig. 3 e). Of note, highest uptake of [ 18 F]AlF-FAPI-74 was observed in the peripheral tumor regions, decreasing towards the central portion of the tumor (Fig. 3 a & d). Among the two animal models, the CT26 model presented the highest tumor-to-muscle-ratio based on %ID/ml (2.4 ± 0.1, 39 min p.i. vs 1.5 ± 1.2, 39 min p.i. ). Contrary, based on SUV the LLC-model presented the highest SUV (mean: 0.59 ± 0.02 max: 1.05 ± 0.05 vs mean 0.36 ± 0.01 max: 1.47 ± 0.03, 40–60 min p.i.) and differentiation between tumor and muscle (mean 0.29 ± 0.02 max: 0.73 ± 0.03 vs mean 0.23 ± 0.01 max: 0.63 ± 0.02, 40–60 min p.i.) resulting in a tumor-to-muscle ratios (40–60 min p.i.) of 2.4 (± 0.04) or 1.5 (± 0.03), tumor-to-blood ratios of 1.2 (± 0.04) or 1.0 (± 0.14) for LLC and CT26 tumors, respectively. The tumor-to-lung ratios (40–60 min p.i.) were calculated for the LLC model to be 2.2 (± 0.09). Radiotherapy effects on tumor growth kinetics Effects of radiotherapy on tumor growth was analyzed in the two different tumor models. Tumor irradiation was performed during exponential growth, once tumors had reached 5–6 mm in diameter. Both tumor models exhibited similar tumor growth kinetics for non-irradiated groups, however, CT26 tumors displayed more variation in growth compared to LLC tumors, as indicated by the larger SD (Fig. 4 b&d). In the LLC model, both radiation regimens (2x6 Gy and 1x12 Gy) induced significant tumor growth delays, although effects were more pronounced in the 1x12 Gy group (Fig. 4 a&b). In the CT26 model, both radiation regimens induced potent and durable growth delay in a comparable way (Fig. 4 c&d), with some animals of the latter group displaying complete tumor regression. Radiotherapy-induced changes in [ 18 F]AlF-FAPI-74 tumor uptake When comparing tumor uptake of [ 18 F]AlF-FAPI-74 in untreated animals, LLC tumors displayed the highest tumor uptake, given in fold-change of muscle signal (Fig. 5 b-d). In this model, the fractionated radiation regimen (2x6Gy) enhanced the overall SUV r two-fold reaching statistically significant differences (p = 0.04) compared to the non-irradiated controls. However, only minor variations in the PET-signal were observed when comparing untreated animals with the single-high dose (1x12 Gy) treated group (p = 0.9) (Fig. 5 b). By using BTV75 as an alternative quantitative approach, the outcomes showed similar trends, with two-fold enhanced signal in the (2x6 Gy) group, also reaching statistical significance (p = 0.04). Similar effects of radiation were observed in the BALB/c CT26 model (Fig. 5 d) however in this model, differences between the untreated group and the fractionated radiation group (2x6Gy) did not reach statistical significance for both SUV mean (p = 0.079) and relative BTV75 values (p = 0.73) (Fig. 5 d). Interestingly, BTV75 values in the 1x12Gy radiation group were lower than in the untreated group (Fig. 5 d). In both models, the fractionated regimen of 2x6 Gy displayed the highest tumor specific BTV75 PET signal relative to muscle, although statistical significance was only reached in the LLC model. Detailed calculations on tumor uptake values from each individual animal are given in Table S1 . When looking at the spatial distribution of [ 18 F]AlF-FAPI-74 in tumors, heterogeneous patterns were observed. The highest PET-signal was typically observed in the periphery of tumors, with limited accumulation in the central areas (Fig. 5 a&c). This trend was noticed in both tumor models, and all treatment groups. Heterogenous tracer uptake was most evident in the larger non-irradiated tumors, where PET signal was clearly visible as a ring around the tumor, as seen in Fig. 5 a&c and in Fig S6 Ex vivo analyses To validate findings based on quantitative PET-imaging, the content of intra-tumoral CAFs in tumor tissue specimens was analyzed also by immunohistochemistry and flow cytometry. IHC analyzes revealed that LLC tumors displayed very poor stroma development and quite low CAF infiltration, as illustrated by the limited infiltration of αSMA + cells (brown signal in micrographs in Fig. 6 a) and negligible expression of extracellular matrix (analyzed by Masson´s trichrome, not shown). Computer-assisted quantification of αSMA expression showed comparable levels between untreated and 1x12Gy irradiated tumors (mean 2,74% and 3,34% positive cells from total amount of cells respectively), but significantly enhanced levels in the 2x6Gy group (mean 5,07% positive cells, p = 0.02) (Fig. 6 a-b). In the CT26 model, tumor irradiation did not affect levels of αSMA expression. Moreover, the percentage of FAP + cells from the total pull of viable cells was calculated by flow cytometry in both non-irradiated and irradiated tumors (Fig. 6 c). From the fraction of viable cells in non-irradiated LLC tumors in C57BL/6J mice, nearly 40% were FAP + . Notably, the fraction of FAP + cells increased to 55% in the 2x6 Gy irradiation group, reaching statistically significance, whereas the values in the 1x12 Gy group remained similar to the untreated animals (47%). Colon carcinoma CT26 tumors in BALB/cJ mice displayed lower proportion of FAP + cells in the viable population compared to the LLC lung tumors (approx. 20%), but no changes in FAP + cells were observed between the experimental groups. 4 Discussion In this study, the main aim was to explore effects of radiation on tumor fibroblasts, and to evaluate the use of [ 18 F]AlF-FAPI-74 with PET as biomarker to study dynamics of FAP + stromal cells in the context of cancer therapy. With the intention of applying a broad approach, we have reproduced results in two different syngeneic murine tumor models; the Lewis lung adenocarcinoma model LLC, and the colon carcinoma CT26. Regarding radiotherapy, we have used two different regimens; a single-high dose (1x12Gy) and a fractionated medium-dose regimen (2x6Gy). The results indicate that focused external beam radiotherapy, especially when given by fractionated (medium-high) doses, may induce a moderate elevation in intratumoral FAP + cells. Moreover, this radiation-induced FAP + elevation is more prominent in the LL/2-luc/C57Bl6 model than in the CT26/BALB/c colon carcinoma model. Additionally, we demonstrate that FAPI-74 is a reliable biomarker to evaluate the levels of FAP + stromal cells in tumors and to address potential therapy-induced changes in CAFs. In this work, we have used the FAPI-74 variant for PET imaging. Our choice was based on several practical and technical advantages that FAPI-74 offers over other FAPI variants, particularly in clinical and research settings. The FAPI-74 is compatible with both 68 Ga and 18 F labeling, offering flexibility in tracer production. The 18 F-labeled FAPI-74 variant, in particular, benefits from a longer half-life (110 minutes vs. 68 minutes) and thus better clinical applicability. Also, [ 18 F]AlF-FAPI-74 has demonstrated lower positron energy compared to 68 Ga-labeled variants, resulting in better spatial resolution and image quality [ 27 ]. Recent studies have shown that 18 F-FAPI-74 PET/CT provides high diagnostic accuracy, particularly in detecting metastatic lymph nodes and small lesions, which may be underestimated with other tracers due to partial volume effects. Its high tumor-to-background ratio and rapid clearance from non-target tissues make it suitable for a wide range of oncologic applications [ 28 ]. [ 18 F]AlF-FAPI-74 was synthesized with a radioactivity yield of 15% and a radiochemical purity of > 99%. In vitro (plasma) and in vivo stability tests confirmed good isotope retention in the chelator and no metabolization of the compound within the experimental timeframe (~ 1 h). These results support the notion that the PET-signal from static scans acquired 1h after [ 18 F]AlF-FAPI-74 injection corresponds to images from the intact tracer. Next, we explored the pharmacokinetics and organ biodistribution of the tracer in healthy animals. As observed in other preclinical studies with FAPI compounds [ 23 ], [ 18 F]AlF-FAPI-74 was cleared rapidly by the urinary system; displayed low uptake in major organs such as liver, lung, spleen and brain, but accumulated to some degree in the intestines and joints. Accumulation of [18F]AlF-FAPI-74 in the intestines was more evident in C57Bl6 animals than in Balb-c animals, whereas accumulation in joints and/or bone structures was evident in both strains. This phenomenon has also been noticed in previous preclinical studies using [ 18 F]FGlc-FAPI [ 29 ] or Al 18 F-NOTA-FAPI [ 23 ]. In our study, we have used young animals that are skeletally immature (10–12 weeks old). Considering that FAP can be overexpressed in locations with active tissue remodeling, it is plausible that high FAPI uptake in bony structures reflects ongoing bone formation in young animals. Notably, relatively high FAP expression in murine osteoblasts and bone marrow stromal cells have been observed by others [ 15 ]. Hence, accumulation of [ 18 F]AlF-FAPI-74 in the murine skeleton could be assigned to physiological uptake. Of note, a recent study demonstrated that circulating soluble FAP (sFAP) in both mice and humans can bind to FAPI radiotracers, potentially altering their biodistribution [ 30 ]. This binding can reduce the amount of free tracer available to bind to membrane-bound FAP in tumors, thereby lowering tumor uptake and increasing background signal, especially in blood-rich organs. The presence of sFAP can prolong the circulation time of the radiotracer, leading to slower clearance and higher blood pool activity. This can complicate image interpretation and reduce the tumor-to-background contrast, which is critical for accurate diagnostics. In our experiments, the signal in blood-rich organs was low at the time that static scans were acquired, and therefore we estimate low impact of sFAP in our analysis. Several PET radiopharmaceuticals with common binding motifs against FAP (FAP inhibitors) have been tested in humans with promising results [ 31 ]. One of the first developed FAPI compounds, [ 68 Ga]Ga-FAPI-04, was successfully tested as imaging agent in 28 different cancers types in humans, displaying limited background signal and providing high image contrast of tumors [ 32 ]. Relevant for our study, the NOTA-chelator conjugated FAPI-74 was synthesized and first tested in humans by Giesel et al. [ 21 ]. High-contrast images of primary tumors, lymph nodes and distant metastasis were achieved, which could support target-volume-definition for guiding radiotherapy delivery. Importantly, no uptake exceeding the perfusion-dependent background was observed in major organs, including intestines and bone structures. As indicated earlier, differences in physiological expression of FAP between rodents and humans may stem from the