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. 2024 Feb 20;14(1):13–21. doi: 10.62347/HVLF3619

Molecular imaging reveals the heterogeneous progression of tumor cells and tumor stroma: a practice of FDG PET and FAPI PET in diagnosing PSMA-negative bone metastases of progressive prostate cancer

Lizhi Zhu 1,*, Peng Chen 2,*, Zhongqiu Guo 1, Fangdu Li 3, Xiu Luo 1, Xia Du 4, Liying Zhang 3, Changjing Zuo 1, Xiao Li 1
PMCID: PMC10944373  PMID: 38500747

Abstract

Tumors are often with complex and heterogeneous biological processes, such as glycometabolism and fibrosis, which are the main biochemical pathways that determine therapeutic effects. Specifically, this study aims to assess the diagnosing performance of 18F-FDG and 68Ga-FAPI-04 PET for different stages of progressive bone metastases with PSMA-negative pathology. Bone metastatic mouse model of prostate cancer was constructed via intra-bone injection of PSMA-negative prostate cancer PC3 cells. Cellular uptakes of 18F-FDG and 68Ga-FAPI-04 were separately performed on PC3, NIH-3T3 (FAP-positive) and a mixture. 68Ga-PSMA-11, 18F-FDG and 68Ga-FAPI-04 PET/CT imaging were performed at 2, 4 weeks after tumor cell transplantation. Furthermore, PSMA and FAP expression in bone metastases were assessed by immunohistochemistry, and then compared with the imageological findings. On the cellular level, the independent tracer uptake on the basis of glycometabolism and fibrosis was observed. For animal imaging, 68Ga-PSMA-11 imaging showed weak or absent tracer uptake in PSMA-negative bone metastatic lesions. In contrast, 68Ga-FAPI-04 PET of bone metastases had a higher uptake and tumor-to-muscle (T/M) ratio than 18F-FDG PET that was relative steady during the observation, but T/M ratio of fibrosis gradually decreased with increasing tumor growth, which ranged from 5.11 ± 1.26 at 2 weeks to 3.54 ± 0.23 at 4 weeks, revealing the delayed formation of tumor stroma in rapid proliferation. In addition, PET imaging results were corroborated by immunohistochemical assessment. In conclusion, molecular imaging approach revealed the heterogeneous progression of tumor cells and tumor stroma of bone metastasis of prostate cancer, and further confirming the necessity of multi-molecular imaging in cancer imaging.

Keywords: Molecular imaging, heterogeneity, prostate cancer, prostate-specific membrane antigen, fibroblast activation protein, bone metastases, glycometabolism, fibrosis

Introduction

Prostate cancer (PCa) is the second most commonly diagnosed cancer worldwide and the fifth leading cause of cancer death in men worldwide [1]. The most common site of metastasis from prostate cancer is bone, occurring in 65%-80% of patients with advanced disease [2]. Bone metastases (BMs) in prostate cancer are associated with pathologic fractures, pain, and reduced survival [3]. PCa that has metastasized to the bone and visceral tissues is highly lethal with 5-year survival rates remaining at 30% [4]. The clinical course of PCa is highly heterogeneous, ranging from indolent to aggressive phenotypes [5]. Heterogeneity exists both among patients, with variable rates of progression, and within individual tumors, which can exhibit a mixture of molecular subtypes. For example, some patients show an indolent course with slow progression, while others rapidly develop lethal metastatic disease. PCa can contain mixtures of molecular subtypes, including TMPRSS2-ERG fusion, SPOP mutation, and others [6]. This heterogeneity, together with the overall prolonged natural history of PCa compared to other solid tumors, creates challenges in diagnosis, staging, and assessing treatment response [7]. Hence, precise diagnostic strategies are warranted to guide therapeutic decisions and improve management across the heterogeneous manifestations of PCa [8]. Molecular imaging approaches that capture different aspects of tumor phenotype may provide enhanced personalized information to meet this need [9].

