18F-AlF-FAPI nanotracer visualized the quick response of sarcoma to radiotherapy by PET/CT

doi:10.21203/rs.3.rs-3113339/v1

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Research Article

18F-AlF-FAPI nanotracer visualized the quick response of sarcoma to radiotherapy by PET/CT

https://doi.org/10.21203/rs.3.rs-3113339/v1

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Background

MRI or CT-based RECIST is the current clinical standard for evaluating the efficacy of radiation therapy. Typically, several months are required after treatment to determine the extent of tumor control, with the possibility of malignant progression. In this study, we synthesize a novel nanoscale ¹⁸F-AlF-labeled FAPI radiotracer and assess its capacity to monitor instant radiotherapy response by PET/CT in tumor xenografted mouse models and a patient with sarcoma, utilizing ¹⁸F-FDG, ⁶⁸Ga-FAPI PET/CT imaging, and MRI imaging as controls.

Results

Current research has generated an ¹⁸F-AlF-FAPI radiotracer with an unique pharmacological architecture. The radiotracer ¹⁸F-AlF-FAPI was a colloid with a diameter of 100–200 nm. The diameter of AlF clusters ranges between 10 and 80 nm, and the majority of ¹⁸F-AlF-FAPI molecules comprise between 2 and 5 AlF clusters. In comparison to ⁶⁸Ga-FAPI, ¹⁸F-AlF-FAPI has a distinct excretion mechanism and a significantly smaller background signal, resulting in a higher tumor-to-background ratio (TBR). After a single dose of 10 Gy of non-lethal X-ray therapy, the xenografted tumor in the mouse exhibited a high uptake of ¹⁸F-AlF-FAPI, followed by tumor progression. In a patient with sarcoma who underwent complete carbon ion radiotherapy (CIRT) treatment and tumor regression, tumor uptake of ¹⁸F-AlF-FAPI was barely detectable, highlighting the potential of ¹⁸F-AlF-FAPI probe-based PET/CT for visualization of quick response to CIRT radiotherapy within one month. Additionally, the tumor site in this case was around 1,4 times larger in ¹⁸F-AlF-FAPI PET imaging than in MRI and ¹⁸F-FDG PET/CT imaging. The physician finally expanded the target volume delineation for CIRT treatment based on the positive region and heterogeneity, indicating the potential of ¹⁸F-AlF-FAPI nanotracer in target volume delineation.

Conclusions

In PET/CT imaging, the novel ¹⁸F-AlF-FAPI nanotracer had a higher TBR and a lower background than ⁶⁸Ga-FAPI due to its distinct formation. ¹⁸F-AlF-FAPI uptake was found to be favorably linked with tumor progression in tumor-xenografted mice and sarcoma patients. Compared to ¹⁸F-FDG, ⁶⁸Ga-FAPI PET/CT imaging, and MRI imaging, ¹⁸F-AlF-FAPI PET/CT imaging revealed greater potential for identifying the rapid response of sarcoma to radiotherapy within one month. ¹⁸F-AlF-FAPI PET/CT imaging has also shown potential in radiotherapy target volume delineation.

18F-AlF-FAPI

PET/CT

nanotracer

carbon ion radiotherapy

radiation response

Radiation therapy is one of the leading cancer treatment modalities, and carbon ion radiotherapy is a novel radiotherapy technique with high linear energy transfer (LET) and distinctive biological effects. Unfortunately, because tumors are heterogeneous, patients frequently experience different tumor responses to radiation. For many types of cancers, the current clinical standard for assessing response to RT is RECIST (Response Evaluation Criteria in Solid Tumors), in which changes in tumor size are measured with MR and CT diagnostic imaging¹. The extent of tumor control (complete reduction in tumor size, tumor shrinkage, or cessation of tumor progression) is often not measurable until months after treatment. The ability to measure early physiological biomarkers that indicate treatment failure prior to long term changes in tumor size would represent a significant advancement in early treatment response detection.

Functional molecular imaging based on PET/CT is a quick quantitively molecular visualization tool to measure physiological biomarkers, which has been applied in physical plan of biological intensity modulated radiotherapy. Researcher has also tried many radiotracers as indicator of tumor response to radiotherapy including ¹⁸F-FDG, choline. Although PET tracer showed its fantastic potential in monitoring of quick response of radiotherapy, the RECIST criteria base on MRI was still the standard for post-radiotherapy in clinic. Exploring of PET radiotracer to visualize the early tumor response play critical role in timely switch to an alternative treatment approach and lead to a better prognosis.