fact that we are comparing organisms at different developmental stages. In tumor-bearing animals, [ 18 F]AlF-FAPI-74 uptake in the tumor region was slightly higher than uptake values in muscle, and most of the tracer accumulated in the periphery of the tumor. These data are consistent with a poorly developed stroma in subcutaneously transplanted tumors, characterized by nearly undetectable extracellular matrix deposition and low abundance of FAP-expressing fibroblasts [ 9 , 33 ]. Our results align well with observations in other preclinical models using tumor cells not genetically modified to overexpress FAP, where the only source of FAP arises from endogenously recruited levels of tumor-activated fibroblasts [ 19 ]. In a recent study by Liu M et al., authors investigate radiation-induced changes in FAPI tumor uptake using the [ 18 F]AlF-NOTA-FAPI-04 variant and using subcutaneously transplanted LLC cells as a model [ 34 ]. Similar to our study, authors observed weak FAPI-04 signal in tumors compared to FDG, but on the contrary, they observed a reduction in FAP expression and FAPI-04 signal in tumors after 1x15 Gy irradiation. In our study, enhanced FAP expression/signal is only observed after 2x6 Gy treatment, indicating that FAP responses to radiation may markedly depend on the radiation regimens used in the experiments. The effects of ionizing radiation on CAFs have been previously investigated in animal models, however, in most studies, tumorigenic effects of irradiated CAFs have been studied after co-implantation of (in vitro) irradiated CAFs along with tumor cells [ 9 , 35 – 37 ]. In fact, studies demonstrating direct effects of radiotherapy on CAFs in situ are very scant [ 38 , 39 ]. This study is one of the very first to investigate dynamics of tumor fibroblasts following radiotherapy treatment. Our data demonstrate low abundance of αSMA + CAFs in the stroma of subcutaneously transplanted LLC and CT26 tumors. Ex vivo analyses on resected tumors indicate that radiotherapy treatment may induce moderate elevation on the amount of CAFs (i.e. accumulation of FAP + CAFs) in tumors, but only when radiation is applied in a fractionated manner with medium-high radiation doses. In vivo image analyses using [ 18 F]AlF-FAPI-74 show similar trends, achieving statistically significant differences only in the (2x6Gy) RT group and only in the LLC/C57Bl6J model. In line with our observations in preclinical models, Verset et al. [ 40 ] observed higher αSMA/tumor epithelial area ratios after neoadjuvant radio(chemo)therapy in rectal cancer specimens from patients, indicating that radiotherapy, when applied in specific regimens, may enhance the number of CAFs in tumor lesions. An alternative explanation to this observation is that the surviving fraction of CAFs after treatment is higher than the tumor epithelial fraction, thus ending in elevated CAFs/tumor cells ratios, but without affecting CAF infiltration or proliferation in tumors. A clear limitation in our study is related to the use of subcutaneously transplanted tumor models, as these models do not recapitulate normal tumor-host tissue interactions similarly to endogenously formed tumors. In these models, the stroma is very little developed and consequently, the number of FAP-expressing cells, normally fibroblasts, is very low. This setback does not improve much by using orthotopically transplanted tumors since even after transplantation of tumor cells lines in the organ of origin, the high proliferative rate imped the formation of proper tumor stroma [ 41 ]. In preclinical settings, the majority of published studies on FAP-targeting PET radiotracers use genetically engineered tumor cells overexpressing FAP [ 42 , 43 ], which are artificial models that do not represent bona fide FAP expression in tumor lesions. Alternative animal models that more faithfully recapitulate the tumor tissue structure normally seen in humans are patient-derived xenografts, genetically engineered animals or environmentally induced models [ 33 ]. Moreover, we have imaged animals and analyzed tissue at one single time point, i.e. 1 week post-radiotherapy. Experiments were designed this way due to inherent limitations in tumor growth rates, which are quite fast when using LLC and CT26 tumor cell lines. Measurements performed at different time points and at longer incubation periods would have given a more accurate view of the CAF dynamics following treatment. Despite the indicated limitations, this study is one of the first demonstrating radiation-induced effects on CAFs i in vivo and demonstrates good performance of [ 18 F]FAPI-74 to study in vivo CAF dynamics in the context of therapy. Further work in more advanced and clinically relevant animal models is necessary to confirm the results presented in this study and to generate data that can be faithfully translated into clinical settings. Conclusions This study provides compelling preclinical evidence supporting the use of [ 18 F]AlF-FAPI-74 PET imaging as a non-invasive tool to monitor CAFs dynamics in response to radiotherapy. Our findings demonstrate that CAFs constitute a relatively sparse population in subcutaneously transplanted tumor models, yet their abundance can be moderately increased following fractionated radiotherapy, particularly in the LLC/C57BL6 model. This radiation-induced elevation in FAP + CAFs was consistently observed across multiple analytical platforms, including PET imaging, immunohistochemistry, and flow cytometry. Importantly, [ 18 F]AlF-FAPI-74 exhibited favorable radiochemical properties, high in vivo stability, and a biodistribution profile suitable for tumor imaging, with low background uptake in most organs and rapid renal clearance. The tracer's ability to detect subtle changes in FAP expression post-irradiation highlights its potential as a sensitive biomarker for assessing stromal responses to cancer therapy. Despite the limitations inherent to subcutaneous tumor models, our results underscore the feasibility of using FAPI-based PET imaging to study therapy-induced stromal remodeling. These findings pave the way for future investigations in more physiologically relevant tumor models and clinical settings, where CAF-targeted imaging could inform treatment planning, monitor therapeutic efficacy, and potentially guide CAF-directed interventions. Abbreviations CAFs: Cancer-associated fibroblasts FAP: Fibroblast activation protein TME: tumor microenvironment LLC: Lewis Lung Carcinoma RT: Radiation therapy FAPI: Fibroblast activation protein inhibitor SUV: Standard uptake values BTV: Biological tumor volume Declarations Ethics approval and consent to participate All animal experiments were approved by the Norwegian Food Safety Authority (FOTS ID 18956 and 27939). This study does not include human participants, human data or human tissue/cells. Consent for publication Not applicable Data availability declaration Datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interest Authors declare no competing interests. FAPI-74 precursor was provided free of charge by SOFIE Biosciences without further influence on study design and data analysis. Funding KL, SK, YG were financed by the Trond Mohn and Tromsø Research Foundations; RB and TH by the Regional Health Authorities (Helse-Nord; grants # HNF 1373-17; 1423-18); The Norwegian Cancer Society and The Aakre Foundation at UiT. MK is financed by the Starting Grant of the Trond Mohn and Tromsø Research Foundations. Author contribution All authors contributed to the design of the study and in data procurement. KL and RB were responsible for in vivo experiments; YG, SK and AMA developed protocols for tracer radiosynthesis and performed stability tests; TH developed and conducted protocols for in vivo tumor irradiations; MK conducted protocols for whole animal imaging procedures and data analysis; KL and MK analyzed PET/MR images; IMZ was responsible for conception of the study, designed experiments and evaluated the results. Acknowledgements The staff from the PETcore facility and the animal housing facility AKM is acknowledged for their excellent work and daily follow-up of the animals. We are also deeply thankful to SOFIE Bioscience for providing the FAPI-74 precursor used in this study. Additionally, Michel Herranz and Lorenzo Ragazzi for aiding in animal handling, radiotracer injection and image acquisitions, and to Ana Paola Lombardi for her assistance in doing aSMA tissue staining and QuPath-assisted digital quantification. References Biffi, G. and D.A. 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Mori, Y., et al., FAPI PET: Fibroblast Activation Protein Inhibitor Use in Oncologic and Nononcologic Disease. Radiology, 2023. 306 (2): p. e220749. Kratochwil, C., et al., (68)Ga-FAPI PET/CT: Tracer Uptake in 28 Different Kinds of Cancer. J Nucl Med, 2019. 60 (6): p. 801-805. Gengenbacher, N., M. Singhal, and H.G. Augustin, Preclinical mouse solid tumour models: status quo, challenges and perspectives. Nat Rev Cancer, 2017. 17 (12): p. 751-765. Liu, M., et al., Properties of [(18)F]FAPI monitoring of acute radiation pneumonia versus [(18)F]FDG in mouse models. Ann Nucl Med, 2024. 38 (5): p. 360-368. Wang, Y., et al., Cancer-associated Fibroblasts Promote Irradiated Cancer Cell Recovery Through Autophagy. EBioMedicine, 2017. 17 : p. 45-56. Pereira, P.M.R., et al., iNOS Regulates the Therapeutic Response of Pancreatic Cancer Cells to Radiotherapy. Cancer Res, 2020. 80 (8): p. 1681-1692. Meng, J., et al., Targeting senescence-like fibroblasts radiosensitizes non-small cell lung cancer and reduces radiation-induced pulmonary fibrosis. JCI Insight, 2021. 6 (23). Tommelein, J., et al., Radiotherapy-Activated Cancer-Associated Fibroblasts Promote Tumor Progression through Paracrine IGF1R Activation. Cancer Res, 2018. 78 (3): p. 659-670. Garate-Soraluze, E., et al., 4-1BB agonist targeted to fibroblast activation protein alpha synergizes with radiotherapy to treat murine breast tumor models. J Immunother Cancer, 2025. 13 (2). Verset, L., et al., Impact of neoadjuvant therapy on cancer-associated fibroblasts in rectal cancer. Radiother Oncol, 2015. 116 (3): p. 449-54. Ding, F., et al., (68)Ga-FAPI-04 vs. (18)F-FDG in a longitudinal preclinical PET imaging of metastatic breast cancer. Eur J Nucl Med Mol Imaging, 2021. 49 (1): p. 290-300. Zhou, H., et al., Synthesis and preclinical evaluation of novel (18)F-labeled fibroblast activation protein tracers for positron emission tomography imaging of cancer-associated fibroblasts. Eur J Med Chem, 2024. 264 : p. 115993. Hu, K., et al., Radiosynthesis and Preclinical Evaluation of Bispecific PSMA/FAP Heterodimers for Tumor Imaging. Pharmaceuticals (Basel), 2022. 