Prostate-specific membrane antigen (PSMA) is commonly over-expressed in PCa [10], enabling for PSMA-targeted positron emission tomography (PSMA PET). Currently, recurrent PCa is the primary indication for PSMA PET, and the majority of published clinical data concentrated on this issue. In contrast to technetium-99m-methyl diphosphonate (99mTc-MDP) bone scan, PSMA PET is feasible to detect metastatic PCa earlier in patients who have undergone radical prostatectomy, modifying management in about half of the patients [11-13]. However, some patients have inter- or intra-lesional heterogeneity at presentation, and some have tumor characteristics that are PSMA-negative, leading to false negative imaging findings [14]. Prior research indicated that PSMA PET results were negative when PSMA-negative tumor area was larger than 50% on immunohistochemistry (IHC), despite very high level of prostate-specific antigen (PSA) expression [15]. In addition, BMs could be PSMA-negative in up to 15% cases [16]. False-negative results in PSMA PET may mislead staging and assessment of therapeutic efficacy in PCa patients. Given this heterogeneity and risk of false negatives with single modality imaging, there is a need for personalized diagnostic strategies in PCa. Molecular imaging approaches that provide complementary biological information could help address this need. Multimodal imaging combining targets such as PSMA, glucose metabolism, and tumor microenvironment markers may improve characterization of heterogeneous PCa phenotypes.

Despite its widespread use, 18F-FDG PET has limited utility for PCa imaging due to the typically low glucose metabolism of prostate tumors [17]. However, changes in glycometabolism during PCa progression, such as upregulation of GLUT transporters, provide a rationale for evaluating 18F-FDG PET in advanced disease [18]. Furthermore, some studies have shown that 18F-FDG PET can detect lesions in certain cases of PCa that are negative on 68Ga-PSMA PET [19,20]. The underlying mechanism for this discrepancy is unclear, but may involve neuroendocrine differentiation or other unknown factors inducing increased glycolysis. While 18F-FDG is suboptimal for routine PCa imaging, these findings suggest it could serve as a complementary modality for patients with biochemical evidence of disease but negative 68Ga-PSMA PET scans. Overall, 18F-FDG PET may be a useful supplemental imaging tool in the subset of PCa patients with low or absent PSMA expression.

In addition to tumor cells, the prostate cancer microenvironment undergoes important changes during progression. Cancer-associated fibroblasts (CAFs) play an important role in the TME because they communicate with PCa cells, affecting their metabolism and vulnerability to medicines [21]. There is compelling evidence to support the role of CAFs in the tumorigenesis and progression of PCa. These roles are predominantly played via contact-dependent and paracrine-signaling processes that are shared by normal and activated fibroblasts that surround PCa cells [22]. A key marker of CAF activation is upregulation of fibroblast activation protein (FAP) expression. Imaging with FAP inhibitors may enable characterization of the tumor microenvironment. Recently, quinoline-based FAP inhibitors (FAPI) have demonstrated promising outcomes in the diagnosis of several cancer types including PCa, making them the hotspot of research in nuclear medicine and molecular imaging. Although the role of FAP in PCa has not yet been extensively evaluated, it appears as the potential to identify the lesion that is of negative or low PSMA expression [23].

Above all, glycometabolism and fibrosis were the two main progression in progressive development of tumor cells and tumor stroma. In the present study, bone metastasis mouse model of PCa bearing PSMA-negative prostate cancer cells was constructed as a typical model with heterogeneity. Three PET imaging methods (i.e., 18F-FDG, 68Ga-PSMA-11 and 68Ga-FAPI-04) were performed in mouse models at different stages of tumor growth. The feasibility of FDG and FAPI PET as the diagnostic tool for the evaluation of progressive BMs with PSMA-negative prostate cancer was assessed.

Materials and methods

Preparation of 68Ga-PSMA-11 and 68Ga-FAPI-04 probe

The PSMA (HBED-CC-PSMA) and FAPI (DOTA-FAPI-04) precursor were purchased from Nice-Labeling Technology Co., Ltd. 68Ga-PSMA-11 and 68Ga-FAPI-04 were prepared in house. For 68Ga-PSMA-11 preparation, the precursor PSMA-11 was dissolved in 1 mL 0.05 M NH4OAc, and then mixed with 4 mL freshly eluted 68GaCl3 (in 0.05 M HCl). The reaction system was heated to 100°C and maintained for 10 min. Radiochemical purity (RCP) was measured with a HPLC with radio-detector. An analysis column (Wondasil C18 Superb 5 µm 4.6 × 250 mm) was used with a flow rate of 1 mL/min, and parameters were set as solvent A: water/0.1% TFA, solvent B: acetonitrile/100%, gradient details: 0-13 min, 33%-67% B. For 68Ga-FAPI-04 probe preparation, the same procedure as 68Ga-PSMA-11 was performed.

18F-FDG was purchased from Atom Kexing Pharmaceuticals, and quality control was performed with a TLC with radio-detector. 95% acetonitrile was used as the mobile phase, and silica gel thin layer chromatography plate was used as the stationary phase. The Rf of FDG was around 0.4-0.5.