Carcinoma-associated fibroblasts (CAFs) play critical roles in resistance to radiotherapy and have been implicated in treatment failure and associated poor clinical outcomes following radiotherapy^2,3. Fibroblast activation protein (FAP) is strongly expressed by the reactive carcinoma-associated fibroblasts (CAFs) of epithelial cancers. Quinoline-based FAP-inhibitors (FAPIs) specifically bind to the enzymatic domain of FAP and are then internalized ⁴. Methods for conjugation of quinoline-based FAP-ligands with chelators suitable for radiolabeling with various radio-metals were developed^5,6. FAPI has been labeled with the positron emitter ⁶⁸Ga⁷, ¹⁸F ⁸and ⁶⁴Cu ⁹.These novel FAP-targeted tracers demonstrated tumor to non-tumor contrast-ratios that were equal or even higher than those attained with FDG-PET/CT ⁷. Moreover, precise and comprehensive target volume delineation for radiotherapy was improved by adding FAPI PET/CT information ^8,10,11. There was a high tumor-to-background ratio of more than 3 in most lesions and TNM was changed in 50% while for patients with metastases new findings occurred in 47%. For almost every patient undergoing irradiation, target volume delineation was improved by FAPI-PET/CT ¹². FAPI were also modified and optimized for use as theragnostic tracers ⁶.

In our previous study, we have report the superiority of ⁶⁸Ga-FAPI-04 over ¹⁸F-FDG PET/CT in imaging metastatic esophageal squamous cell carcinoma ¹³, and compared with ¹⁸F-FDG PET/CT, the primary tumor, enlarged pelvic lymph nodes and right maxillofacial lesion showed higher uptake in ⁶⁸Ga-FAPI PET/CT ¹⁴. We also report ⁶⁸Ga-FAPI and ¹⁸F-FDG PET/CT images in a patient with extrapulmonary tuberculosis mimicking malignant tumor¹⁵. In this study, we aimed to explore the potential of ¹⁸F-AlF-FAPI as an indicator to visualize the tumor response to carbon ion radiotherapy.

The preclinical favorable precursor FAPI-04 was labeled with ¹⁸F-AlF (Aluminum-Fluoride) by complexation with the NOTA-moiety of ligand. ¹⁸F-AlF-FAPI showed its unique superiority in drug form and signal-to-noise ratio, which depicts that carbon ion significantly decrease the FAP expression in sarcoma that was resistant to photon radiotherapy. Our present study sheds light on the critical potential of ¹⁸F-AlF-FAPI to quantically visualize the tumor response to carbon ion radiotherapy.

General

Aluminium chloride were purchased from Energy Chemical (Shanghai China). Sodium acetate were purchased from Sigma (Sigma - Aldrich,Mexico ).0.9% Sodium Chloride Injection were purchased from Baxter Healthcare (Shanghai) Co.,Ltd. Dimethyl sulfoxide were purchased from Sigma (Sigma - Aldrich,France).

Anhydrous Ethanol were purchased from Nanjing Chemical Reagent Co.,Ltd. The precursor, NOTA-FAPI-04, was obtained from Huayi (Changshu, Jiangsu, China). Cartridges were purchased from Waters (Waters, MA,USA). Millex-GV syringe filter units were purchased from Millipore (Millipore, MA, Puerto Rico).

Auto Radiosynthesis

Siemens Explora GN module was used and modified for the automatic synthesis of [¹⁸F]AlF-NOTA-FAPI-04 (Fig. 1). The recipe for synthesis was programmed in Siemens Explora GN software (Build 1.0, ChemController 2.0 embedded). The [¹⁸F]fluoride was produced on-site by irradiating [¹⁸O]H₂O with 11-MeV protons in a Siemens Eclipse HP cyclotron system. [¹⁸F]fluoride was added to the collection tube after the radiation exposure, and it was then passed through a Sep-Pak light QMA cartridge (Waters Accel Plus QMA Light cartridge, preconditioned with 5 mL 0.5 M NaOAc pH 3.9 and 20 mL of water). A 0.5 mL addition of sodium acetate helped elute the trapped [¹⁸F]fluoride. Before adding 1 mL of a solution of FAPI-04 (4 mM), the mixture was incubated for 5 minutes at room temperature with 6 µL of AlCl₃ in water (10 mM) and 300 µL of dimethyl sulfoxide (DMSO, Simga-Aldrich, Darmstadt, France). The reaction was conducted at 100°C for 15 minutes, cooled to room temperature, and then transferred to Alum N (Waters Corporation Milford, Massachusertts USA) and SPE (Waters Oasis HLB Plus Light cartridge). Before sterile filtration, 10 mL of 0.9% saline was used to elute the SPE. The purified product was collected in 3.5 mL of 0.9% saline and 1.5 mL of anhydrous ethanol before being run through a sterile 0.22 µm filter.