15 (3). Supplementary Files STable1SUVquantification.xlsx SFig1biodistributionSUV.pdf SFig2biodistributionSUVmax.pdf SFig3suplementLLCSUVmax.pdf Sfig4suplementCT26SUVmax.pdf SFig5TumormuscleuptakeinIDml.pdf SFig6.SupplementperipheraluptakeA4.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7065428","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":486274333,"identity":"cbb744d3-0055-4a3f-b98c-c83b58a069a6","order_by":0,"name":"Kristin Lode","email":"","orcid":"","institution":"Oslo University Hospital: Oslo Universitetssykehus","correspondingAuthor":false,"prefix":"","firstName":"Kristin","middleName":"","lastName":"Lode","suffix":""},{"id":486274334,"identity":"35f8f1ca-6a75-412b-847a-903c1ece25ba","order_by":1,"name":"Sindhu Kancherla","email":"","orcid":"","institution":"UiT The Arctic University of Norway: UiT Norges arktiske universitet","correspondingAuthor":false,"prefix":"","firstName":"Sindhu","middleName":"","lastName":"Kancherla","suffix":""},{"id":486274335,"identity":"cbba8daa-33f1-4f87-9caa-0be4b385c16c","order_by":2,"name":"Yngve Guttormsen","email":"","orcid":"","institution":"UiT The Arctic University of Norway: UiT Norges arktiske universitet","correspondingAuthor":false,"prefix":"","firstName":"Yngve","middleName":"","lastName":"Guttormsen","suffix":""},{"id":486274336,"identity":"6e61437f-e940-402a-9378-74ed390a19c2","order_by":3,"name":"Rodrigo Berzaghi","email":"","orcid":"","institution":"UiT The Arctic University of Norway: UiT Norges arktiske universitet","correspondingAuthor":false,"prefix":"","firstName":"Rodrigo","middleName":"","lastName":"Berzaghi","suffix":""},{"id":486274337,"identity":"095c0c10-d9d7-4826-931d-4bc8f31ea123","order_by":4,"name":"Vera Susana Maia","email":"","orcid":"","institution":"UiT The Arctic University of Norway: UiT Norges arktiske universitet","correspondingAuthor":false,"prefix":"","firstName":"Vera","middleName":"Susana","lastName":"Maia","suffix":""},{"id":486274338,"identity":"940c76ad-b1c0-48b4-8e4f-64024d7f52b9","order_by":5,"name":"Angel Moldes-Anaya","email":"","orcid":"","institution":"UNN Tromsø: Universitetssykehuset Nord-Norge HF","correspondingAuthor":false,"prefix":"","firstName":"Angel","middleName":"","lastName":"Moldes-Anaya","suffix":""},{"id":486274339,"identity":"043321c1-55c5-490d-9807-45af5c5855bc","order_by":6,"name":"Turid Hellevik","email":"","orcid":"","institution":"UNN Tromsø: Universitetssykehuset Nord-Norge HF","correspondingAuthor":false,"prefix":"","firstName":"Turid","middleName":"","lastName":"Hellevik","suffix":""},{"id":486274340,"identity":"eb8ec828-5437-4f4c-917f-d89f0839bf04","order_by":7,"name":"Mathias Kranz","email":"","orcid":"","institution":"UiT The Arctic University of Norway: UiT Norges arktiske universitet","correspondingAuthor":false,"prefix":"","firstName":"Mathias","middleName":"","lastName":"Kranz","suffix":""},{"id":486274341,"identity":"fe4bd702-2248-401a-9720-a61382fd4713","order_by":8,"name":"Inigo Martinez-Zubiaurre","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACNgbGBigTzLABihCthQ3MSGNgI6gHSTMIHGYgaA2fdHPziw8M2+TN5ze3Pfjw53xin3wD24MP+MyWOdhmOYPhtuGcY4zthjN4bie2sTGwG87Ap0Uisc2Yh+E24ww2xjZpHgmwFjZpHiK02IO1/DE4R5SW5sdALYlgLQwJB4izhXGGwe3kGWyJbZI9B5KN20AMfH6Rn5H++MOHitu2M5iPP5P48cdOdn7z4WMS+EIMbBGDAYoAPD3gBMwEjBwFo2AUjIIRDwDfoEQIR8x8BgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-5408-9088","institution":"UiT The Arctic University of Norway: UiT Norges arktiske universitet","correspondingAuthor":true,"prefix":"","firstName":"Inigo","middleName":"","lastName":"Martinez-Zubiaurre","suffix":""}],"badges":[],"createdAt":"2025-07-07 12:23:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7065428/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7065428/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87362468,"identity":"26cd4ac4-ef30-4810-9e11-ef5d87856443","added_by":"auto","created_at":"2025-07-23 05:55:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":335920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRadiosynthesis and stability of [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]AlF-FAPI-74. \u003c/strong\u003eA) Scheme of the [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 radiosynthesis performed in a semiautomatic GE Tracerlab FX2 N module; B) HPLC radiochromatogram of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 formulated product identifying the radiotracer as a single peak at a retention time of approx. 12 min. C) In vitro stability demonstrated as an HPLC radiochromatogram of plasma samples prepared after incubation of [18F]AlF-FAPI-74 with mice plasma at 37℃ during 60 min; D) In vivo stability in C57Bl6 (blue) and E) BALB/c (red) mice strains demonstrated as an HPLC radiochromatogram of plasma samples prepared from blood collected 60 min post-injection.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig1radiochem.png","url":"https://assets-eu.researchsquare.com/files/rs-7065428/v1/0eb3f950fca73d58bc0a3c76.png"},{"id":87362479,"identity":"313c520a-e689-4a13-8af0-087b2334aa02","added_by":"auto","created_at":"2025-07-23 05:55:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3147932,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDynamic uptake and organ biodistribution following i.v. injection of [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]AIF-FAPI-74\u0026nbsp; in healthy\u0026nbsp; mice\u003c/strong\u003e. A) Dynamic PET biodistribution of \u003cstrong\u003e[\u003c/strong\u003e\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 in BALB/c and C57BL/6 mice over a 60-minute time course post-intravenous injection. Time-activity curves (% injected dose per milliliter, %ID/mL) are shown for major organs including the brain, lungs, liver, spleen, kidneys, intestines, stomach, urinary bladder, muscle, and blood. Each panel compares tracer kinetics between the two mouse strains, revealing similar biodistribution patterns. Notably, renal elimination is evidenced by high tracer accumulation in the kidneys and urinary bladder, with a steep increase over time particularly in C57BL/6 mice. Hepatic uptake is also prominent, with relatively sustained liver retention observed in both strains. Low brain uptake is consistent with limited blood-brain barrier permeability of the tracer. Minor uptake in muscle and gastrointestinal organs indicates limited nonspecific retention in peripheral tissues. Blood clearance profiles suggest rapid systemic distribution and excretion. These findings provide insight into the tracer’s pharmacokinetics and organ-specific handling, underscoring the importance of strain selection in preclinical imaging studies. Data represent mean values from n = 4 animals per group. B) Exemplary time series (%ID/ml scale, 0-1, 9-10, 10-15, 25-30, 40-45 and 55-60 min p.i.) of one representative Balb/c (top panels) and C57BL6 (lower panels) mouse, showing similar biodistribution (n=4 per strain). Uptake in bone structures (skull, chest, backbone) was observed in both models.\u003c/p\u003e","description":"","filename":"Fig2biodistributionandPETIDml.png","url":"https://assets-eu.researchsquare.com/files/rs-7065428/v1/7c74785e1bca8042eb83d002.png"},{"id":87362469,"identity":"b4adfd0e-e52c-4531-9bc3-b6db6be5bf7a","added_by":"auto","created_at":"2025-07-23 05:55:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":473605,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDynamic uptake of [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]-AIF-FAPI-74 in subcutaneously implanted lung adenocarcinoma (LL/2-Luc2) and colon carcinoma (CT26) tumors.\u003c/strong\u003e Time-activity curves and tumor-to-background ratios of the radiotracer in syngeneic murine tumor models over a 60-minute dynamic PET imaging period. (A–C) Data from Lewis lung carcinoma (LLC) tumor-bearing C57BL/6 mice show radiotracer uptake in the tumor and muscle tissue (A), along with corresponding tumor-to-muscle, tumor-to-blood, and tumor-to-lung ratios (B–C), illustrating favorable tumor-to-background contrast over time. (D–F) Similarly, in CT26 colon carcinoma-bearing BALB/c mice, radiotracer uptake is shown in the tumor and muscle (D), with associated tumor-to-muscle and tumor-to-blood ratios plotted over time (E–F). Rapid tracer accumulation within tumors is observed for both models, with retention plateauing at later time points, indicating stable intratumoral localization. Tumor-to-organ ratios progressively increase, suggesting ongoing clearance from non-target tissues and improving imaging contrast.\u003c/p\u003e","description":"","filename":"Fig4FAPI.png","url":"https://assets-eu.researchsquare.com/files/rs-7065428/v1/0e24da80b704cfd0cff8bc0f.png"},{"id":87362477,"identity":"5e1cb010-2370-4125-8738-ee1bf15935dd","added_by":"auto","created_at":"2025-07-23 05:55:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3259940,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTumor growth responses to radiotherapy. \u003c/strong\u003eEffect of focused RT on tumor growth was evaluated over time. In the top panels, lines represent tumor growth from single individuals, whereas curves in bottom panels represent mean values for each experimental group (± SD). The black vertical dotted line in figures indicates timepoint for CT-guided RT to tumors, i.e. day 8 or 10 after tumor-cell injections in C57BL/6J and BALB/c mice, respectively.\u003c/p\u003e","description":"","filename":"Figure3FAPI.png","url":"https://assets-eu.researchsquare.com/files/rs-7065428/v1/ba7f9b8581763b3b894a9f2f.png"},{"id":87362478,"identity":"b02d31b8-4634-44f5-adef-ac01520ea4a2","added_by":"auto","created_at":"2025-07-23 05:55:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":909816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in tumor-specific [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]-AIF-FAPI-74 uptake following radiotherapy.\u003c/strong\u003e Tumor uptake of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 in irradiated and non-irradiated LLC (a and b) and CT26 (c and d) tumors, respectively. Tumor-specific uptake was quantified based on tumor delineations on MR images prior to PET co-registration, using tracer uptake in contralateral muscle as reference. Tumor-specific uptake was normalized to background signal in contralateral muscle. Values are average values of VOI of the whole tumor as SUV, whereas BTV75 represent the “tumor-biological-value-75” of the highest 75 percentile tracer accumulation in the tumor VOI. Bars represent mean values ± SD from four animals.\u003c/p\u003e","description":"","filename":"Figure5FAPI.png","url":"https://assets-eu.researchsquare.com/files/rs-7065428/v1/bddda761152bf42c0595186b.png"},{"id":87362480,"identity":"7685584b-61c7-4c57-bd7a-a00ff2c2506a","added_by":"auto","created_at":"2025-07-23 05:55:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4597922,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEx vivoanalyses of tumor-infiltrating CAFs by flow cytometry and immunohistochemistry.