Cell culture and cellular uptake

PSMA-negative PCa cell line PC3 and embryonic mouse fibroblast cell line NIH-3T3 were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) or RPMI 1640 medium (RP1640, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37°C in 5% CO2.

For cellular uptake of 68Ga-PSMA-11, 18F-FDG or 68Ga-FAPI-04, 1 × 105 PC3 or NIH-3T3 in logarthmic growth phase were co-cultured with 0.37 MBq radiopharmaceuticals for one hour. Medium was removed, and cells were washed with PBS for three times. Cellular uptake was quantified with a gamma-counter.

For cellular uptake of 18F-FDG or 68Ga-FAPI-04 that was performed on cell mixture, PC3 and NIH-3T3 cells were mixed together in a 1:1 ratio and plated in 24-well plates at a total density of 1 × 105 cells/well. Cells were allowed to adhere overnight before radiotracer incubation. On experiment day, culture media was replaced with fresh media containing 0.37 MBq of 18F-FDG or 68Ga-FAPI-04 and incubated for 1 hour at 37°C. After incubation, media was removed and cells were gently washed 3 times with PBS. Cells were harvested and radioactivity was measured using a gamma-counter.

Bone metastasis model preparation

Male nude mice (8-10 weeks old, 18-20 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. Animals were housed under a 12-h light/12-h dark cycle and allowed free access to food and water. For mouse models, the leg of the anesthetized mouse was flexed. A 27-gauge syringe needle was inserted into the marrow cavity parallel to the long axis of the femur, followed by injection of 20 µL of PC3 cell suspension (1 × 106 cells per mouse). All mouse experiments were approved by and performed following the guidelines of the Institutional Animal Care and Use Committee at Changhai hospital.

18F-FDG, 68Ga-PSMA-11 and 68Ga-FAPI-04 PET/CT scanning

18F-FDG and 68Ga-FAPI-04 PET/CT imaging was performed at 2, 4 weeks after tumor cell transplantation, respectively. 68Ga-PSMA-11 PET/CT was performed at 4 weeks after tumor transplantation. 18F-FDG, 68Ga-PSMA-11 and 68Ga-FAPI-04 PET/CT scans were carried out within two days, with at least 12 h between scans to allow for physical decay of the previous tracer before the next scan. Before PET measurements, mice were anesthetized by intraperitoneal injection of Lidocaine (3%, wt%). For 18F-FDG PET/CT, mice were fasted for 6 h before injection of 18F-FDG into the tail vein. The dosage of 18F-FDG injection was 5.55-7.40 MBq. Data were acquired using a PET/CT scanner (Biograph64, Siemens, Germany) after 1 h of intravenous administration. Image acquisition parameters were set as follows: for CT, tube voltage is 120 kV; Tube current: 35 mA; Pitch: 1.0; Reconstruction layer thickness: 1 mm. PET data acquisition was obtained for 5 minutes per bed position. For 68Ga-PSMA-11 and 68Ga-FAPI-04 PET/CT scan, the injection dose and procedure were the same as above, except that the radiopharmaceutical were 68Ga-PSMA-11 and 68Ga-FAPI-04, respectively.

Image analysis

TureD system of post-processing workstation was used for corrected for attenuation and image reconstruction to form transverse plane, coronal plane and sagittal plane and generate 3D projection image. Regions of interest (ROIs) were manually outlined on the muscle, and bone metastases. The PET/CT workstation provides SUV (standardized uptake value) as the quantitative value. One experienced nuclear medicine physician was responsible for measuring the SUVmax value and calculating tumor-to-background ratios.

Hematoxylin & eosin staining and immunohistochemistry

All mice that underwent PET imaging were euthanized by cervical dislocation. The right hindlimb was dissected and fixed in 10% neutral-buffered formalin, and decalcified in formic acid. Paraffin-embedded sections were stained with hematoxylin & eosin (H&E) and immunohistochemistry (IHC) according to standard protocol. The expression of PSMA and anti-fibroblast activated protein α (FAPα) was quantified by using the IHC toolbox plugin in ImageJ software. The following primary antibodies were used: anti-PSMA mAb (1:250, ab133579, Abcam, Shanghai, China), anti-FAP mAb (1:100, ab53066, Abcam, Shanghai, China).

Quantification and statistical analysis

Data were presented as the mean ± standard deviation of at least n = 3. Comparisons were performed using an unpaired t test. A P value less than 0.05 was considered statistically significant. Data were analyzed using GraphPad Prism version 9.3 (GraphPad Prism Software).

