ACE2 PET in healthy and diseased conditions

Angiotensin converting enzyme 2 (ACE2) played a critical role in regulating renin‐angiotensin‐aldosterone system (RAAS). In this research, 68Ga‐cyc‐DX600 was synthesized as PET tracer of ACE2 imaging. ACE2 positron emission tomography/magnetic resonance (PET/MR) was preliminary administered on twelve healthy volunteers, and the images were normalized and registered to establish the standard model of ACE2 PET. In diseased conditions, 68Ga‐cyc‐DX600 PET and 18F‐FDG PET were compared for COVID‐19 (one in acute phase and three in post‐COVID), anemia (n = 1) and malignancies (n = 2) to evaluate the diagnostic efficiency. 68Ga‐cyc‐DX600 PET was of a definite ACE2 dependence. For the tracer uptake of ACE2 PET/MR of female and male, differences existed in salivary glands, upper respiratory tract and kidneys, meanwhile, age, and body mass index (BMI) were also the confounding factors. RAAS‐related tissue and organs were of the relatively higher tracer uptake, such as SUVmean of cardiac chamber (3.786 ± 1.495), liver (5.342 ± 2.267), spleen (4.465 ± 2.508), and kidney (4.906 ± 1.619 for female and 8.431 ± 5.179 for male). For COVID‐19, ACE2 PET revealed ACE2 fluctuations, particularly in the susceptible organs, including liver, spleen and testis. In the case of anemia, the activated local RAS in the bone marrow was of diffuse high tracer uptake. ACE2 PET of malignancies added supplementary information to FDG PET. 68Ga‐cyc‐DX600‐based ACE2 PET models were established for visually monitoring of whole‐body ACE2 expression. The feasibility of ACE2 PET in supervising disease was primarily proved in COVID‐19, anemia and malignancies as providing a comprehensive view on the disease process and functional recovery.


INTRODUCTION
2][3] ACE2 is widely expressed in the kidney, heart, testis, lung, pancreas, bladder, stomach, ileum, and liver. 4,5ACE2 takes effect in normal physiological regulation and pathological remodeling of diseases, particularly in terms of counter-regulation of the local and systemic renin-angiotensin-aldosterone system (RAAS), which is responsible for converting Ang (angiotensin) I and Ang II into Ang 1-9 and Ang 1-7, respectively.Self-regulation of RAAS takes part in vasodilatation, anti-proliferation, anti-fibrosis, and anti-inflammatory, and is partially reflected as ACE2 fluctuation. 2,68][9] As the world grapples with emerging challenge in epidemic prevention arising from viral mutation, a number of symptoms remained after the survival, including fatigue, cough, chest tightness, sore throat, anosmia, dysgeusia, memory/cognitive impairment, joint/muscle pain, sleeping alterations, and so on, arose and affected hosts in an unsuspected way. 10,11This protracted disease symptom beyond acute phase has been termed as "long COVID," although there is not a consensus on the exact definition and pathophysiological mechanism. 12At a molecular level, the dysfunction of organs and systems is the nature of long COVID and of a closer relationship with sequelae.ACE2 dysfunction plays a key role in the development of long COVID, as well as other ACE2-regulated diseases, [13][14][15][16] therefore, exploring and monitoring ACE2 functional recovery may guide the management of long COVID-19 survivors. 17wing to the over-expression of ACE2 protein in kinds of tumors, cancer has already been identified as an individual risk factor for COVID-19. 18ACE2/Ang-(1-7)/MasR axis is cancer-associated and takes antitumor effect via inhibiting cancer cell proliferation and metastasis, tumor-associated angiogenesis and epithelialmesenchymal transition; hence ACE2 was deemed as a new tumor biomarker. 19Furthermore, the human protein atlas showed that top five tumors with high ACE2 expression were colorectal cancer, kidney cancer, gastric cancer, pancreatic cancer, and liver cancer. 20Besides that, ACE2 dysfunction has been implicated in many diseases progression, including but not limited to cardiovascular disease, kidney-related diseases, hypertension, diabetes, and so forth. 21,22 clinic, the existing ACE2 detection techniques include western blot, immunohistochemistry and enzyme-linked immunosorbent assay 23 ; nevertheless, these methodologies could not realize visualization of ACE2 fluctuation in vivo or elucidate the intrinsic relationship between ACE2 change and clinical symptom complex.Targeting to the trans-membrane domain and metalloproteinase active site, ACE2-specifific molecular imaging protocol would therefore be of profound help in tracking the endocytosisrelated ACE2 fluctuation. 24In this research, we firstly focused on setting up a standardized 68 Ga-cyc-DX600 positron emission tomography (PET) model, and then applied ACE2 PET in some diseased conditions (acute-or post-COVID, anemia, tumors) to verify the feasibility of clinical transformation and to explore the clinical findings.