\u003c/strong\u003e A) Immunohistochemistry micrographs of LLC and CT26 tumors showing intratumoral aSMA+ CAFs (brown color) from tumor samples retrieved 1 week after completion of radiation treatment. Arrows indicate areas in the tumor periphery with enhance aSMA+ CAFs. B) Digitally quantified +aSMA expression on whole tumor tissue slides (n=6 per strain). C) \u0026nbsp;Flow cytometry analyses to quantify FAP+ cells in enzymatically digested fresh tumor tissue specimens retrieved 1 week after completion of radiation treatments. Bars represent mean values ± SD. Sham-irradiated tumors (non-irradiated) and tumors treated with 2x6Gy and 1x12 Gy were compared(n=6).\u003c/p\u003e","description":"","filename":"Figure6FAPI.png","url":"https://assets-eu.researchsquare.com/files/rs-7065428/v1/95b119d8f374e7b8331bc80b.png"},{"id":90991056,"identity":"01b51e06-b311-408f-a188-05391a6894d2","added_by":"auto","created_at":"2025-09-10 11:22:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20078112,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7065428/v1/2207a8b2-c6ff-4be2-a762-9d69859b8517.pdf"},{"id":87362470,"identity":"688e8b56-8237-477e-af7b-3d3deae22165","added_by":"auto","created_at":"2025-07-23 05:55:52","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":67004,"visible":true,"origin":"","legend":"","description":"","filename":"STable1SUVquantification.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7065428/v1/6a09b33915163af36fa43ad0.xlsx"},{"id":87362475,"identity":"658fba46-7f7b-46a3-89cc-b12f00777fda","added_by":"auto","created_at":"2025-07-23 05:55:52","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":275018,"visible":true,"origin":"","legend":"","description":"","filename":"SFig1biodistributionSUV.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7065428/v1/fe55224a78260dbc0168cc49.pdf"},{"id":87362472,"identity":"98984fff-8c3f-47a7-ae96-58b6d3abd546","added_by":"auto","created_at":"2025-07-23 05:55:52","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":92027,"visible":true,"origin":"","legend":"","description":"","filename":"SFig2biodistributionSUVmax.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7065428/v1/99b3c1821dde0db69b01b7e7.pdf"},{"id":87362473,"identity":"654ec5bc-fe6c-4007-a291-fcc6cc5e74a1","added_by":"auto","created_at":"2025-07-23 05:55:52","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":56676,"visible":true,"origin":"","legend":"","description":"","filename":"SFig3suplementLLCSUVmax.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7065428/v1/36095558e42997e3650b549c.pdf"},{"id":87362474,"identity":"69665f4e-74d3-4935-aaa9-df8dc6ac9a0b","added_by":"auto","created_at":"2025-07-23 05:55:52","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":45030,"visible":true,"origin":"","legend":"","description":"","filename":"Sfig4suplementCT26SUVmax.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7065428/v1/b82c8677fbb2a8cadae4cc86.pdf"},{"id":87362483,"identity":"bae9ade8-2b0a-4373-b0e0-a962b5326539","added_by":"auto","created_at":"2025-07-23 05:55:53","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":408669,"visible":true,"origin":"","legend":"","description":"","filename":"SFig5TumormuscleuptakeinIDml.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7065428/v1/151f91d90eb0d861a8df8cbb.pdf"},{"id":87362494,"identity":"a044846d-7d85-4af7-8db2-c2bd2f48a1f3","added_by":"auto","created_at":"2025-07-23 05:55:53","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1661800,"visible":true,"origin":"","legend":"","description":"","filename":"SFig6.SupplementperipheraluptakeA4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7065428/v1/ed2eb58c7d153019a9d55df5.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePreclinical evaluation of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 for PET imaging to study cancer-associated fibroblast responses to radiotherapy\u003c/p\u003e","fulltext":[{"header":"1 Background","content":"\u003cp\u003eAmong all the stromal cells that reside in the tumor microenvironment (TME), cancer-associated fibroblasts (CAFs) are one of the most abundant and critical components, providing not only physical support for tumor cells, but also playing a key role in promoting or restraining tumorigenesis in a context dependent manner [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The presence of CAFs in the TME is frequently correlated with increased angiogenesis, invasion and metastasis, and thus associated with worse prognosis in a wide variety of solid malignancies [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Besides, CAFs are recognized mediators of immunosuppression in the TME [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Of note, recent reports highlight the participation of CAFs in therapy resistance [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In the context of radiotherapy (RT), the ultimate role of CAFs in therapeutic outcomes remain unresolved [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. While some studies claim that ionizing radiation have detrimental effects on CAFs by inducing growth arrest and impaired motility [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], others argue that exposing fibroblasts to radiation promotes their conversion into a more activated and aggressive phenotype [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Hence, further research is needed to increase our understanding of CAF responses to ionizing radiation, and to elucidate the potential role that CAFs may play in tumor radio-resistance.\u003c/p\u003e\u003cp\u003eGiven its important role in cancer progression and therapy resistance, the tumor stroma represents an attractive target for delivering diagnostic and therapeutic compounds [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Several approaches have been applied to target CAFs with novel radiolabeled probes based on antibodies, peptides and small molecule inhibitors in different cancer types [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Currently, some of the most practiced strategies are represented by radiotracers targeting fibroblast activation protein (FAP) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. FAP is a membrane bound proline-specific serine protease with dipeptidyl peptidase and endopeptidase activities [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], known to degrade denatured type-I collagen, alpha-2 antiplasmin and FGF21 \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. High FAP expression is associated with pathologic remodeling of the extracellular matrix, a process that is inherent to the development of solid malignancies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Reactive stromal fibroblasts, i.e. CAFs in solid tumors, are featured by abundant surface expression of FAP, and their presence is frequently associated with bad prognosis, whereas low or no expression is observed on normal fibroblasts in healthy tissues in humans [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, FAP can also be expressed on stromal fibroblasts during non-malignant processes, such as tissue remodeling, fibrosis, wound healing and inflammation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDevelopment of the selective FAP inhibitor UAMC-1110 has led to promising radiolabeled FAP inhibitors (FAPIs) that have been tested in different tumor entities [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Quinoline-based FAP inhibitors specifically bind to the enzymatic domain of FAP prior to cellular internalization. Different methods for conjugation of quinoline-based FAP ligands with chelators suitable for radiolabeling have been developed [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, we are using a FAPI-74 variant that includes a NOTA chelator. Other FAPI based radiotracers have been successfully used as tumor-specific imaging biomarkers in preclinical and clinical models [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Here, we investigate the impact of radiotherapy on CAFs \u003cem\u003ein vivo\u003c/em\u003e, using PET/MR imaging of a FAP-targeting radiotracer in two different preclinical tumor models. Results indicate low but significant tumor uptake of FAPI-74, and some background PET-signal in joints. Fractionated medium-dose radiotherapy is inducing two-fold enhanced tracer-uptake, visible as hyperintense tumor-specific PET signals.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cp\u003eCell cultures\u003c/p\u003e\u003cp\u003eMurine cell lines of Lewis lung carcinoma (LLC) expressing luciferase (LL/2-Luc2) and colon carcinoma (CT26) were purchased from ATCC (Virginia, USA; Cat # CRL-1642-LUC2 and # CRL-2638). LLC cells were cultured in RPMI high glucose (Sigma Life Science; Cat #D5796) supplemented with 10% FBS, 100 U/mL penicillin, 100 \u0026micro;g/mL streptomycin and 2 \u0026micro;g/mL blasticidine, whereas CT26 cells were cultured in RPMI-1640 (Sigma Life Science; Cat # R8758) supplemented with 10% FBS, 100 U/mL penicillin and 100 \u0026micro;g/mL streptomycin.\u003c/p\u003e\u003cp\u003eIn vivo models\u003c/p\u003e\u003cp\u003eFemale C57BL/6J and BALB/cJ mice (age 6\u0026ndash;8 weeks, 21.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 g, Charles River Sulzfeld, Germany), were acclimatized in the local animal facility for minimum five days prior to experimentation. Animals had access to water and standard chow (Scanbur, BK, Norway) \u003cem\u003ead libitum\u003c/em\u003e. All procedures and experiments involving animals were conducted according to regulations by the Federation of European Laboratory Animal Science Association (FELASA) and the Norwegian law FOR-2017-04-05-451 and approved by the Norwegian Food and Safety Authority (Project FOTS ID 18956 and 25795). The LL/2-Luc (luciferase-expressing Lewis lung adenocarcinoma-LLC), which is syngeneic to C56Bl6J mice, and the CT26 (colon adenocarcinoma), which is syngeneic to Balb/c mice were used in this study. Here, the LL/2-luc cell line is referred to as LLC (Lewis Lung Carcinoma) for simplification. Prior to inoculation, all cell lines were tested and proven pathogen free by Idexx Bioanalytics (Mice Comprehensive test). For inoculation, cells were prepared in sterile RPMI and Geltrex\u0026trade; (Gibco, Cat # A1413201) at 1:1 ratio. For tumor-cell inoculation, 100 \u0026micro;L cell suspension (5x10\u003csup\u003e5\u003c/sup\u003e cells) were injected subcutaneously into the right hind flank of mice under anesthesia. During growth, tumors were measured at least three times per week using a digital caliper, and volumes calculated using the modified ellipsoidal formula (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:V=\\frac{1}{2}(length\\times\\:{width}^{2})\\)\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAnimal tumor irradiation\u003c/p\u003e\u003cp\u003eAnimals were subjected to image-guided radiotherapy in a dedicated small-animal irradiator (Precision X-ray, North Branford) when tumors had reached 5\u0026ndash;6 mm in diameter (8\u0026ndash;10 days upon cell inoculation). Animals were anesthetized prior to CT imaging and RT-delivery by continuous isoflurane gas (0.5 L/min oxygen with 4% isoflurane) in induction chambers. Anesthesia was maintained throughout the full procedure by continuous isoflurane gas via nose cone (0.4 L/min oxygen with 2.0% isoflurane).