Results

Synthesis and radiolabeling

As the clinical used radiopharmaceuticals, 18F-FDG, 68Ga-PSMA-11 and 68Ga-FAPI-04 were automatically synthesized with a radiochemical purity of higher than 95%, guaranteeing the real represent of pathology-related tracer uptake. The typical radio-HPLC spectrum of 68Ga-FAPI-04 and 68Ga-PSMA-11 were as Figure 1A, 1B, and the typical radio-TLC spectrum of 18F-FDG was as Figure 1C.

Figure 1.

Figure 1

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The typical radio-HPLC spectrum of 68Ga-FAPI-04 (A) and 68Ga-PSMA-11 (B), and the typical radio-TLC spectrum of 18F-FDG (C).

Independent tracer uptake of 18F-FDG and 68Ga-FAPI-04

PC3 cells stand for the PSMA-negative model cell, and embryonic mouse fibroblast cell line NIH-3T3 stand for the FAP-positive model cell. The above two cells were independent with concomitant matrix in verifying PSMA- or FAP-related tracer uptake. All of the co-culture with radiopharmaceuticals were performed for one hour. 68Ga-PSMA-11 uptake was minimal and equivalent in PC3 (0.25 ± 0.05%) and NIH-3T3 (0.29 ± 0.03%) cells, and no difference was observed (Figure 2A), confirming lack of PSMA expression.

Figure 2.

Figure 2

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Cellular uptake of 68Ga-PSMA-11, 18F-FDG and 68Ga-FAPI-04. A. Cellular uptake of 68Ga-PSMA-11; B. Cellular uptake of 18F-FDG and 68Ga-FAPI-04; C. Cellular uptake of 18F-FDG and 68Ga-FAPI-04 in cell mixture.

18F-FDG uptake was higher in PC3 cells (3.55 ± 0.37%) compared to NIH-3T3 cells (1.51 ± 0.57%), while 68Ga-FAPI-04 uptake was lower in PC3 cells (0.91 ± 0.15%) versus NIH-3T3 cells (3.91 ± 0.35%) (Figure 2B). A FAP-negative biochemical nature of PC3 was proved. For mixed cultures containing equal numbers of PC3 and NIH-3T3 cells, 18F-FDG and 68Ga-FAPI-04 uptake levels were intermediate between the individual cell lines (Figure 2C). This supported the independence of glycolytic metabolism versus fibroblast targeting.

Image analysis

Figures 3 and 4 presents the typical PET/CT images, including 68Ga-PSMA-11, 18F-FDG and 68Ga-FAPI-04 in a bone metastatic model, and corresponding quantitative data of mice at 2- and 4-week post tumor cell transplantation. As expected, 68Ga-PSMA-11 imaging showed very weak or absent radiotracer uptake in BMs at 4 weeks after transplantation. Weak uptake of tumor was due, in part, to nonspecific accumulation caused by increased tumor blood flow and vascular permeability. For the 18F-FDG PET/CT images acquired at 60 min post injection, high radiotracer uptake was observed in the site of bone metastasis at 2 weeks after transplantation, with SUVmax of 0.87 ± 0.20. Two weeks later, 18F-FDG PET/CT showed irregular mass shadow of high density, with increased radiotracer uptake in the site of bone metastasis, with SUVmax of 0.92 ± 0.41 (Figure 4B). The kidney and bladder displayed relatively high radioactivity accumulation at 60 min post injection. The radioactivity accumulation of the bladder was represented as the total excretion at 60 min post injection. Compared with FDG PET imaging at 2 weeks (T/M ratio was 3.48 ± 0.57), a high contrast of a T/M ratio of 4.38 ± 1.10 was achieved at 4 weeks post transplantation (Figure 4C).

Figure 3.

Figure 3

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(A) Change in PC3 xenograft tumor volume after transplantation. The typical PET imaging of 68Ga-FAPI-04 (B), 68Ga-PSMA-11 (C) and 18F-FDG (D) in mouse model bearing bone metastases with negative-PSMA expression at different time points after transplantation.

Figure 4.

Figure 4

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A. The SUVmax of 68Ga-FAPI-04 in the bone metastases and muscle. B. The SUVmax of 18F-FDG in the bone metastases and muscle. C. The tumor-to-background (muscle) ratio of 68Ga-FAPI-04 and 18F-FDG in the mouse at different time points after transplantation.