Cell Culture

SK-LMS-1 cells were cultivated in Dulbecco modified Eagle medium containing 10% fetal calf serum at 37℃ in 5% carbon dioxide.

Animal model

All animal studies were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC). For in vivo tests, 1x10⁷ SK-LMS-1 cells were subcutaneously inoculated into the right armpit of 8-week-old BALB/c nu/nu mice (Shanghai JIHUI). SK-LMS-1 tumor xenograft mice is a model in which the tumor cells are FAP-negative with activated mouse fibroblasts. Tumor xenograft mice were investigated when the tumor had grown to approximately 1 cm³.

Distribution study by γ-counter

¹⁸F-AlF-labeled FAPI-04 (0.74 MBq/mouse) was injected into SK-LMS-1 xenograft mice via the tail vein to evaluate the whole-body biodistribution. The animals (n = 3 for each time point) were sacrificed by euthanasia after the indicated time points (from 30 min to 2 h). For the bone and muscle, part of the rear limb was collected. The distributed radioactivity was measured in all dissected organs and in blood using a γ-counter (GC-911, ZONKIA). Radioactivity counts were normalized by calibration using the ¹⁸F standard solution. The values are expressed as %ID per gram of tissue(%ID/g).

microPET/CT imaging

PET imaging was performed using a small-animal PET scanner (Inveon, Siemens). The mice were intravenous injected with ¹⁸F-AlF-FAPI-04 (3.7 MBq/mouse). Within 120 min, a dynamic scan was performed after injection, and a static scan was also performed 1 h after injection. The animals were maintained under 2% isoflurane anesthesia throughout the scanning period. The PET raw data were dynamically sorted into 27 frames (8*1, 8*2, 6*6 and 5*20 min) and were reconstructed iteratively using the three-dimensional ordered-subset expectation maximization/maximum algorithm (OSEM3D/MAP, Siemens) and were converted to SUV images.

X-ray radiation of mouse

The mice were anesthetized using an O₂/isoflurane (2% isoflurane, 1.2 L/min O₂). A 225 kVp X-ray beam (PXi precision X-RAD 225, dose rate = 2.042 Gy/min, 225 kV, 13.3 mA, 50 cm SSD) was used as the photon beam. For irradiation experiments, a single 10 Gy X-ray irradiation dose was delivered to the tumor in mice. 7 days after irradiation, a ¹⁸F-AlF-FAPI-04 PET/CT scan was performed.

Autoradiography

After injection of ¹⁸F-AlF-FAPI-04 in saline, the tumor was removed and cut into slices using a microtome. Quantification and Heterogeneity analysis of tumors were done using Autoradiography (Typhoon FLA 9500, GE Healthcare Life Sciences).

Patients

A 37-year-old woman with a history of recurrent radiation-induced sarcoma following radiotherapy for nasopharyngeal carcinoma, received chemotherapy and anti-body immunotherapy for 18 months with efficacy evaluation recorded as partial response (PR). The patient was given radical carbon ion radiotherapy (CIRT) in 2021 at our hospital at a dose of 70 GyE/20Fx for one month.

Clinical PET/CT imaging

The ¹⁸F-AlF-FAPI-04/FDG PET/CT scans were performed on a Biograph 16 PET/CT scanner (Siemens Healthcare, Erlangen, Germany) according to standard clinical scanning protocols. All patients fasted for at least 6 hours before the scan, and none had a blood glucose levels > 8.7 mmol/L. A whole-body scan was acquired approximately 1 hour after intravenous administering of ** MBq/kg of AlF-FAPI-04 and 5.18 MBq/kg of ¹⁸F-FDG. The CT scans were performed first (120 kVp, 150 mAs) and reconstructed to a 512 × 512 matrix. Then, a 3D row-action maximization likelihood algorithm was used to reconstruct the PET images. The PET images were converted into SUV units by normalizing the activity concentration to the dosage of injected ¹⁸F-AlF-FAPI-04/FDG and patient body weight.