METHOD
This prospective study was performed in concordance with the Declaration of Helsinki and approved by ethics committee of Shanghai Jiangong Hospital (approval no.JGEC2022-001).Written informed consents were obtained from all study participants.

Radiopharmaceuticals
The precursor cyc-DX600-DOTA for ACE2 imaging was purchased from Shanghai Junna Medical Biotechnology Co., Ltd. 68Ga-cyc-DX600 was prepared in house as previously reported 25,26 and met the requirement of quality control. 18F-FDG was purchased from Shanghai Atom Kexing Pharmaceutical Co., Ltd.

Normalization of ACE2 PET
For the normalization of maximum intensity projection (MIP) images, male and female healthy persons were treated separately.A standard template was firstly selected from the whole-body ACE2 PET scans; following that, a registration algorithm implemented by ANTsPy was then used to register other PET images to the standard template.
For each subject, MIP image was obtained by projecting the maximum intensity value of the registered 3D image to the coronal plane.Lastly, MIP images were summed and then divided by a factor (the number of times overlapped for each pixel with intensity ≥ 0.01) to obtain the averaged MIP images.
For the normalization of cross-sectional pictures, all the registered PET images were summed and divided by 12 to obtain the averaged PET of four transverse images that do not include the gender-specific organs.In addition, for the PET image and magnetic resonance imaging (MRI) image that were already registered, the PET image was directly superimposed on the MRI image to obtain the sections crossing the nasopharynx, heart, liver, and kidneys.

Image interpretation of ACE2 PET/MR
Image quality of ACE2 PET/MR was evaluated and approved by three experienced nuclear medicine physicians mainly according to the targeting performance of PET tracer to ACE2-expressing tissue and organs.
For normal physiological uptake, SUV max and SUV mean of tissues or organs including gray matter, white matter, salivary glands, the upper respiratory tract, lung (lung), myocardium (Myo), cardiac chambers (Cha), liver (Liv), spleen (Spl), stomach (Sto), pancreas (Pan), intestines (Int), colon (Col), kidney (Kid), spine (Spi), aorta as blood pool (Aor), and muscle (Mus) were measured on fused PET/MR images by drawing three spherical regions of interest (3-5 mm 3 ) over each organ, so as to calculate reference ranges of targeted organs in healthy people.For male subjects, prostate, testicles and penis were measured; for female subjects, breast, uterus and ovary were measured.
For diseased states involving multiple sites or systems, such as anemia and COVID-19, visual abnormalities, such as diffuse/focal increased or decreased tracer uptake, and/or numerical anomalies that were lower than 80% or higher than 120% of the reference ranges or baseline were described as "suspect."For tumor, the abnormal signal lower or higher than normal organ background was recorded on basis of T2 weighted MR images, and SUV max and SUV mean of suspected foci were measured on fused images.PET/MR images were reviewed independently and all "suspect" were defined by three nuclear medicine physicians, and discrepancies were resolved by consensus. 18F-FDG PET images served as a comparison or reference for image interpretation of ACE2 PET/MR.

Statistics
Figures were plotted using Origin 2022b as described in each figure legends.The references of SUV were represented as mean ± standard, and compared using paired t-test between male and female subjects.Organ-specific SUV with no gender difference was treated as a single group of subjects in the following correlation analysis with age and body mass index (BMI).All the statistics were performed with SPSS Statistics 23.0, and any differences with p-value less than 0.05 were defined as statistically significant.