\u003c/p\u003e\u003cp\u003eAnatomical CT images were acquired for each tumor and imported into the SmART-plan advanced treatment planning system (Precision X-ray, North Branfor) for tumor delineation, pretreatment dose-calculations and plan evaluations, as previously described [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Next, RT was delivered to tumors by the treatment plan, using two opposing photon beams, with maximum energy 225 kV, dose-rate 3.1 Gy/min, and collimator-size \u0026Oslash;=10 mm. Radiation-regimens included one single-high dose of 12 Gy (1x12 Gy), or two intermediate doses of 6 Gy (2x6 Gy) delivered 24 hrs apart.\u003c/p\u003e\u003cp\u003eRadiosynthesis of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74\u003c/p\u003e\u003cp\u003eChemicals for radiotracer production were purchased from VWR (Oslo, Norway), unless otherwise stated. Fluoride (\u003csup\u003e18\u003c/sup\u003eF-) batches (~\u0026thinsp;10 GBq) were locally produced via the nuclear reaction 18O(p,n)\u003csup\u003e18\u003c/sup\u003eF in the \u0026ldquo;PETtrace 860\u0026rdquo; medical cyclotron (GE Healthcare, Uppsala, Sweden), using a proton beam (16.5 MeV) to bombard a Niobium target prefilled with [\u003csup\u003e18\u003c/sup\u003eO]H\u003csub\u003e2\u003c/sub\u003eO (Rotem Industries, Israel).\u003c/p\u003e\u003cp\u003eRadiosynthesis of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 was performed following the procedure by Dahl [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and Giesel [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] et al, with some modifications. Briefly, the [\u003csup\u003e18\u003c/sup\u003eF]AlF moiety was inserted to the FAPI-74-NOTA precursor (SOFIE Biosciences, VA, US) in a Tracerlab FX2N automatic fixed-tube module as follow: 10 GBq of [18F-] ion were trapped on an anion exchange cartridge (Waters Accel Plus QMA Light cartridge) (Waters, Oslo, Norway) preconditioned with NaOAc (5 mL, 0.5 M, pH 3.9) and rinsed with diH2O. The fluoride [18F-] ion was then eluted with a 3:4 (v/v) mixture of NaOAc (0.5M, pH 3.9) and DMSO. The solution was further incubated in the secondary reactor vessel with the precursor FAPI-74 (60 \u0026micro;g in 20 \u0026micro;L diH2O9), together with AlCl3 (10mM)) and sodium ascorbate (44.16 mM in 0.5 M NaOAc, pH 3.9)). This mixture was allowed to react (95\u0026deg;C, 15 min), then cooled down (to 20\u0026deg;C) and diluted with sodium ascorbate in 0.5M phosphate buffer, pH 8.19. Final purification was performed by solid-phase extraction in an Oasis HLB Plus Light cartridge (Waters, Oslo, Norway). Final product was eluted with ethanol and formulated in sodium ascorbate (50.8 mM, 7 mL) spiked with phosphate buffer to a final pH of 7.4.\u003c/p\u003e\u003cp\u003eSimplified quality control of the formulated product was performed by high-pressure liquid chromatography (HPLC) coupled with photodiode array (PDA) detection set to detect at λ\u0026thinsp;=\u0026thinsp;254 nm (Agilent Technologies, Waldbronn, Germany) and a FlowRam radioflow (RAD) detector (Lablogic, Sheffield, UK) in series. Analysis of the formulated product [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74, the non-radioactive standard [\u003csup\u003e19\u003c/sup\u003eF]AlF-FAPI-74 and the precursor FAPI-74 were performed on an X-Bridge BEH C18 (4.6X150 mm, 3.5 \u0026micro;m) (Waters, Oslo, Norway) analytical column by gradient elution with a mobile phase (0.1% NH3 and 95% acetonitrile). during 30 min, using a Flowrate of 1 mL/min.\u003c/p\u003e\u003cp\u003eIn vitro and in vivo [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 metabolic stability tests\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e metabolic stability of [\u003csup\u003e18\u003c/sup\u003eF]FAPI-74 was first studied in mouse plasma obtained by full-blood centrifugation (2500 rpm, 5 min, 4\u0026deg;C). Next, plasma was incubated with ~\u0026thinsp;10 MBq of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 in a shaking water bath (37\u0026deg;C) with circulation, and samples (100 \u0026micro;L) collected 5, 15, 30, 45, 60 and 120 min after adding the radiotracer, followed by inclusion of 100 \u0026micro;L ice-cold acetonitrile per sample to deproteinize the plasma. Upon a reaction-time for indicated timepoints, samples were subjected to vortex mixing followed by ultrasound bath ( 5 min) and final centrifugation (4\u0026deg;C, 10 min, 14500 rpm). A volume of 100 uL from the clear supernatants were analyzed by HPLC, following the procedure described above.\u003c/p\u003e\u003cp\u003eFor checking the \u003cem\u003ein vivo\u003c/em\u003e stability of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74, sample preparations were carried out in a similar manner as for the \u003cem\u003ein vitro\u003c/em\u003e stability study, except that animals (n\u0026thinsp;=\u0026thinsp;3 per timepoint per strain) were injected i.v. with 100\u0026thinsp;\u0026plusmn;\u0026thinsp;20 MBq [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 (in 50 \u0026micro;L) through a tail-vein catheter, blood collected by cardiac puncture (1 mL) at different time points (15, 30 and 60 min) post-injection, with further sample processing as described for the \u003cem\u003ein vitro\u003c/em\u003e stability test.\u003c/p\u003e\u003cp\u003eIn vivo imaging procedures of whole-body PET and MRI\u003c/p\u003e\u003cp\u003eAnimals were anesthetized with isoflurane (4% induction in O\u003csub\u003e2\u003c/sub\u003e) and subjected to simultaneous whole-body PET and MRI (MR Solutions 7.0T PET/MR, Guildford, UK) one day prior to first dose of RT (baseline pre-RT FAPI values), and one-week post-RT. For PET/MR imaging,7 MBq (\u0026plusmn;\u0026thinsp;3 MBq) in 100 \u0026micro;L [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 were injected via retro-orbital or tail-vein under anesthesia (Isoflurane, 0.4 L/min, 2% in O\u003csub\u003e2\u003c/sub\u003e). Radioligand biodistribution was compared between the two routes of administration showing near identical biodistribution patterns (not shown). This observation has also been demonstrated by others [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Following tracer-injection, animals were returned to their cages and remained awake until imaging, 40 min post-injection. For imaging, animals were placed in a prone position in a multi-mouse bed (Mouse hotel, MINERVE, Esternay, France) holding two mice. To acquire static images, PET scans were performed for 20 min in all animals. The list-mode data were rebinned into 24x5s, 8x60s, 10x300s time frames and reconstructed into 3D datasets using OSEM (two iterations, 32 subsets) and DICOM data exported for further analysis.\u003c/p\u003e\u003cp\u003ePET data analyses\u003c/p\u003e\u003cp\u003eIn vivo organ biodistribution was assessed and quantified on PET/MR images. In each animal, 3D organs were carefully delineated on T1-weighted MR images followed by PET image co-registration. The muscle VOI was defined around the whole thigh muscle of the mouse hindleg excluding the bone (hypointense/dark in MRI). Average activities (in kBq/mL) were extracted from all organ volume-of-interest (VOIs) and converted into standardized uptake values (SUV) or percentage injected dose per mL (%ID/mL), using the decay-value corrected for total injected dose. This value is equivalent to percentage injected dose per gram (%ID/g) and calculated for \u003cem\u003ein vivo\u003c/em\u003e organ biodistribution [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTumor volumes were segmented using PMOD (v4.3, PMOD Technologies LLC, Switzerland) based on T1 weighed MRI, and SUV or %ID/g for the VOIs were obtained. For tumors, regions with higher accumulation of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 were defined on the PET data by applying a threshold of 75% of the maximum VOI value to obtain the tumor-biological-active-volume (TBV75). BTV75 was defined by retaining the tumor sub-volume comprising the top 25% of PET signal intensities, i.e., all voxels within the tumor VOI with uptake\u0026thinsp;\u0026ge;\u0026thinsp;75% of the maximum SUV. This region was used to highlight areas of highest FAPI tracer accumulation and to evaluate radiotherapy-induced changes in biologically active tumor regions. The VOI of muscle in the contralateral leg from the tumor was used to obtain SUV values of the physiological perfusion of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74, to calculate tumor-to-background ratio. Quantitative data are presented as fold-change uptake in tumor relative to contralateral muscle.\u003c/p\u003e\u003cp\u003eEx vivo tumor analyses of CAFs\u003c/p\u003e\u003cp\u003eFor immunohistochemistry evaluations, tumors were excised 7d post-RT, fixed in paraformaldehyde (4% in saline buffer) immediately after resection and embedded in paraffin blocks. The Discovery Ultra Research instrument (Roche 05987750001) was used for automated preparation and immunohistochemical staining of tumor tissue sections (4 \u0026micro;m thick). Anti-mouse αSMA antibody (D4K9N, Cell Signaling) for identifying and quantifying levels of CAFs in tumors was used at 1:100 dilution for staining. The antibody was validated for IHC-P (formalin fixed and paraffin-embedded murine tissue) by the supplier. Optimization of dilutions, incubation times, antigen retrieval and temperatures were performed in-house (Supplementary figure ). Staining and antibody specificity was verified by an internal tissue control containing several normal and cancer tissues. Negative controls were conducted by omitting the primary antibody. Positive staining on specimens were quantified digitally using the QuPath software (Version v.0.5.1, tool: Positive Cell Detection).