Introduction

Angiotensin-converting enzyme 2 (ACE2) is a zinc metalloproteinase with encoding gene located on X chromosome and belongs to type I transmembrane protein. The structure of ACE2 protein consists of a signal peptide, a transmembrane domain and a metalloproteinase active site containing a zinc-binding domain. As a homolog of angiotensin converting enzyme (ACE) and an important constituent in renin–angiotensin–aldosterone system (RAAS), ACE2 mainly degrades Ang II to produce heptapeptide angiotensin 1–7 (Ang 1–7), regulates blood pressure and fluid balance [1,2,3]. ACE2 was initially thought to be expressed only in the heart, kidney, and testis but was later proved of low-level expression in the eyeballs, brain, lungs, and digestive tracts [4].

Besides the normal expression, ACE2 is figured out as a key regulatory molecule in the pathological remodeling of many diseased conditions [5]. Currently, ACE2 has become a research hotspot as a target protein of host cells in SARS-CoV-2 infection [4, 5]. Before the COVID-19 pandemic, it has been proved that ACE2 protein is specifically up-regulated in some tumorous tissues [6]. The up-regulation of ACE2 expression together with the tumor-induced decline in immunity constitute the pathological basis of tumor burden that serves as one of the risk factors of coronavirus infection [7, 8]. In clinic, ACE2 protein is over-expressed in kinds of tumors and involved in a variety of biochemical behaviors, such as tumor genesis, growth, metastasis, and tumor inhibition [6]. For instance, GEPIA online analysis and HPA database analysis of tumor data showed that ACE2 mRNA and the subsequent expression of ACE2 protein were high in gastrointestinal tumors, such as colon cancer and gastric cancer. Similarly, ACE2 mRNA expression was relatively high in lung cancer and renal papillary cell carcinoma as well, but lower than that in gastrointestinal tumors [9]. These above characteristics made up the principle of ACE2-based tumorous localization and differentiation.

The existing detection tool for ACE2 expression in tumors and other pathological processes included but not limited to western blot (WB), immunohistochemistry (IHC), polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) [10], all of which cannot elucidate the systematic ACE2 fluctuation as a whole and cannot be visualized in vivo, in particular, lacking the ability of dynamic and quantitative assessment of whole body. In consideration of the sensitivity of PET imaging and specificity of molecular imaging, ACE2 PET may be meaningful in the detection, localization, and functional evaluation for multiple tumors, especially when combined with CT or MRI; meanwhile, ACE2 PET is promising to differentiate the tumors between subtypes, and determine the primary foci and metastasis.

So far, ACE2-targeted molecular imaging has been proposed to evaluate pathological changes of ACE2 expression, such as SARS-CoV-2 infection and tumors [11,12,13]. In this study, 68Ga-cyc-DX600 PET will be used as the diagnostic mode of tumorous ACE2 and verified with ex vivo analysis of tumorous tissues. Mice models included the tumors in RAAS organs and others, such as lung cancer, breast cancer, prostate cancer, and so on. Additionally, in an esophageal cancer patient, ACE2 PET was compared with FDG PET in evaluating malignancy.

Method

Preparation and characterization of radiopharmaceuticals

68Ga-cyc-DX600 was prepared in house with DOTA-modified cyclic DX600 peptide (cyc-DX600-DOTA, DX600 peptide with condensed disulfide bond of cysteine) as the precursor following the reported protocol [11, 12]. In detail, the newly eluted 68Ga3+ in 4 mL 0.05 M HCl was mixed with precursor in 1 mL 0.25 M NH4Ac, heated to 100 ℃ and maintained for 10 min. Radiochemical purity (RCP) was measured with HPLC system (1260 Infinity, Agilent Technologies) equipped with a radioactive detector (Flow-count, Eckert & Ziegler) to determine the labeling rate, as well as the stability in PBS and 5% fetal bovine serum for one hour incubation at room temperature.