For 68Ga-FAPI-04 imaging, the BMs had a high uptake of 68Ga-FAPI-04 at 2 and 4 weeks after transplantation (Figure 4A). Other organs demonstrated low nonspecific binding that quickly decreased, resulting in low background signal and favorable tumor-to-background ratios. T/M ratios gradually decreased with increasing tumor size, which ranged from 5.11 ± 1.26 at two weeks to 3.54 ± 0.23 at four weeks (Figure 4C).

In addition, the T/M ratio of 68Ga-FAPI-04 was higher than that of 18F-FDG (5.11 ± 1.26 vs. 3.48 ± 0.57, t = 1.657, P = 0.173) at 2 weeks post transplantation. The comparison of the T/M ratio of 68Ga-FAPI-04 with that of 18F-FDG was reversed at 4 weeks post tumor transplantation (3.54 ± 0.23 vs. 4.38 ± 1.10, t = 1.054, P = 0.351) (Figure 4C). In these cases, the high T/M ratio could clearly distinguish BMs from background visually, although there was no statistically significant difference between these two groups at both time points.

H&E and immunohistochemistry

In gross pathological anatomy, a mass was seen in the femur on the side of the PCa tumor cells inoculated, which was grayish-white and bulged outward. In histopathology of bone metastasis, a large number of epithelial cells in the bone marrow cavity and outside the bone cortex. The tumor cells were arranged in fascicles with obvious atypia, hyperchromatic with a large nucleus (Figure 5A, 5D). IHC staining showed that the expression of PSMA was negative in bone metastasis tissues (Figure 5B, 5E), while FAP was highly expressed, and the positive staining area of FAP was 21.51 ± 2.64% (Figure 5C, 5F).

Figure 5.

Figure 5

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Hematoxylin & eosin and immunohistochemical staining of FAP and PSMA expression in bone metastatic lesion of PCa. A, D. Photomicrograph of H&E-stained bone metastatic lesion (40 × and 200 ×, respectively); B, E. Bone metastatic lesion demonstrated negative PSMA expression on immunohistochemistry (40 × and 200 ×, respectively); C, F. Fibroblast activation protein-α (FAP-α) staining in bone metastatic tissues, showing high expression of FAP-α in tumor stroma (40 × and 200 ×, respectively).

Above all, tumor progression was complicated and dynamic, hence, a single molecular imaging was miss-leading in understanding tumor status. The medical pictures of glycometabolism and fibrosis that were acquired at 2 and 4 weeks manifested the asynchronism of tumor progression and tumorous stroma formation.

Discussion

For individuals with PCa, early diagnosis of distant metastases is crucial for staging and therapeutic planning, but the diagnostic results relied on a sole imaging modality was somewhat unilateral in patient management. In the past few years, the use of 68Ga-PSMA-11 PET/CT rapidly spread worldwide. Numerous studies have highlighted the high sensitivity of 68Ga-PSMA-11 PET/CT in PCa patients as compared to other imaging modalities [24]. Of note, a few PCa types do not adequately express PSMA. However, there are limited studies of other imaging methods available for the detection of PCa patients that are of negative PSMA uptake [25,26]. Therefore, despite patients’ having high PSA levels, PSMA-based imaging may not indicate any pathological lesions [27].

18F-FDG is frequently thought to perform poorly in PCa imaging because of its unique metabolic properties that result in a low affinity for glucose in metastatic lesions of PCa [28]. In our present study, 18F-FDG PET/CT imaging could sensitively detect BMs lesions that were PSMA-negative in early-stage, and could continuously monitor the progressive changes of BMs. These results are consistent to the imaging findings of Chen et al. in patients with biochemical recurrent PCa [25], implying that 68Ga-PSMA-11-negative patients with biochemical recurrence were likely to benefit from 18F-FDG PET. Despite the potential for FDG imaging in PSMA-negative tumors, the reason behind these imaging findings is unclear. One reason may be involved in other yet-unknown physiological or pathological conditions, such as neuroendocrine differentiation in prostate cancer. Bakht et al. revealed that high levels of glucose uptake were correlated to a suppression of PSMA and an elevation of neuroendocrine biomarkers [29]. Conversely, several reports showed that neuroendocrine differentiation of PCa was not associated with increased 18F-FDG uptake [30]. Albeit plausible, this mechanism awaits further research and validation. However, we still recommend FDG PET as an essential tool for PSMA-negative imaging, especially for progressive PCa diagnosis and treatment monitoring.