Automatic synthesis and characterization of ¹⁸F-AlF-FAPI-04 showed distinctive morphology

FAPI-04 was chosen for this study because of its potential as a targeting moiety to image FAP expression in CAF (Fig. 1a). According to a modified recipe based on earlier reports^16,17, the radiosynthesis of the percussor FAPI-04-NOTA with ¹⁸F-AlF was carried out on a Siemens Explora GN module (Fig. 1b). The entire radiosynthesis took about 15 minutes at 100°C in the reaction vessel, producing a ¹⁸F-AlF-FAPI-04 tracer with a radioactivity yield of 40.3%, a radiochemical purity of > 99%, and a molar activity of 4.59 GBq/µmol. Figure 1c displays the overall synthesis progress that was tracked.

The radiotracer sample was imaged by a transmission electron microscope (TEM), which is shown in Fig. 2a, after the radioactivity of the tracer had decay to a level that was safe for humans. Interesting was the fact that ¹⁸F-AlF-FAPI-04 radiotracer was a form of colloid, with a diameter of 100–200 nm. AlF clusters have a diameter of between 10 and 80 nm and most molecules of ¹⁸F-AlF-FAPI-04 contain 2 to 5 AlF clusters. Because of the NOTA coordinate ability, one ¹⁸F-AlF-FAPI-04 include several FAPI precursor, which means this radiotracer was a type of radiotracer with multiple targeting moiety in a single composite. Based on the form of ¹⁸F-AlF-FAPI-04, our theory was that ¹⁸F-FAPI will have high affinity to FAP expressed on CAFs and higher signal-to-noise ratio (SNR) or target-to-background ratio (TBR) in human body (Fig. 2b).

The radiochemical purity was determined using the analytical HPLC chromatograms. At 5.62 minutes, only one radioactive peak was found (Fig. 3a). F⁻ ions were added to the ¹⁸F-AlF-FAPI-04 solution and then the solution was monitored by HPLC to further confirm the peak of ¹⁸F-AlF-FAPI-04. The presence of additional added F⁻ ions at 1.8 min indicates that the ¹⁸F-AlF-FAPI-04 contributed to the 5.62 min peak in the radiochromatogram. At 1, 2, and 4 hours after adding saline and FBS, the stability of ¹⁸F-AlF-FAPI-04 was once again checked. At 1.8 min (the F⁻ ion position), there was no discernible signal, indicating a 4-hour stability of ¹⁸F-AlF-FAPI-04 in saline. While the addition of FBS appears to cause an obvious escape of ¹⁸F⁻ from ¹⁸F-AlF-FAPI-04, this is likely due to the stronger binding forces between Al³⁺ and anionic groups on proteins like phosphate groups, which disrupt the coordinate of Al³⁺ with NOTA and F⁻. The approximate half-life determination test was used to confirm the radionuclide (Fluorine-18) identity. The final product's pH was consistently found to be 6.5. The Chinese Pharmacopeia's standards for bacterial toxins were met by all three batches, which were also discovered to be sterile.

Distribution and excretion way of ¹⁸F-AlF-FAPI-04 in mouse

Mice were observed using a small animal PET/CT after receiving intravenous injections of ¹⁸F-AlF-FAPI-04 and ⁶⁸Ga-FAPI solution via the tail vein. Since ⁶⁸Ga-FAPI was primarily found in the bladder, it was likely excreted primarily by the kidney (Fig. 4a). The intestine and gallbladder had little signal. At the same time, we discovered that the ⁶⁸Ga-FAPI injected mouse had a high background signal. Additionally, ⁶⁸Ga-FAPI tends to deposit in the spiral cord and bone (Fig. 4b and insert). While the ¹⁸F-AlF-FAPI-04 injected mouse displayed a clear signal in the gall bladder, intestine, and urine, indicating an alternate route for ¹⁸F-AlF-FAPI-04 excretion other than through the urine (Fig. 4a). Due to its sparse distribution in the spinal cord and bones, ¹⁸F-AlF-FAPI-04 may be used to track bone metastasis (Fig. 4b and insert). Additionally, when compared to ⁶⁸Ga-FAPI PET/CT imaging, ¹⁸F-AlF-FAPI-04 PET/CT imaging displayed a weak background signal.