18F-FDG was purchased from Atom Kexing Radiopharmaceuticals Ltd with strict quality control. Radiopharmaceuticals with labeling rate higher than 95% were used in imaging research immediately, and the specific radioactivity of 68Ga-cyc-DX600 was controlled as about 3.7 MBq/µg in the imaging research on various tumor-bearing mice models or patients.

To verify the maintain of ACE2 targeting ability on cellular level, 125I-labeled DX600 and 125I-labeled cyc-DX600-DOTA were synthesized via Iodogen-catalyzed iodization with I-125. Cellular binding was performed on 1 × 105 HEK-293T/hACE2 cells that expressed humanized ACE2 protein. After the co-culture with 1 nmol radiopharmaceuticals for 30 min or 1 h, the excess radiopharmaceuticals were washed away by cold PBS for three times, and the bound peptide or precursor was quantified with a gamma-detector.

Establishment of xenografted models

The animal experiments involved in this study strictly complied with China’s national laws, regulations, and standards related to experimental animals, including Regulations on the Management of Experimental Animals (Revised version on March 1, 2017) and Guidelines for Ethical Review of Experimental Animal Welfare (GB/T 35,892–2018). This project was approved and guided by the Ethics Committee of Ningbo University (Approval No.: NBU20210040).

NOD-SCID mice (male, 6 weeks old) were used in this research. HEK-293 cells and HEK-293T/hACE2 cells were used to establish the paired models to verify the ACE2 specificity of 68Ga-cyc-DX600. The angiotensinogen degradation and circulation involved the liver, kidney, lung, and others, so MIA-PaCa-2 human pancreatic cancer (PanC) cell, 786-O human renal clear cell adenocarcinoma (ccRcc) cell, SK-N-SH human neuroblastoma (NB) cell, 22Rv1 human prostate cancer (PCa) cell, A549 human non-small cell lung cancer (NSCLC) cell, HepG2 human hepatoma (HCC) cell, as well as 4T1 mouse breast cancer (BC) cell were selected to establish the subcutaneous xenograft tumor models.

Cells at logarithmic growth stage were selected, digested, and re-suspended with 0.01 M PBS. Tumor cells suspension was mixed with basement membrane matrix in equal volume, and then stored temporarily in ice-cold water. The needle was inserted completely into the subcutaneous position horizontally to inject 100 µL mixture (1 × 106 cells), and then held for 30 s to solidify the mixture. The successful rate and growth rate of xenografts depended on the characteristics of tumor cells, such as proliferating potential and invasiveness. There was a period of 10 to 40 days for xenografts to reach 250 ± 50 mm3.

68Ga-cyc-DX600 PET/CT of model mice

NOD-SCID mice (n = 3, male, 6 weeks old) without tumor burden were utilized to characterize the metabolic features. For each mouse, 3.7 MBq 68Ga-cyc-DX600 was intravenously injected. In order to maintain a same injected specific activity, mice from the same group were simultaneously injected, anesthetized, and scanned at 15, 30, 45, 60, 90, and 120 min postinjection (min P.I.) using the clinical used PET/CT scanner (Biograph64, Siemens, Germany). PET/CT scans started with a low-dose CT scan with tube voltage: 120 kV; tube current: 35 mA; pitch: 1.0; reconstructed layer thickness: 1 mm; and followed by PET scan of the whole body within one bed for 3 min.

After the optimization of scan timepoints, scans at 60 min P.I. were performed for the subsequent experiments. For ACE2 PET of tumor models, 3.7 MBq 68Ga-cyc-DX600 was intravenously injected into each mouse. Mice were kept anesthetic and scanned with the same protocols as above. For the block experiments performed on models bearing HEK-293T/hACE2 xenograft tumor, 20 µg DX600 peptide (c.a. 20-fold of precursor dosage) were injected at 1 h before the injection of 68Ga-cyc-DX600.