Although the molecular markers of tumor cells are significantly different and even deficient, the tumor microenvironment has similar characteristics. CAFs are a pivotal component of stroma, accounting for 90% of the total tumor mass in desmoplastic tumors. The PCa-promoting characteristics of CAFs have been shown in numerous research, including raising tumorigenic potential, improving androgen sensitivity, and favoring the metastatic process [31-33]. Because CAFs are genetically more stable than cancer cells, diagnostic and therapeutic targeting of CAFs in the stroma has distinct advantages and potential. In this study, we hypothesized that FAP imaging could serve as a potential diagnostic tracer targeting CAFs for PSMA-negative PCa, and a robust uptake of the FAPI tracer was observed in the PSMA-negative BMs. The imaging results were corroborated by immunohistochemical studies using FAPα antibody. Additionally, high tumor-to-background contrast was paramount for tumor imaging. In the present study, a relative high T/M ratio in FAPI imaging, up to 5.11 ± 1.26 were observed. Furthermore, we also found that T/M ratio of 68Ga-FAPI-04 imaging was higher than that of 18F-FDG imaging in the early-stage of BMs development, implying that stroma formation accompanied with BMs and was more active in early stage, but the delayed formation of tumor stroma in rapid proliferation was observed as well. Hence, the additive value of FAPI imaging was exhibited by the enhanced T/M ratios, increasing the confidence in the lesion diagnosis.

Nevertheless, little is known regarding FAP expression in various disease stages of PCa. According to a recent study by Hintz et al., FAP expression is a characteristic of metastatic castration-resistant PCa regardless of genetic subtype, therapeutic strategy, or metastatic site. Moreover, they found that a significant increase in FAP mRNA expression in metastatic disease compared to primary PCa [34]. Similarly, in a separate clinical trial, Kesch et al. indicated that 68Ga-FAPI-04 PET/CT was highly positive in subjects with advanced castration-resistant PCa [35]. These data were consistent with our results and further provided a rationale for FAPI imaging as a potential diagnostic tool for PCa, particularly in patients with heterogeneous tumor phenotypes. Notably, the nature of FAP expression is very non-specific in a mixed illness environment and at early diagnosis because it is related to chronic inflammation, including but not limited to, rheumatoid arthritis, fibroses, and wound healing. Hence, we stressed that due to its lack of specificity, single-step imaging with FAP may not be helpful for the early identification of PCa. Patients with PSMA-negative or PSMA-expression heterogeneity may benefit more from FAP imaging for PCa diagnosis, as well as the potential of FAPI-targeted theranostic in PCa. Large-scale randomized clinical trials are further required to generate definitive evidence.

FDG PET is known to have limited specificity for cancer due to uptake in sites of inflammation and infection. Similarly, FAP expression occurs in wound healing, fibrosis and other conditions, reducing specificity of FAPI PET when interpreted alone. Caution is warranted when evaluating either scan, or correlation with clinical context is key for accurate interpretation. For PCa, FDG and FAPI PET may be the most suitable for assessing advanced disease where PSMA imaging falls short. FDG could identify morphological and metabolic alterations associated with aggressive tumors [36]. FAPI may detect stromal remodeling during progression to castration resistance [35]. However, questions remain regarding appropriate clinical use cases, influence of therapies, and ability to improve patient outcomes. Larger prospective studies are needed to define applications and limitations.

In summary, while FDG and FAPI PET hold promise for characterizing prostate cancer in certain scenarios, care is required when applying these modalities clinically due to non-specificity issues. Additional research is needed to realize their full potential while mitigating limitations.

This study has several potential limitations. Firstly, the experimental model was the transplantation of PSMA-negative PCa cells into the femur of mice, which were poorly able to sufficiently mimic the bone metastatic progression after the occurrence of orthotopic prostate tumor formation, as in the human disease. Secondly, FDG and FAPI imaging for different pathological subtypes and different course of prostate cancer were not evaluated due to objective limitations of the mouse model.

Conclusions

This study revealed the feasibility of 18F-FDG and 68Ga-FAPI-04 PET/CT as the complementary diagnostic tool for bone metastasis of PCa with negative PSMA-expression. Due the complexity of tumor progression, FDG and FAP-targeted imaging may be valuable as a candidate application for the PCa patients with PSMA-negative or PSMA-expression heterogeneity, especially for tumor cell proliferation and tumor stroma.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (81871390, 82272040), and the First Affiliated Hospital of Naval Medical University (Shanghai Changhai Hospital) “Guhai Plan in the 14th Five-Year Plan period” (GH145-22).

Disclosure of conflict of interest

None.

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