In order to determine the distribution of ¹⁸F-AlF-FAPI-04 in mice, the organs of mice that received the injection were removed and their radioactivity was measured using a gamma counter. The presence of distinct signals in the urine and bile further supports the theory that ¹⁸F-AlF-FAPI-04 is primarily excreted through the urine and feces. Additionally, following intravenous administration, the excretion rate of ¹⁸F-AlF-FAPI-04 in the urine gradually increased and the signal in the bile gradually decreased (Fig. 4c). This result supports the theory that the multitarget molecular form of ¹⁸F-AlF-FAPI-04 shown in Figs. 2a and 2b actually exists. The way that bile excretion was a characteristic of large molecules. In addition, almost every major organ in the body showed no detectable signal, with the exception of some weak signals in the joints and bones. This indicates that the radiochemistry of ¹⁸F-AlF-FAPI-04 in the mouse body was good.

Heterogenic distribution of ¹⁸F-FAPI in xenografted sarcoma in mouse

A tumor-xenografted mouse was injected with the ¹⁸F-AlF-FAPI-04 radiotracer and imaged by microPET/CT to determine the distribution of the radiotracer in the tumor. In Fig. 5a, a typical image was displayed. The radiotracer ¹⁸F-AlF-FAPI-04 was not taken up by all of the tumor, and there was a clear distribution heterogeneity. In this case, the radiotracer was concentrated in three areas and weaker signals could be seen elsewhere (Fig. 5a and insert). Autoradiography could support these findings further. About 4–6 slices of each tumor were observed (Fig. 5c). Even though the radiotracer signal was present throughout the tumor, it was not uniform and displayed a clear heterogenic distribution, which strongly implies that each region has a different radioresistance.

Additionally, we continuously tracked the tumor xenograft's uptake of ¹⁸F-AlF-FAPI-04. Tumor uptake was found to range from 13.77 to 18.81%ID/g (n = 6), with high retention from 30 to 120 minutes following intravenous injection (Fig. 5b). The signal in the tumor quickly rose to a high level within the first 30 minutes of injection via tail vein. And at one hour after injection, the signal in the tumor reached its maximum value, ~ 19% ID/g. then the signal starts to gradually fade away. This outcome showed when it was best to acquire high-quality imaging data with a high SUVmax.

FAP expression of xenografted sarcoma under X-ray radiation imaged by ¹⁸F-AlF-FAPI-04 PET/CT imaging

The differential uptake of ¹⁸F-AlF-FAPI-04 in tumors treated with or without radiation was observed using PET/CT and autoradiography in order to research the usefulness of ¹⁸F-AlF-FAPI-04 PET/CT imaging in evaluating tumor response to radiation (Fig. 6a). After receiving 10 Gy of nonlethal photon radiation for 7 days, the tumor in the treated mouse displayed a stronger uptake of the radiotracer ¹⁸F-AlF-FAPI-04 than the untreated mouse, which was clearly discernible by autoradiography (Fig. 6a). Similar results were seen on the ¹⁸F-AlF-FAPI-04 PET/CT image, with the tumor showing a stronger signal following radiation. These results indicate the presence of FAP or FAP + CAFs in radiation-induced tumors. The tumor still has the capacity to multiply and expand at the same time (Fig. 6b).

FAP expression of human sarcoma under CIRT imaged by ¹⁸F-AlF-FAPI-04 PET/CT imaging

A patient with radiation sarcoma was included in order to examine the response of radioresistant sarcoma to carbon ion radiotherapy and the potential of ¹⁸F-AlF-FAPI-04 PET/CT imaging in carbon ion radiotherapy. This patient, a 37-year-old female with nasopharyngeal carcinoma, underwent chemotherapy and radiation treatment in 2015. In 2018, pathology discovered that the patients' left mandibles had radiation sarcoma. Chemotherapy and anti-PD-1 checkpoint therapy were then used to treat the sarcoma, with partial success. The patients were given radical carbon ion radiotherapy (CIRT) in 2021 at our hospital at a dose of 70 GyE/20Fx.