Image reconstruction was performed in the postprocessing workstation TureD system to form the maximal intensity projection (MIP) images and hybrid PET/CT images in transverse, coronal, and sagittal planes. PET/CT workstation provided a quantification value of tracer uptake as maximum Standardized Uptake Value (SUVmax) of ACE2 PET. Regions of interest (ROIs) were drawn on the tumors and other organs. A nuclear medicine physician was responsible for the identifying, outlining and measuring of ROIs, which were checked by another physician later.

Biodistribution, immunohistochemistry, and western blotting

Immediately after the completion of ACE2 PET scan of mice with tumor burden of HEK-293 or HEK-293T/hACE2 cells, mice were sacrificed via hyperanesthesia to harvest the blood and tumors, and then the main organs were washed with 10% paraformaldehyde twice. All the samples were weighed and then measured with a gamma-counter. The organ-specific biodistribution was expressed as the percent of injected dosage in unit tissue weight (%ID/g).

Immunohistochemical analysis of HEK-293-based subcutaneous tumors was performed to observe the ACE2 expression, so as to verify the source of high uptake in ACE2 PET. Deparaffinized sections were boiled in 1 × target retrieval solution (Dako, Capenteria, CA) for 20 min. After blocking in 5% normal goat serum diluted in 0.01 M PBS, the sections were incubated with anti-ACE2 antibody (ab108209, Abcam, Burlingame, CA, USA) overnight at 4 ℃. The sections were washed with 0.01 M PBS for three times, and then the second antibody was added and incubated at room temperature for 30 min. After washing with 0.01 M PBS again, the sections were stained with DAB, counterstained with hematoxylin, dehydrated, cleared, and mounted. Slides were evaluated under 200 × objective and digitized using a color camera mounted to the microscope.

For the tumorous tissues, western blotting was performed according to a standard protocol using anti-ACE2 antibody. Fresh tumorous tissues were lysed in radioimmunoprecipitation assay buffer containing 1 mM phenylmethylsulfonyl fluoride. After removing cell debris by centrifugation (11,000 rpm) for 10 min at 4 ℃, the supernatant protein solution was adjusted to equal concentration and then mixed with loading buffer. Samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, blocked with 5% milk in TBST. After washing the membranes with TBST for three times, horseradish peroxidase-conjugated secondary antibody was added and incubated together 2 h at room temperature, and then washed again with TBST. Band intensity of ACE2 proteins was normalized to that of GADPH, and signals were detected by enhanced chemiluminescence.

Patients enrollment and PET imaging of subjects

Four patients with different tumor types were enrolled in this research to further verify the efficiency of ACE2 PET in tumor imaging. As a standard imaging modality in tumor evaluation, FDG PET was performed on each subject as well with an interval of less than one week.

Patients were scanned with PET/MR scanner (Biograph mMR; Siemens Healthcare, Erlangen, Germany) with 68Ga-cyc-DX600 intravenously injected at 1.85 MBq/kg body weight. One subject was scanned twice from 50 to 70 min P.I. and from 80 to 100 min P.I., the other three patients were scanned from 80 to 100 min P.I. only; similarly, 18F-FDG was intravenously injected at 3.7 MBq/kg body weight and scanned from 50 to 70 min P.I. Seven bed positions were scanned with 3 min per bed. The MRI protocol was T2-weighted sequence with fat suppression for whole-body positioning: transversal, TR 3000 ms, TE 89 ms, flip angle 90°, 33 slices, slice thickness 6 mm, FOV 400 × 400, voxel size 1.3 × 1.3 × 6.0 mm3. Notably, FDG PET of the lung cancer patient was scanned with a PET/CT scanner (Biograph64, Siemens, Germany) in a routine protocol.