The normalization of ACE2 PET
Twelve healthy volunteers, consisting of six female subjects with a medium age of 31 (25-52) and mean BMI of 20.9, and six male subjects with a medium age of 40 (24-53) and mean BMI of 25.1, were enrolled for the normalization of whole-body ACE2 PET. Figure 1A,B respectively provided the normalized ACE2 PET of female and male subjects.Corresponding to biological ACE2 expression, RAASrelated organs and blood circulation were of high tracer uptake, representing by liver, spleen, and cardiac chambers (plasma ACE2).Due to the difference on metabolic rate, kidneys were of wide inter-individual variability.

F I G U R E 1
The normalized angiotensin converting enzyme 2 (ACE2) PET, including maximum intensity projection (MIP) of female A and male B, as well as cross-sectional ACE2 positron emission tomography/magnetic resonance (PET/MR) of salivary glands and nasal cavity C, heart and lungs D, stomach, liver, and spleen E and kidneys and intestines F.
Besides, the salivary glands, myocardium and testis also had relatively high tracer uptake, attributing to pathological basis of susceptibility for SARS-CoV-2 infection.Similarly, the low tracer uptake was on account of low ACE2 expression under normal physiological conditions, as well as the organ-specific obstacle of tracer delivery, for example, the inability of the tracer entering the brain possibly led to the absence of tracer.The tissue or organs with high incidence of corona virus infection were visualized in cross-sectional pictures (Figure 1C-F).
For SUV max (Figure 2A) and SUV mean (Figure 2B) of most organs, there was no difference on tracer uptake between female and male subjects, except for salivary glands, upper respiratory tract and kidneys.Although white matter was of the difference, baseline was too low to be distinguishable.As a reference in adjusting tracer uptake or comparing with standard values, the detailed mean ± standard deviation of SUV max and SUV mean were listed after the box charts of each organ, meanwhile, the heatmap of SUV max (Figure 2C) and SUV mean (Figure 2D) between organ-specific SUV and confounding factors (gender, age and BMI) were summarized via showing the range of coefficients of correlation.

Patients characteristics
Seven participants with diseased conditions were enrolled for evaluation of the clinical application of ACE2 PET.
A patient that was previously in the group of healthy volunteers enrolled as acute COVID-19.He was diagnosed with the positive nucleic acid (Omicron BF.7) for 5 days, and was undergoing hyperpyrexia, cough and joint pain.Long COVID participants complained of several unexplained sequelae after 2 months recovery from acute COVID-19 (Omicron BA.2), such as cough, fatigue, sleeping alterations, chest tightness, sore throat or joint pain.A patient with severe hepatitis B cirrhosis with persistent anemia was included for bone local RAAS evaluation.Meanwhile, a suspected lung cancer patient and a patient with liver metastases from rectal cancer who has underwent several chemotherapies, triple radiofrequency ablations and one TACE were enrolled to verify the diagnosing efficiency of ACE2 PET in tumor.The detailed characteristics were summarized in Table 1.

3.3
Clinical performance of 68 Ga-cyc-DX600 PET/MR for diseases

Different phase of COVID-19
The clinical value of ACE2 PET was preliminary verified for visually monitoring ACE2 changes and recovery at the molecular level, so as to shed light on the intrinsic relationship between ACE2 and clinical symptoms.A 36-year-old man who was suffering from persistent hyperpyrexia (38.9 • C), fatigue and sore all over for 3 days after the The gender-grouped horizontal boxes (red for female and blue for male) of SUV max A and SUV mean B with values presented as mean ± SD on the right.The heatmap of correlation coefficients between organs and confounding factors of SUV max C and SUV mean D.
exposure to Omicron BF.7 strain underwent ACE2 PET/MR at the first day after symptom relief to evaluate the virus-induced changes.Compared with images prior to infection (Figure 3A), ACE2 expression in liver, spleen and testis demonstrated prominent declines, meanwhile increases in upper respiratory tract, aorta, cardiac chamber and pancreas were recorded, but other organs showed a relatively moderate fluctuation (Figure 3B-E).As the main metabolic and immune organs, the deposition of virus together with inflammation burden attributed to virus-induced substantial ACE2 downregulation.
In cases of post-COVID patients, a 71-year-old COVID-19 survivor with chief complaint of fatigue, sore throat and sleeping alterations for 3 months underwent ACE2 PET/MR examination.There was no structural abnormality on T2WI, but liver and spleen were of diffuse higher uptake on fused PET/MR images (Figure 4A), which may be correlative with high total bile acids and prothrombin time international normalized ratio (INR) of the patient when other underlying diseases were excluded.During COVID-19 recovery period, details about individual differences as well as definitive mechanism require further investigation, compensatory ACE2 rebound in liver and spleen revealed systemic compensation and recovery of RAAS.Additionally, diffuse higher uptake in bilateral testis was visually observed in another long COVID participant (Figure 4B).ACE2 PET possibly provided some molecular clue and evidence for the current research findings that SARS-CoV-2 infection could lead to testicular atrophy and hypogonadism.