Prior to receiving CIRT treatment, the patient was imaged using MRI, ¹⁸F-AlF-FAPI-04, and ¹⁸F-FDG PET/CT imaging, as shown in Fig. 7. On an MRI, the tumor can be seen clearly in the left upper cervical paravertebral space. In comparison to MRI and ¹⁸F-FDG PET/CT imaging, the tumor region was approximately 1.4 times larger in ¹⁸F-AlF-FAPI-04 imaging. Information was provided in detail in Table 1. The tumor's boundary differs from what is seen on MRI imaging. MRI revealed the tumor's physical border, and ¹⁸F-AlF-FAPI-04 characterized the FAP positive CAFs (Fig. 7b). The critical area that required additional treatment was visible on the ¹⁸F-AlF-FAPI-04 PET/CT image because the FAP positive CAFs were strongly associated with resistance to radiotherapy and chemotherapy. The doctor extended the target volume delineation for CIRT treatment based on the results of the ¹⁸F-AlF-FAPI-04 PET/CT imaging because of the imaging data.

The ¹⁸F-AlF-FAPI-04 PET/CT and MRI were performed as follow-up procedures one month after the CIRT therapy. The lesion volume increased but the degree of enhancement decreased, according to MRI. When ¹⁸F-AlF-FAPI-04 PET/CT was used, the metabolic volume of lesions significantly decreased, as did the SUV value. These results imply a significant potential for ¹⁸F-AlF-FAPI-04 PET/CT in early predicting tumor response to CIRT therapy.

Table 1

Detailed information of sarcoma before CIRT treatment imaged by MRI, ¹⁸F-FAPI and ¹⁸F-FDG PET/CT imaging
Parameter	MR	¹⁸F-FDG	¹⁸F-FAPI
Dmax	3.2 cm	3.2 cm	4.6 cm
SUVmax	/	12.2	10.5
TBRmax	/	5.6	4.0
SUVmean	/	4.6	4.5
TBRmean	/	3.5	2.9
MTV	/	25.2 cm³	64.8 cm³

Table 2

Detailed information of sarcoma one month after CIRT treatment imaged by MRI and ¹⁸F-FAPI PET/CT imaging
Parameter	Pre-treatment	Post-treatment	Variance ratio
MR (Dmax)	3.2 cm	4.2 cm	+ 31.3%
¹⁸F-FAPI SUVmax	10.5	9.1	-13.3%
¹⁸F-FAPI TBRmax	4.0	1.6	-60.0%
¹⁸F-FAPI SUVmean	4.5	3.4	-24.4%
¹⁸F-FAPI TBRmean	2.9	1.3	-55.2%
¹⁸F-FAPI MTV	64.8 cm³	47.9 cm³	-26.1%

Radiotherapy is one of the most important cancer-treatment strategies, and more than half of cancer patients undergoing antitumor therapy require radiotherapy. However, primary and acquired resistance shortly after treatment remain significant challenges that must be addressed in order to improve radiotherapy efficacy¹⁸. The CAFs itself was radioresistant. More important was that CAFs have been shown to confer radiotherapy resistance to cancer cells via paracrine actions of secreted growth factors¹⁹ and the metabolic reprogramming²⁰. CAFs could promote irradiated cancer cell recovery and tumor regrowth after radiotherapy through inducing autophagy²¹. CAFs are naturally resistant to radiotherapy, people have been exploring to eliminating CAFs through antibody dependent recognition of specific cell surface markers, normalization of activated CAFs, targeting CAFs paracrine signaling pathways and targeting CAF-derived extracellular matrix proteins. However, until now there is no targeting drugs has been verified effective in clinical²². In this present study, we further verified the FAP positive CAFs were radioresistant to photon radiation (Fig. 6). Moreover, CIRT could eliminate the most of FAP positive CAFs one month after treatment (Fig. 7). Most important was that the ¹⁸F-FAPI PET/CT imaging clearly quantically visualize the elimination, showing a great potential of ¹⁸F-FAPI PET/CT to present the quick response of tumor to CIRT. To our knowledge, until now there is no reports of such a direct intuitive way to quantically visualize the efficacy of CIRT to FAP positive CAFs.