Statistics

All the 68Ga-cyc-DX600 uptakes were quantified as SUVmax of ACE2 PET. The ratio of blood pool (heart) to right thigh muscle (H/Ms) was quantified to record the tendency of blood clearance. A linear regression was performed between WB results and SUVmax. Difference assessments specified for cellular binding and in vivo tracer uptake were performed with Student’s t-test. All the statistics were performed with IBM SPSS Statistics 26, and any differences with p-value less than 0.05 were defined as statistically significant.

Results

Fig. 1 depicts the synthesis of 68Ga-cyc-DX600 and I-125 labeled compounds that started from the DX600 peptide. The disulfide bond increased the structural stability of precursor, and the modification with DOTA empowered the peptide with possibility in transferring to a theranostic radiopharmaceutical. After the modification, cyc-DX600-DOTA was of a theoretical molecular weight of 3418.77, and radio-HPLC (Fig. 1a and b) manifested that the labeling rate of 68Ga-cyc-DX600 was higher than 99%. The labeling procedure was completed within 20 min with a routine radiochemical yield of 75–80%.

Fig. 1
figure 1

Schematic diagram of preparation and the characterization of radiopharmaceuticals, including the radio-HPLC spectrum (a and b) of 68Ga-cyc-DX600, radio-TLC of 125I-DX600 (c) and 125I-cyc-DX600-DOTA (d), stability of 68Ga-cyc-DX600 (e), and cellular binding of 125I-DX600 and 125I-cyc-DX600-DOTA to HEK-293T/hACE2 cells (f)

Radio-TLC (Fig. 1c and d) manifested that the labeling rates of I-125 labeled DX600 and cyc-DX600-DOTA were acceptable (> 95%) for cellular binding assay. After incubation for 1 h at room temperature, 68Ga-cyc-DX600 was stable in 0.01 M PBS with RCP of 96.1 ± 1.2%, as well as 91.2 ± 2.5% in 5% FBS (Fig. 1e).

The in vitro combination efficiency (Fig. 1f) of 125I-labeled DX600 and 125I-labeled cyc-DX600-DOTA proved the comparable binding efficiency to ACE2 antigen, 32.3 ± 3.5% vs 31.5 ± 2.9% (p > 0.05) at 30 min and 38.1 ± 3.7% vs 36.6 ± 3.4% (p > 0.05) at 60 min.

Based on MIP images of 68Ga-cyc-DX600 PET (Fig. 2a), tracers quickly distributed to whole body and were gradually cleared from blood circulation. The elimination from the body via urinary system happened soon after the injection. Hence, heart and kidneys, as the blood pool and metabolic organs, were of the highest SUVmax during the 120 min observation (Fig. 2b), but the tracer of heart was nearly cleared out at the end of observation. Although the clearance of tracers continued after 60 min P.I., there was no statistically difference of SUVmax for most organ at the following time points. For example, SUVmax of heart decreased from 1.15 ± 0.08 at 15 min P.I. to 0.71 ± 0.13 at 60 min P.I. (p < 0.05), but there was no statistical difference between SUVmax_60 and SUVmax_90 (0.71 ± 0.13 vs 0.58 ± 0.11, p > 0.05), as well as SUVmax_90 and SUVmax_120 (0.58 ± 0.11 vs 0.43 ± 18, p > 0.05). NOD-SCID mice provided a clear biological background for ACE2-targeted tumor imaging. In addition, the ratio of H/Ms was of the tendency to be stable as 3.8 between 45 and 60 min P.I., as well as the background (muscle) was relatively stable after 60 min P.I. (Fig. 2c). In consideration of the half-time of Ga-68 and the metabolic characteristics of 68Ga-cyc-DX600, 60 min postinjection was set as the scanning point for the ACE2-related imaging of tumor models.