\u003c/p\u003e\u003cp\u003eIn addition, presence of FAP\u003csup\u003e+\u003c/sup\u003e cells within irradiated tumors was compared to non-irradiated tumors by flow cytometry. For this purpose, tumors were collected 7 days post-radiotherapy, minced and enzymatically digested. The resulting cell suspensions were stained with viability dye and murine anti-FAP antibody (Bioss Antibodies, Cat# bs 5758-A680). Data was obtained by flow cytometry from cells gated according to their scatter properties (FSC-A vs SSC-A) and doublet exclusion (FSC-A vs FSC-H). Cell debris were excluded from the analyses based on scatter signals. Data acquired by flow cytometry were analyzed by FlowJo software (Version v.10.10; TreeStar, OR, USA).\u003c/p\u003e\u003cp\u003eStatistical analyses\u003c/p\u003e\u003cp\u003eAll values are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed using GraphPad Prism (GraphPad Software Inc., La Jolla, CA). Comparison of data with three or more experimental groups were conducted using one-way ANOVA followed by Dunnett post hoc corrections for multiple comparisons. Comparison of data with two groups were conducted using unpaired two-tailed student\u0026rsquo;s t-test. Level of significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"3 Results","content":"\u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 radiosynthesis \u0026amp; \u003cem\u003ein vivo/in vitro\u003c/em\u003e stability tests\u003c/p\u003e\u003cp\u003eFAPI-74 was efficiently radiolabeled by [\u003csup\u003e18\u003c/sup\u003eF]AlF insertion onto the NOTA chelator (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), which resulted in a radiochemical yield of 14.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4% (n\u0026thinsp;=\u0026thinsp;8) and \u0026gt;\u0026thinsp;99% purity. The radiolabeled compound eluted in a single HPLC peak in the radio-analysis at a retention time of 12 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) and demonstrated high molar activities (213\u0026thinsp;\u0026plusmn;\u0026thinsp;44 GBq/\u0026micro;mol).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEvaluation of metabolic stability of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 \u003cem\u003ein vitro\u003c/em\u003e, in mouse plasma, showed full stability up to 120 min n (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In vivo stability of the radiotracer was assessed in healthy female BALB/cJ and C57BL/6J mice and showed full stability up to 60 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed\u0026amp;e, respectively), allowing flexibility in the timing of image acquisition, as the radiotracer remains unchanged over a longer period. The \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e stability tests revealed that the radiolabeled compound, upon exposure to blood plasma, still eluted in one single peak upon a retention time of 12 min.\u003c/p\u003e\u003cp\u003eDynamic PET scans \u0026amp; organ biodistribution in healthy animals\u003c/p\u003e\u003cp\u003eOrgan biodistribution \u003cem\u003ein vivo\u003c/em\u003e was assessed by dynamic PET/MR image analysis and quantitative average tracer uptake expressed in kBq/mL for each animal. The VOI data was subsequently used to calculate percentage injected dose pr mL (%ID/mL) using the decay corrected total injected dose. Interestingly, organ uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) in C57BL/6J mice was calculated to be slightly higher in brain, spleen, kidneys, urimary bladder and muscle than in the albino BALB/cJ mice, based on PET dynamic data (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Radiotracer-uptake in urinary bladder demonstrated the largest difference between these two strains, but with large standard deviations between animals (coefficient of variance 129.7% or 138.4%, BALB/cJ or C57BL/6J (Fig \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Furthermore, the radiotracer showed quick clearance from the organs through the kidneys, resulting in major uptake in the urinary bladder (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea \u0026amp; b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 uptake in tumor lesions\u003c/p\u003e\u003cp\u003eTo assess tumor-associated [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 uptake, tumor volumes were carefully delineated in a series of MR images, and co-registered with the corresponding PET images (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea \u0026amp; d). The injected dose was decay corrected to the starting time for PET scanning, and the SUV (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-c \u0026amp; e-f), the SUV max/mean (Fig \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u0026amp;S4) and the %ID/ml (Fig \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e) was calculated. To compensate for physiological tissue perfusion, a VOI in the contralateral leg muscle was delineated and used as reference region in each animal, calculating the ratio tumor-to-muscle. Furthermore, tumor-to-blood ratios were calculated for both models to observe the blood clearance and the tumor-to-lung ratio for the LLC model (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec \u0026amp; d). In general, tumor uptake was low and heterogenous in both the LLC and the CT26 models, but with values higher than muscle background the CT26 tumor model (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Of note, highest uptake of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 was observed in the peripheral tumor regions, decreasing towards the central portion of the tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea \u0026amp; d). Among the two animal models, the CT26 model presented the highest tumor-to-muscle-ratio based on %ID/ml (2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1, 39 min p.i. vs 1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2, 39 min p.i. ). Contrary, based on SUV the LLC-model presented the highest SUV (mean: 0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 max: 1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 vs mean 0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 max: 1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, 40\u0026ndash;60 min p.i.) and differentiation between tumor and muscle (mean 0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 max: 0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 vs mean 0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 max: 0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, 40\u0026ndash;60 min p.i.) resulting in a tumor-to-muscle ratios (40\u0026ndash;60 min p.i.) of 2.4 (\u0026plusmn;\u0026thinsp;0.04) or 1.5 (\u0026plusmn;\u0026thinsp;0.03), tumor-to-blood ratios of 1.2 (\u0026plusmn;\u0026thinsp;0.04) or 1.0 (\u0026plusmn;\u0026thinsp;0.14) for LLC and CT26 tumors, respectively. The tumor-to-lung ratios (40\u0026ndash;60 min p.i.) were calculated for the LLC model to be 2.2 (\u0026plusmn;\u0026thinsp;0.09).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRadiotherapy effects on tumor growth kinetics\u003c/p\u003e\u003cp\u003eEffects of radiotherapy on tumor growth was analyzed in the two different tumor models. Tumor irradiation was performed during exponential growth, once tumors had reached 5\u0026ndash;6 mm in diameter. Both tumor models exhibited similar tumor growth kinetics for non-irradiated groups, however, CT26 tumors displayed more variation in growth compared to LLC tumors, as indicated by the larger SD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb\u0026amp;d). In the LLC model, both radiation regimens (2x6 Gy and 1x12 Gy) induced significant tumor growth delays, although effects were more pronounced in the 1x12 Gy group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u0026amp;b). In the CT26 model, both radiation regimens induced potent and durable growth delay in a comparable way (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u0026amp;d), with some animals of the latter group displaying complete tumor regression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRadiotherapy-induced changes in [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 tumor uptake\u003c/p\u003e\u003cp\u003eWhen comparing tumor uptake of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 in untreated animals, LLC tumors displayed the highest tumor uptake, given in fold-change of muscle signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-d). In this model, the fractionated radiation regimen (2x6Gy) enhanced the overall SUV\u003csub\u003er\u003c/sub\u003e two-fold reaching statistically significant differences (p\u0026thinsp;=\u0026thinsp;0.04) compared to the non-irradiated controls. However, only minor variations in the PET-signal were observed when comparing untreated animals with the single-high dose (1x12 Gy) treated group (p\u0026thinsp;=\u0026thinsp;0.9) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). By using BTV75 as an alternative quantitative approach, the outcomes showed similar trends, with two-fold enhanced signal in the (2x6 Gy) group, also reaching statistical significance (p\u0026thinsp;=\u0026thinsp;0.04).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilar effects of radiation were observed in the BALB/c CT26 model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed) however in this model, differences between the untreated group and the fractionated radiation group (2x6Gy) did not reach statistical significance for both SUV\u003csub\u003emean\u003c/sub\u003e (p\u0026thinsp;=\u0026thinsp;0.079) and relative BTV75 values (p\u0026thinsp;=\u0026thinsp;0.73) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Interestingly, BTV75 values in the 1x12Gy radiation group were lower than in the untreated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). In both models, the fractionated regimen of 2x6 Gy displayed the highest tumor specific BTV75 PET signal relative to muscle, although statistical significance was only reached in the LLC model. Detailed calculations on tumor uptake values from each individual animal are given in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eWhen looking at the spatial distribution of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 in tumors, heterogeneous patterns were observed. The highest PET-signal was typically observed in the periphery of tumors, with limited accumulation in the central areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u0026amp;c). This trend was noticed in both tumor models, and all treatment groups. Heterogenous tracer uptake was most evident in the larger non-irradiated tumors, where PET signal was clearly visible as a ring around the tumor, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u0026amp;c and in Fig \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e\u003c/p\u003e\u003cp\u003eEx vivo analyses\u003c/p\u003e\u003cp\u003eTo validate findings based on quantitative PET-imaging, the content of intra-tumoral CAFs in tumor tissue specimens was analyzed also by