Another interesting aspect of this study was the unique structure of the ¹⁸F-FAPI radiotracer, which was created using the AlF-¹⁸F radiolabeling technique. The appearance of ¹⁸F-AlF-FAPI in solvent (50 percent alcohol in saline) was first presented (Fig. 2), and each tracer contained several AlF particles. The hydrophobic region of FAPI integrated as a result of hydrophobic force was a possible mechanism. because of the integrated formation and size, the in vivo behavior of ¹⁸F-AlF-FAPI was thus quite different compared to ⁶⁸Ga-FAPI (Fig. 4). In muscle, ¹⁸F-AlF-FAPI demonstrated very little non-specific uptake, producing very little background signal. Additionally, the tracer's uptake by the spiral cord and bone decreased to zero, which may help researchers find bone metastases. The unique formation of ¹⁸F-AlF-FAPI radiotracer also changed the excretion way of FAPI that the radiotracer excreted through feces and urine (Fig. 4). Thus, according to clinical data (Fig. 7 and Table 2), the TBRmax in this radiation sarcoma patient before treatment are 4, which is close to that of ¹⁸F-FDG (TBRmax = 5.6).

Our present result further confirms the value of PET/CT imaging based on FAPI radiotracer in radiation. Previous has reported the possibility of FAPI PET/CT guided delineation for radiotherapy in lung cancer^8,10, head and neck cancer²³. In this present study, we make it real clinical application and demonstrate a real case that using ¹⁸F-AlF-FAPI PET/CT to guard carbon ion radiotherapy. Based on the ¹⁸F-AlF-FAPI PET/CT, physicians changed the irradiation plan and extended irradiation region. Considering of the heterogeneity of ¹⁸F -FAPI distribution and the radioresistance of CAF, ¹⁸F-FAPI PET/CT imaging provides a good tool for radiotherapy boost delineation. The FAP expression was a strong correlation with tumor resistance to radiotherapy and chemotherapy^2,3. High CAFs component was found to be correlated with advanced pT and pN stage (pathological TNM stage system) and poor prognosis,^24,25,26. FAP-positive cells in the stroma can change the immune system's antitumor response²⁷. Therefore, the high FAP expression area should be the target area of radiotherapy boost.

CAFs play critical roles in resistance to radiotherapy and have been implicated in treatment failure and associated poor clinical outcomes following radiotherapy. CIRT present its potential to further improve the outcome of radiotherapy. Herein, ¹⁸F-FAPI PET/CT imaging result in this primary research indicated the potential of CIRT to effectively eliminate the CAFs. And ¹⁸F -FAPI PET/CT also showed its value in clinic. Based the ¹⁸F-FAPI PET/CT imaging, physicians could make a better delineation plan combined with MRI or ¹⁸F-FDG PET/CT results, and have another powerful tool to monitor the response of tumor to treatment.

We explored a type of Fluorine-18 labeled Al[¹⁸F]F-FAPI with a high radioactivity yield of 40.3% and present its unique structure and thus lower background and higher TBR in patient. Based on ¹⁸F-FAPI PET/CT, Physicians extended the target volume delineation for CIRT treatment. ¹⁸F-FAPI PET/CT results also showed the high elimination effects of CIRT radiotherapy on radioresistant sarcoma, which present its value to monitor the tumor response to treatment. Al[¹⁸F]F-FAPI based PET/CT showed a great potential in radiotherapy.

LET: linear energy transfer; CAF: Carcinoma-associated fibroblasts; RECIST: Response Evaluation Criteria in Solid Tumors; FAPI: Quinoline-based FAP-inhibitors; AlF:Aluminum-Fluoride.

Supplementary Information

No additional information.

Acknowledgements

Not applicable.

Author information

Zili Li, Caiyue Ren and Mingyu Liu contributed equally to this work.

Jingyi Cheng and Yun Sun shared senior authorship.

Authors and Affiliations

Department of Nuclear medicine, Shanghai Proton and Heavy Ion Center, Fudan University Cancer Hospital, Shanghai 201321, China

Zili Li, Caiyue Ren, Mingyu Liu, Jiangang Zhang, Jianping Zhang, Ming Qi, Yangbo Xue, Xiaoping Xu, An Yao, Shaoli Song, Jingyi Cheng and Yun Sun

Department of Research & Development, Shanghai Proton and Heavy Ion Center, Fudan University Cancer Hospital, Shanghai 201321, China

Yun Sun

Shanghai Key Laboratory of radiation oncology (20dz2261000) , Shanghai 201321, China