Fig. 2
figure 2

Time-dependence of 68Ga-cyc-DX600 PET MIP images (a) and corresponding organ-specific quantification on metabolism (b) and the ratios of heart to muscle (c)

Fig. 3 manifests the diagnostic efficiency of 68Ga-cyc-DX600 PET/CT. The difference on ACE2 expression can be visually determined on MIP images, transverse, coronal, and sagittal planes. The xenografts based on HEK-293T/hACE2 (Fig. 3a) or HEK-293 (Fig. 3b) cells can be distinguished with distinct SUVmax (2.12 ± 0.19 vs 0.27 ± 0.09, p < 0.05). In addition, the positive detection of 68Ga-cyc-DX600 PET on HEK-293T/hACE2-bearing mice was effectively blocked with abundant pre-injection of DX600 peptide, and SUVmax of xenograft decreased from 2.12 ± 0.19 to 0.55 ± 0.20 (p < 0.05), an uptake that was slightly higher than ACE2-negative xenografts (0.27 ± 0.09) or background (0.16 ± 0.04) (Fig. 3c).

Fig. 3
figure 3

68Ga-cyc-DX600 PET/CT and verification of ACE2 targeting. a, b The PET/CT images of HEK-293T/hACE2 and HEK-293 xenografts, including MIP images, transverse, coronal, and sagittal planes, and the corresponding HE and IHC staining; c MIP images of 68Ga-cyc-DX600 PET/CT of HEK-293T/hACE2 xenografts pre-blocked with DX600; d the summary of SUVmax of ACE2-positive, ACE2-negative and pre-blockage ACE2-positive xenografts; e the difference of biodistribution of mice modes bearing ACE2-positive or ACE2-negative tumors

Although with the comparable cellular structure and tissue structure as proved by HE staining, the distinct ACE2 expression of HEK-293 and HEK-293T/hACE2-based xenografts were confirmed by IHC on ACE2, further manifested the efficiency of 68Ga-cyc-DX600 PET/CT in differentiating tumorous ACE2 conditions.

Based on the quantification of biodistribution, tumorous ACE2 expression lead no significant difference on tracer uptake of main organs (Fig. 3e). For the tumors acquired at 70 min P.I., there were a significant difference on tracer uptake (5.66 ± 0.50%ID/g for ACE2-positive, and 1.11 ± 0.23% for ACE2-negative). Notably, there was still a baseline for ACE2-negative tumors that was resulted from the plentiful blood supply of xenografts.

To further verify the diagnosing efficiency in ACE2 of various tumor types, the subcutaneous xenograft models can avoid confounding factors to the utmost extent. The diagnostic value of 68Ga-cyc-DX600 PET/CT was further verified in a series of models bearing xenografts (Fig. 4a). The SUVmax of xenografts were 2.12 ± 0.35 for MIA-PaCa-2-based human pancreatic cancer xenograft, 0.55 ± 0.13 for 786-O-based human renal clear cell adenocarcinoma xenograft, 1.39 ± 0.35 for SK-N-SH-based human neuroblastoma xenograft, 0.49 ± 0.15 for 22Rv1-based human prostate cancer xenograft, 0.51 ± 0.11 for A549-based human non-small cell lung cancer xenograft, 0.65 ± 0.20 for HepG2-based human hepatoma xenograft, as well as 0.15 ± 0.08 for 4T1-based mouse breast cancer xenograft, and these SUVmax positively correlated with ACE2 expression as proved by western blot (Fig. 4b). The correlation coefficient was 0.903 between the SUVmax and band intensity ratio of WB (ACE2/GAPDH) for each type of models (p < 0.05) (Fig. 4c).

Fig. 4
figure 4

A series of typical MIP images of 68Ga-cyc-DX600 PET/CT for the subcutaneous xenografts (a), the corresponding WB results of above xenografts (b), and the relationship between ACE2 expression and SUVmax of ACE2 PET (c)

The clinical value of 68Ga-cyc-DX600 PET was preliminary verified on a series of tumor types, including lung cancer, esophageal cancer, peritoneal metastases, and bladder cancer. The characteristics of four patients and the detailed findings of ACE2 PET were summarized in Table 1.