immunohistochemistry and flow cytometry. IHC analyzes revealed that LLC tumors displayed very poor stroma development and quite low CAF infiltration, as illustrated by the limited infiltration of αSMA\u003csup\u003e+\u003c/sup\u003e cells (brown signal in micrographs in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) and negligible expression of extracellular matrix (analyzed by Masson\u0026acute;s trichrome, not shown). Computer-assisted quantification of αSMA expression showed comparable levels between untreated and 1x12Gy irradiated tumors (mean 2,74% and 3,34% positive cells from total amount of cells respectively), but significantly enhanced levels in the 2x6Gy group (mean 5,07% positive cells, p\u0026thinsp;=\u0026thinsp;0.02) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-b). In the CT26 model, tumor irradiation did not affect levels of αSMA expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMoreover, the percentage of FAP\u003csup\u003e+\u003c/sup\u003e cells from the total pull of viable cells was calculated by flow cytometry in both non-irradiated and irradiated tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). From the fraction of viable cells in non-irradiated LLC tumors in C57BL/6J mice, nearly 40% were FAP\u003csup\u003e+\u003c/sup\u003e. Notably, the fraction of FAP\u0026thinsp;+\u0026thinsp;cells increased to 55% in the 2x6 Gy irradiation group, reaching statistically significance, whereas the values in the 1x12 Gy group remained similar to the untreated animals (47%). Colon carcinoma CT26 tumors in BALB/cJ mice displayed lower proportion of FAP\u003csup\u003e+\u003c/sup\u003e cells in the viable population compared to the LLC lung tumors (approx. 20%), but no changes in FAP\u0026thinsp;+\u0026thinsp;cells were observed between the experimental groups.\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn this study, the main aim was to explore effects of radiation on tumor fibroblasts, and to evaluate the use of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 with PET as biomarker to study dynamics of FAP\u0026thinsp;+\u0026thinsp;stromal cells in the context of cancer therapy. With the intention of applying a broad approach, we have reproduced results in two different syngeneic murine tumor models; the Lewis lung adenocarcinoma model LLC, and the colon carcinoma CT26. Regarding radiotherapy, we have used two different regimens; a single-high dose (1x12Gy) and a fractionated medium-dose regimen (2x6Gy). The results indicate that focused external beam radiotherapy, especially when given by fractionated (medium-high) doses, may induce a moderate elevation in intratumoral FAP\u0026thinsp;+\u0026thinsp;cells. Moreover, this radiation-induced FAP\u0026thinsp;+\u0026thinsp;elevation is more prominent in the LL/2-luc/C57Bl6 model than in the CT26/BALB/c colon carcinoma model. Additionally, we demonstrate that FAPI-74 is a reliable biomarker to evaluate the levels of FAP\u0026thinsp;+\u0026thinsp;stromal cells in tumors and to address potential therapy-induced changes in CAFs.\u003c/p\u003e\u003cp\u003eIn this work, we have used the FAPI-74 variant for PET imaging. Our choice was based on several practical and technical advantages that FAPI-74 offers over other FAPI variants, particularly in clinical and research settings. The FAPI-74 is compatible with both \u003csup\u003e68\u003c/sup\u003eGa and \u003csup\u003e18\u003c/sup\u003eF labeling, offering flexibility in tracer production. The \u003csup\u003e18\u003c/sup\u003eF-labeled FAPI-74 variant, in particular, benefits from a longer half-life (110 minutes vs. 68 minutes) and thus better clinical applicability. Also, [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 has demonstrated lower positron energy compared to \u003csup\u003e68\u003c/sup\u003eGa-labeled variants, resulting in better spatial resolution and image quality [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Recent studies have shown that \u003csup\u003e18\u003c/sup\u003eF-FAPI-74 PET/CT provides high diagnostic accuracy, particularly in detecting metastatic lymph nodes and small lesions, which may be underestimated with other tracers due to partial volume effects. Its high tumor-to-background ratio and rapid clearance from non-target tissues make it suitable for a wide range of oncologic applications [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 was synthesized with a radioactivity yield of 15% and a radiochemical purity of \u0026gt;\u0026thinsp;99%. \u003cem\u003eIn vitro\u003c/em\u003e (plasma) and \u003cem\u003ein vivo\u003c/em\u003e stability tests confirmed good isotope retention in the chelator and no metabolization of the compound within the experimental timeframe (~\u0026thinsp;1 h). These results support the notion that the PET-signal from static scans acquired 1h after [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 injection corresponds to images from the intact tracer. Next, we explored the pharmacokinetics and organ biodistribution of the tracer in healthy animals. As observed in other preclinical studies with FAPI compounds [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 was cleared rapidly by the urinary system; displayed low uptake in major organs such as liver, lung, spleen and brain, but accumulated to some degree in the intestines and joints. Accumulation of [18F]AlF-FAPI-74 in the intestines was more evident in C57Bl6 animals than in Balb-c animals, whereas accumulation in joints and/or bone structures was evident in both strains. This phenomenon has also been noticed in previous preclinical studies using [\u003csup\u003e18\u003c/sup\u003eF]FGlc-FAPI [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] or Al\u003csup\u003e18\u003c/sup\u003eF-NOTA-FAPI [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In our study, we have used young animals that are skeletally immature (10\u0026ndash;12 weeks old). Considering that FAP can be overexpressed in locations with active tissue remodeling, it is plausible that high FAPI uptake in bony structures reflects ongoing bone formation in young animals. Notably, relatively high FAP expression in murine osteoblasts and bone marrow stromal cells have been observed by others [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Hence, accumulation of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 in the murine skeleton could be assigned to physiological uptake. Of note, a recent study demonstrated that circulating soluble FAP (sFAP) in both mice and humans can bind to FAPI radiotracers, potentially altering their biodistribution [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This binding can reduce the amount of free tracer available to bind to membrane-bound FAP in tumors, thereby lowering tumor uptake and increasing background signal, especially in blood-rich organs. The presence of sFAP can prolong the circulation time of the radiotracer, leading to slower clearance and higher blood pool activity. This can complicate image interpretation and reduce the tumor-to-background contrast, which is critical for accurate diagnostics. In our experiments, the signal in blood-rich organs was low at the time that static scans were acquired, and therefore we estimate low impact of sFAP in our analysis.\u003c/p\u003e\u003cp\u003eSeveral PET radiopharmaceuticals with common binding motifs against FAP (FAP inhibitors) have been tested in humans with promising results [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. One of the first developed FAPI compounds, [\u003csup\u003e68\u003c/sup\u003eGa]Ga-FAPI-04, was successfully tested as imaging agent in 28 different cancers types in humans, displaying limited background signal and providing high image contrast of tumors [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Relevant for our study, the NOTA-chelator conjugated FAPI-74 was synthesized and first tested in humans by Giesel et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. High-contrast images of primary tumors, lymph nodes and distant metastasis were achieved, which could support target-volume-definition for guiding radiotherapy delivery. Importantly, no uptake exceeding the perfusion-dependent background was observed in major organs, including intestines and bone structures. As indicated earlier, differences in physiological expression of FAP between rodents and humans may stem from the fact that we are comparing organisms at different developmental stages.\u003c/p\u003e\u003cp\u003eIn tumor-bearing animals, [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 uptake in the tumor region was slightly higher than uptake values in muscle, and most of the tracer accumulated in the periphery of the tumor. These data are consistent with a poorly developed stroma in subcutaneously transplanted tumors, characterized by nearly undetectable extracellular matrix deposition and low abundance of FAP-expressing fibroblasts [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our results align well with observations in other preclinical models using tumor cells not genetically modified to overexpress FAP, where the only source of FAP arises from endogenously recruited levels of tumor-activated fibroblasts [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In a recent study by Liu M et al., authors investigate radiation-induced changes in FAPI tumor uptake using the [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-FAPI-04 variant and using subcutaneously transplanted LLC cells as a model [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Similar to our study, authors observed weak FAPI-04 signal in tumors compared to FDG, but on the contrary, they observed a reduction in FAP expression and FAPI-04 signal in tumors after 1x15 Gy irradiation. In our study, enhanced FAP expression/signal is only observed after 2x6 Gy treatment, indicating that FAP responses to radiation may markedly depend on the radiation regimens used in the experiments.