Zili Li, Caiyue Ren, Mingyu Liu, Jiangang Zhang, Jianping Zhang, Ming Qi, Yangbo Xue, Xiaoping Xu, An Yao, Shaoli Song, Jingyi Cheng and Yun Sun

Shanghai Engineering Research Center of Proton and Heavy Ion Radiation Therapy, Shanghai 201321, China

Zili Li, Caiyue Ren, Mingyu Liu, Jiangang Zhang, Jianping Zhang, Ming Qi, Yangbo Xue, Xiaoping Xu, An Yao, Shaoli Song, Jingyi Cheng and Yun Sun

Author Contributions

ZLL, CYR and MYL performed experiments and analyzed data. YS analyzed data and wrote the paper; YBX, AY, XPX assisted ZLL with tracer synthesis and characterization. JGZ, JPZ and MQ assisted MYL with animal studies and histology. JYC and CYR collected clinical samples. YS, JYC, SLS design experiments and conceived of this study, analyzed the data, and revised the paper. All authors read and approved the final paper.

Corresponding author

Correspondence to Yun Sun, E-mail: [email protected], Shanghai Proton and Heavy Ion Center, Shanghai, China; Jingyi Cheng, E-mail: [email protected], Shanghai Proton and Heavy Ion Center, Shanghai, China.

Funding

This work was supported by the Natural Science Foundation of Shanghai (21ZR1481800) and Shanghai Pudong New Area Health Commission (PKJ 2020-Y56, PKJ 2018-Y49).

Data Availability

All data are provided in the manuscript and Supplementary files or are available from the corresponding authors upon reasonable request.

Ethics approval and consent for participants

All the experimental protocols were approved by institutional committee of Shanghai Proton and Heavy Ion Center. All methods were carried out in accordance with relevant guidelines and regulations. The study was carried out in compliance with the ARRIVE guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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You are reading this latest preprint version

18F-AlF-FAPI nanotracer visualized the quick response of sarcoma to radiotherapy by PET/CT

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Materials and methods

General

Auto Radiosynthesis

Cell Culture

Animal model

Distribution study by γ-counter

microPET/CT imaging

X-ray radiation of mouse

Autoradiography

Patients

Clinical PET/CT imaging

Results

Automatic synthesis and characterization of ¹⁸F-AlF-FAPI-04 showed distinctive morphology

Distribution and excretion way of ¹⁸F-AlF-FAPI-04 in mouse

Heterogenic distribution of ¹⁸F-FAPI in xenografted sarcoma in mouse

FAP expression of xenografted sarcoma under X-ray radiation imaged by ¹⁸F-AlF-FAPI-04 PET/CT imaging

FAP expression of human sarcoma under CIRT imaged by ¹⁸F-AlF-FAPI-04 PET/CT imaging

Discussion

Conclusion

Abbreviations

Declarations

References

Status:

Version 1

18F-AlF-FAPI nanotracer visualized the quick response of sarcoma to radiotherapy by PET/CT

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Materials and methods

General

Auto Radiosynthesis

Cell Culture

Animal model

Distribution study by γ-counter

microPET/CT imaging

X-ray radiation of mouse

Autoradiography

Patients

Clinical PET/CT imaging

Results

Automatic synthesis and characterization of 18F-AlF-FAPI-04 showed distinctive morphology

Distribution and excretion way of 18F-AlF-FAPI-04 in mouse

Heterogenic distribution of 18F-FAPI in xenografted sarcoma in mouse

FAP expression of xenografted sarcoma under X-ray radiation imaged by 18F-AlF-FAPI-04 PET/CT imaging

FAP expression of human sarcoma under CIRT imaged by 18F-AlF-FAPI-04 PET/CT imaging

Discussion

Conclusion

Abbreviations

Declarations

References

Status:

Version 1

Automatic synthesis and characterization of ¹⁸F-AlF-FAPI-04 showed distinctive morphology

Distribution and excretion way of ¹⁸F-AlF-FAPI-04 in mouse

Heterogenic distribution of ¹⁸F-FAPI in xenografted sarcoma in mouse

FAP expression of xenografted sarcoma under X-ray radiation imaged by ¹⁸F-AlF-FAPI-04 PET/CT imaging

FAP expression of human sarcoma under CIRT imaged by ¹⁸F-AlF-FAPI-04 PET/CT imaging