Table 1 Characteristics of four patients with tumor

A 63-year-old patient with primary tumor of lung cancer (poorly differentiated adenocarcinoma) had accepted multiple chemotherapy and was enrolled to evaluate the therapeutic effects. He underwent a routine 18F-FDG PET/CT and two 68Ga-cyc-DX600 PET/MR at 50 min P.I. and 80 min P.I. successively (Fig. 5). After multiple chemotherapy, only slight FDG uptake (SUVmax = 2.36) were visually observed in lesion in 18F-FDG PET/CT indicating an effective inhibition of tumor activity, while lesion was of high 68Ga-cyc-DX600 uptake at 50 min P.I. (SUVmax = 2.36) and 80 min P.I. (SUVmax = 3.43). Notably, compared with images at 50 min P.I., there was a comparable but a slight higher ratio of target to background at 80 min P.I. (TBR50 = 4.23; TBR80 = 4.73); hence, 68Ga-cyc-DX600 PET provided a long observation window and was operable in a dual-phase scan protocol, and images were both of a relatively clear background, while a relatively favorable ratio of target to background at 80 min P.I. for this patient.

Fig. 5
figure 5

FDG PET and dual-phase ACE2 PET of a 63-year-old man who had accepted multiple chemotherapy for lung cancer (poorly differentiated adenocarcinoma). Slight FDG uptake in lesion was detected on 18F-FDG PET/CT (a); lesion was of high tracer uptake in images both at 50 (b) and 80 (c) min P.I., comparatively, of a better TBR at 50 min P.I

For the 55-year-old man who had been diagnosed with hepatitis B cirrhosis for 10 years, he underwent 18F-FDG PET/MR and 68Ga-cyc-DX600 PET/MR for clinical condition assessment due to fatigue, anorexia, and weight loss in the past 4 months. The mucosa of the distal two thirds of the esophagus was thickened and with high uptake of 18F-FDG (SUVmax = 28.24) in Fig. 6c, subsequent gastroscopy highly suspecting esophageal cancer. Besides, several paratracheal enlarged lymph nodes with high uptake (SUVmax = 21.50) could be observed in 18F-FDG PET/MR (Fig. 6b), suggesting possible metastasis. Comparatively, no obvious uptake of 68Ga-cyc-DX600 was observed in corresponding diseased esophagus and enlarged lymph nodes in Fig. 6f and g, which probably owing to esophageal cancer without an increased ACE2 expression. Noticeably, cross-sectional images at the level of the lung in 68Ga-cyc-DX600 PET/MR demonstrated a high-density nodule with high uptake in the posterior basal segment of left lung lower lobe (SUVmax = 13.34), which may be under-reported or missed in 18F-FDG PET/MR with a mild uptake (SUVmax = 1.60).

Fig. 6
figure 6

A 55-year-old man with hepatitis B cirrhosis who was suspected of esophageal cancer underwent 18F-FDG PET/MR and 68Ga-cyc-DX600 PET/MR successively. a, e MIP images of 18F-FDG PET/MR and 68Ga-cyc-DX600 PET/MR, and corresponding cross-sectional images (CT, PET and fusion images from top to bottom) at the level of the paratracheal enlarged lymph nodes (b and f), thickened esophagus (c and g) and lung nodule (d and h); two-point series lines of SUVmax (i) and SUVmean (j) for 18F-FDG PET/MR and 68Ga-cyc-DX600 PET/MR

Furthermore, for the diffused high uptake throughout the bone in 68Ga-cyc-DX600 PET/MR (SUVmax = 15.61 for the iliac ala), tumor burden and disease-lead anemia were the main judgement, because the laboratory tests of this patient shown decreased hemoglobin (75 g/L) and platelet (78 × 109/L).