\u003c/p\u003e\u003cp\u003eThe effects of ionizing radiation on CAFs have been previously investigated in animal models, however, in most studies, tumorigenic effects of irradiated CAFs have been studied after co-implantation of (in vitro) irradiated CAFs along with tumor cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In fact, studies demonstrating direct effects of radiotherapy on CAFs \u003cem\u003ein situ\u003c/em\u003e are very scant [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This study is one of the very first to investigate dynamics of tumor fibroblasts following radiotherapy treatment. Our data demonstrate low abundance of αSMA\u0026thinsp;+\u0026thinsp;CAFs in the stroma of subcutaneously transplanted LLC and CT26 tumors. Ex vivo analyses on resected tumors indicate that radiotherapy treatment may induce moderate elevation on the amount of CAFs (i.e. accumulation of FAP\u0026thinsp;+\u0026thinsp;CAFs) in tumors, but only when radiation is applied in a fractionated manner with medium-high radiation doses. In vivo image analyses using [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 show similar trends, achieving statistically significant differences only in the (2x6Gy) RT group and only in the LLC/C57Bl6J model. In line with our observations in preclinical models, Verset et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] observed higher αSMA/tumor epithelial area ratios after neoadjuvant radio(chemo)therapy in rectal cancer specimens from patients, indicating that radiotherapy, when applied in specific regimens, may enhance the number of CAFs in tumor lesions. An alternative explanation to this observation is that the surviving fraction of CAFs after treatment is higher than the tumor epithelial fraction, thus ending in elevated CAFs/tumor cells ratios, but without affecting CAF infiltration or proliferation in tumors.\u003c/p\u003e\u003cp\u003eA clear limitation in our study is related to the use of subcutaneously transplanted tumor models, as these models do not recapitulate normal tumor-host tissue interactions similarly to endogenously formed tumors. In these models, the stroma is very little developed and consequently, the number of FAP-expressing cells, normally fibroblasts, is very low. This setback does not improve much by using orthotopically transplanted tumors since even after transplantation of tumor cells lines in the organ of origin, the high proliferative rate imped the formation of proper tumor stroma [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In preclinical settings, the majority of published studies on FAP-targeting PET radiotracers use genetically engineered tumor cells overexpressing FAP [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], which are artificial models that do not represent \u003cem\u003ebona fide\u003c/em\u003e FAP expression in tumor lesions. Alternative animal models that more faithfully recapitulate the tumor tissue structure normally seen in humans are patient-derived xenografts, genetically engineered animals or environmentally induced models [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Moreover, we have imaged animals and analyzed tissue at one single time point, i.e. 1 week post-radiotherapy. Experiments were designed this way due to inherent limitations in tumor growth rates, which are quite fast when using LLC and CT26 tumor cell lines. Measurements performed at different time points and at longer incubation periods would have given a more accurate view of the CAF dynamics following treatment. Despite the indicated limitations, this study is one of the first demonstrating radiation-induced effects on CAFs i \u003cem\u003ein vivo\u003c/em\u003e and demonstrates good performance of [\u003csup\u003e18\u003c/sup\u003eF]FAPI-74 to study \u003cem\u003ein vivo\u003c/em\u003e CAF dynamics in the context of therapy. Further work in more advanced and clinically relevant animal models is necessary to confirm the results presented in this study and to generate data that can be faithfully translated into clinical settings.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides compelling preclinical evidence supporting the use of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 PET imaging as a non-invasive tool to monitor CAFs dynamics in response to radiotherapy. Our findings demonstrate that CAFs constitute a relatively sparse population in subcutaneously transplanted tumor models, yet their abundance can be moderately increased following fractionated radiotherapy, particularly in the LLC/C57BL6 model. This radiation-induced elevation in FAP\u0026thinsp;+\u0026thinsp;CAFs was consistently observed across multiple analytical platforms, including PET imaging, immunohistochemistry, and flow cytometry.\u003c/p\u003e\u003cp\u003eImportantly, [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 exhibited favorable radiochemical properties, high in vivo stability, and a biodistribution profile suitable for tumor imaging, with low background uptake in most organs and rapid renal clearance. The tracer's ability to detect subtle changes in FAP expression post-irradiation highlights its potential as a sensitive biomarker for assessing stromal responses to cancer therapy.\u003c/p\u003e\u003cp\u003eDespite the limitations inherent to subcutaneous tumor models, our results underscore the feasibility of using FAPI-based PET imaging to study therapy-induced stromal remodeling. These findings pave the way for future investigations in more physiologically relevant tumor models and clinical settings, where CAF-targeted imaging could inform treatment planning, monitor therapeutic efficacy, and potentially guide CAF-directed interventions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCAFs: Cancer-associated fibroblasts\u003c/p\u003e\n\u003cp\u003eFAP: Fibroblast activation protein\u003c/p\u003e\n\u003cp\u003eTME: tumor microenvironment\u003c/p\u003e\n\u003cp\u003eLLC: Lewis Lung Carcinoma\u003c/p\u003e\n\u003cp\u003eRT: Radiation therapy\u003c/p\u003e\n\u003cp\u003eFAPI: Fibroblast activation protein inhibitor\u003c/p\u003e\n\u003cp\u003eSUV: Standard uptake values\u003c/p\u003e\n\u003cp\u003eBTV: Biological tumor volume\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Norwegian Food Safety Authority (FOTS ID 18956 and 27939). This study does not include human participants, human data or human tissue/cells.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eData availability declaration\u003c/p\u003e\n\u003cp\u003eDatasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eCompeting interest\u003c/p\u003e\n\u003cp\u003eAuthors declare no competing interests. FAPI-74 precursor was provided free of charge by SOFIE Biosciences without \u0026nbsp; further influence on study design and data analysis.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eKL, SK, YG were financed by the Trond Mohn and Troms\u0026oslash; Research Foundations; RB and TH by the Regional Health Authorities (Helse-Nord; grants # HNF 1373-17; 1423-18); The Norwegian Cancer Society and The Aakre Foundation at UiT. MK is financed by the Starting Grant of the Trond Mohn and Troms\u0026oslash; Research Foundations.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Author contribution\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the design of the study and in data procurement. KL and RB were responsible for in vivo experiments; YG, SK and AMA developed protocols for tracer radiosynthesis and performed stability tests; TH developed and conducted protocols for in vivo tumor irradiations; MK conducted protocols for whole animal imaging procedures and data analysis; KL and MK analyzed PET/MR images; IMZ was responsible for conception of the study, designed experiments and evaluated the results.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe staff from the PETcore facility and the animal housing facility AKM is acknowledged for their excellent work and daily follow-up of the animals. We are also deeply thankful to SOFIE Bioscience for providing the FAPI-74 precursor \u0026nbsp; used in this study. Additionally, Michel Herranz and Lorenzo Ragazzi for aiding in animal handling, radiotracer injection and image acquisitions, and to Ana Paola Lombardi for her assistance in doing aSMA tissue staining and QuPath-assisted digital quantification.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBiffi, G. and D.A. Tuveson, \u003cem\u003eDiversity and Biology of Cancer-Associated Fibroblasts.\u003c/em\u003e Physiol Rev, 2021. \u003cstrong\u003e101\u003c/strong\u003e(1): p. 147-176.\u003c/li\u003e\n\u003cli\u003ePaulsson, J. and P. Micke, \u003cem\u003ePrognostic relevance of cancer-associated fibroblasts in human cancer.\u003c/em\u003e Semin Cancer Biol, 2014. \u003cstrong\u003e25\u003c/strong\u003e: p. 61-8.\u003c/li\u003e\n\u003cli\u003eMonteran, L. and N. 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However, if and how CAF-mediated responses to radiotherapy (RT) affects clinical outcomes remains undetermined. Here, we aimed to investigate impact of RT on CAFs using antigen-specific, non-invasive, molecular PET-imaging. The FAP-specific radiotracer [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 was applied to monitor CAF dynamics following external beam RT in two syngeneic subcutaneous murine tumor models (LLC and CT26). Tumors were irradiated using two radiation regimens (1x12 Gy or 2x6 Gy), and PET/MR imaging was performed 7 days post-RT. Additionally, dynamics of FAP\u0026thinsp;+\u0026thinsp;CAFs in tumors was quantified \u003cem\u003eex vivo\u003c/em\u003e using flow cytometry and immunohistochemistry.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eBiodistribution studies of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 showed radiotracer signal in joint/bone structures and intestines in both mouse strains. Tumor-targeted irradiation led to significant reduction in tumor size. Uptake of [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 in subcutaneous tumors was low but significantly above muscle-background values. Quantification of standardized uptake values (SUV) from static PET-images revealed two-fold increased PET signal in LLC tumors irradiated with 2x6 Gy. Ex vivo analysis confirmed low abundance of FAP\u0026thinsp;+\u0026thinsp;cells in tumors and demonstrated similar RT-induced changes in CAFs across the different models.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOur findings suggest that CAFs represent a relatively sparse cell population in subcutaneously transplanted tumor models, and that radiotherapy may induce a moderate increase in FAP\u003csup\u003e+\u003c/sup\u003e cells in LLC tumors. Additionally, we demonstrate that [\u003csup\u003e18\u003c/sup\u003eF]AlF-FAPI-74 is a reliable biomarker for evaluating levels of FAP\u0026thinsp;+\u0026thinsp;stromal cells in tumors and for addressing potential therapy-induced changes in CAFs.\u003c/p\u003e","manuscriptTitle":"Preclinical evaluation of [18F]AlF-FAPI-74 for PET imaging to study cancer-associated fibroblast responses to radiotherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 05:55:48","doi":"10.21203/rs.3.rs-7065428/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"13a7ac3b-b198-4a63-bb6c-765e946f99e6","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-10T11:14:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-23 05:55:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7065428","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7065428","identity":"rs-7065428","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}