For patient No. 2, there were significantly different performances of esophageal cancer, no matter the primary lesion or metastasis, in 18F-FDG PET/MR and 68Ga-cyc-DX600, which simultaneously reflected in images and values (SUVmax and SUVmean) (Fig. 6i and j). Esophagus primary foci and lymph node metastases had high 18F-FDG uptake, but with negative uptake of 68Ga-cyc-DX600. Adversely, the possible lung metastases were of positive 68 Ga-cyc-DX600 uptake and no obvious/mild uptake in 18F-FDG PET/MR. The quantitative values of ACE2 PET and FDG PET were negatively correlated (for SUVmax: r =  − 0.971, p = 0.006; for SUVmean: r =  − 0.994, p = 0.001).

A 54-year-old man with peritoneal metastases but no definite primary focus who had finished intraperitoneal chemotherapy also underwent PET/MR examinations for efficacy evaluation. Multiple nodules in omentum, mesentery, and pelvic peritoneum of high signal on T2WI were observed, and were of no significant uptake in 68Ga-cyc-DX600 PET/MR (SUVmax = 0.875), but mild increased uptake in 18F-FDG PET/CT. Similar performance of modest tracer uptake (SUVmax = 0.985) in lesions was observed in a bladder cancer with multiple lymph node metastasis in bilateral pelvic wall and inguinal area.

In the view of researchers, SUVmax was not high in term of absolute value in these cases in ACE2 PET, the physiological uptake of the surrounding organs and tissue should be considered as an impact factor, for which the TBR could represented to estimate ACE2-based tumor aggressiveness, providing Supplementary information for routine 18F-FDG PET.

Discussion

In this study, DX600 was developed as an ACE2 PET tracer that with a high structural stability and radiochemical stability. There were some proposed protocols in designing ACE2-targeted imaging tracer, for example of inhibitor-based tracers that utilizing DX600 or MLN4760 [13, 14], the stimulation of corona virus/host interactions that utilizing protein-binding domain of virus [15]. Due to the difference on binding site to ACE2 protein, the binding efficiency, applicable species, and metabolic pathways were diverse to different scenes. This study focused on the verification of 68Ga-cyc-DX600 PET protocol in diagnosing tumors, and verified the applicable value and effectiveness in the several types, both in animal models and in patient volunteers. In this work, the whole pictures of ACE2 expression in patients exhibited the diversity of tumor-related ACE2 expression in the view of primary lesion, metastasis, and adjacent normal tissues. In comparison, although served as golden standard and of a high accuracy, the molecular laboratory tests are inadequate in diagnosing tumorous pathological changes of ACE2, especially in a view of whole-body evaluation.

Tumor-related pathological changes are dynamically affected by tumor progression, treatments, tumor subtypes and other factors. Figuring out the ACE2 expression of tumors is meaningful for theranostics. As a tumor suppressor gene, ACE2 is involved in tumor genesis and development by inhibiting tumor angiogenesis. ACE2 and its upstream and downstream proteins or related proteins in the dynamic balance of Ang 1–7 may be the key targets for antitumor genesis and metastasis-related biochemical therapies [16, 17]. Taking colorectal cancer as an example, the expression level of ACE2 in colorectal cancer tissues of all clinical stages were significantly higher than that in normal tissues, suggesting that ACE2 is suitable for the diagnosis and targeted treatment of colorectal cancer in the whole cycle [18]. In the cases of primary clear cell renal cell carcinoma (ccRCC), the higher ACE2 expression in tumors was associated with better survival outcomes in patients; meanwhile, in vitro and in vivo models confirmed that ACE2 protein was of a direct anti-tumor effect on ccRCC. In addition, ACE2 expression in various tumors is a significant biomarker related to the level of immune invasion, and the up-regulation of ACE2 expression could indirectly improve the efficacy of PD-L1 combination therapy [19]. Furthermore, as an important downstream component of ACE2 bio-activity, the combination of Ang 1–7 with vascular endothelial growth factor receptor tyrosine kinase inhibitor and immune checkpoint inhibitor, providing a new anti-tumor approach for ccRCC therapy [20]. Therefore, relying on the sufficient understanding of ACE2 expression helps to improve tumor-related diagnosis and treatment plans, and the dynamic changes of ACE2 under therapeutic effects can be used as the targeted biochemical process of ACE2 PET for dynamic monitoring on treatments or prognosis.

Studies on the expression, physiological and biochemical effects of ACE2 in tumors were of a long history, revealing the role of ACE2 in angiogenesis, fibroblast and so on [21]; meanwhile, studies on the role of tumor-expressed ACE2 in SARS-CoV-2 infection have also promoted the study on tumorous ACE2 [22]. After the outbreak of COVID-19, Prof. Fan outlined the map of ACE2-related cancer analysis for the first time, confirming that the expression of ACE2 is up-regulated in most tumors, and genetic abnormalities such as ACE2 mutation and abnormal copy number amplification can occur in multiple tumors, including colorectal adenocarcinoma, renal papillary cell carcinoma, pancreatic cancer, gastric adenocarcinoma, and lung adenocarcinoma [6]. The primary outcome of this research manifested that the tumor types of significantly upregulated ACE2 were not limited to RAAS organs and provided more visual manifestations of susceptible conditions. In other words, tumor patients with high ACE2 expression are more prone to COVID-19.

As shown in Fig. 4, the increase of SUVmax of multiple tumors was observed, not only the RAAS-related organs but also the other organs related to ACE2 fluctuation. Similar to this research, studies based on preclinical models have shown that increased ACE2 in gallbladder cancer, non-small cell lung cancer, and hepatocellular carcinoma can prevent tumor growth and epithelial-mesenchymal transformation and inhibit tumor activity [20, 23, 24]; therefore, ACE2 PET is helpful in predicting the prognosis. As a system related to multiple physiological process, the manifestations of ACE2 expression were more complicated in human beings. For example, in patient no. 2, although esophageal cancer was of high ACE2 mRNA expression, lower ACE2 protein expression was observed in metastases. Besides, as well known, high ACE2 protein expression could inhibit tumor proliferation and metastasis and has positive correlation with tumor prognosis, which possibly account for the negative uptake of the primary lesion or lymphatic metastasis in ACE2 PET. The wide presentation of ACE2 in the blood may be the reason for the high uptake of the pulmonary nodule via hematogenous metastasis. Additionally, this explains the negative correlation between tumor metabolism and ACE2 expression in the same subject (Fig. 6). Haznedaroglu et al. proposed the hypothesis that local RAS is able to assist the systemic RAS in exerting the antitumor effect, particularly in hematological malignancy [25]. The decrease of blood cells could activate local bone marrow RAS, thereby upregulating ACE2 expression to promote hematopoiesis, which may account for the high bone uptake in 68Ga-cyc-DX600 PET/MR.

There were some limitations in this research. On the one side, NOD-SCID mice used in this research were not humanized ACE2 ones, so the ACE2 PET-based tumorous ACE2 was of limitations in fully revealing the systemic changes. In fact, in addition to playing an important role in tumor self-regulation, ACE2, as an important regulatory protein involved in multi-system, multi-organ, and multi-disease remodelling, is regulated and reversely regulated by multiple organs in the body [4]. One the other side, dual-phase scans were only performed in one patient due to the poor compliance of others. Although the results showed a better tumor-to-background ratio, ACE2 PET can be further explored to grasp the dynamic changes of systemic RAAS under the influence of drugs and multiple factors, especially for understanding the involvement of organs during treatment.

Conclusion

The feasibility of tumor imaging via 68Ga-cyc-DX600 PET was manifested in kinds of tumor models. As an ACE2-specific imaging for the differential diagnosis of tumors, ACE2 PET added complementary value to conventional nuclear medicine diagnosis, such as FDG PET on glycometabolism.