Monitoring of local RAS
Local RAS in the bone marrow could regulate the proliferation and differentiation of hematopoietic cells under physiological and pathological conditions, for thus, anemia was investigated with ACE2 PET for local RAS.A 53-year-old man who had been diagnosed with hepatitis B cirrhosis for 10 years sought for further treatment due to fatigue, anorexia, and weight loss in the past 2 months.CT suggested a large amount of pleural effusion and ascites, and laboratory test showed the decreased red blood cells count (2.79 × 10 12 /L, reference [4.0-5.5]× 10 12 /L), hemoglobin (75 g/L, reference [120-160] g/L), and platelet count (66 × 10 9 /L, reference [100-300] × 10 9 /L).Noticeably, the significant diffuse high uptake throughout the bone was visibly observed in ACE2 PET/MR, mainly presenting in sternum and bilateral ribs (Figure 5A, SUV max = 13.21),spine bone (Figure 5B, SUV max = 14.98), iliac bone (Figure 5B, SUV max = 15.39), and bilateral thigh bone (Figure 5C, SUV max = 15.01).As the most likely pathology basis of this high ACE2 expression, persistent anemia could activate local RAS, which up-regulates ACE2 expressing to enhance erythropoiesis via the stimulation of erythroid progenitor cells and increases hematopoietic progenitor cell proliferation.

ACE2 PET for tumors
The diagnostic and assessment performance of ACE2 PET for tumor were firstly verified on a patient with liver metastases from rectal cancer who underwent 18 F-FDG PET/CT and 68 Ga-cyc-DX600 PET/MR successively for routine pre-chemotherapy assessment.In 18 F-FDG PET/CT, the liver metastatic lesions manifested as low density on CT and circular increased FDG uptake (SUV max = 6.50), suggesting that the multiple metastatic foci were still under active after radiofrequency ablation and TACE (Figure 6A).Comparatively, liver metastases were of heterogeneous high signal on T2WI and obvious lower uptake of 68 Ga-cyc-DX600 (SUV max = 3.12) than surrounding normal liver (SUV max = 9.34) on ACE2 PET/MR (Figure 6B).The different performances of residual metastases in 18 F-FDG PET/CT and ACE2 PET/MR intuitively demonstrated the correlation of tumor activity and ACE2 expression via molecular imaging methods, and exhibited as the fact that low ACE2 expression could be conductive to high activity and aggressiveness of tumor.
The above case preliminarily evidenced that ACE2 PET has the potential to serve as the molecular imaging tool for dynamic monitoring on treatments or prognosis of ACE2-expressing tumors.Likewise, left upper lung primary lesion (SUV max = 2.30) and right lower lung metastases (SUV max = 2.12) were of similar uptake with normal lung tissue in ACE2 PET of a patient with F I G U R E 3 A patient in acute phase of COVID underwent angiotensin converting enzyme 2 (ACE2) positron emission tomography/magnetic resonance (PET/MR) compared with images pre-COVID or the standard reference.The maximum intensity projection (MIP) images of ACE2 PET/MR prior to COVID A and in the 5th day of acute phase B, and the cross-sectional images at the level of the upper respiratory tract, cardiac chambers, liver, pancreas, and testis.The changes of main organs were presented by before-after plots that divided as steady, sudden increase and sudden decrease C and D, and specific values compared to reference in double Y boxes E with the patient-specific SUV marked with a yellow circle.suspected lung cancer (Figure 7B), while these lesions were of high FDG uptake in 18 F-FDG PET/CT (Figure 7A, left: SUV max = 6.33; right: SUV max = 4.68), for which the subsequent pathology of bronchoscopy highly demonstrated as the lung squamous cell carcinoma, a common sub-type with high ACE2 expression.

DISCUSSION
Due to the wide distribution of ACE2 and the involvement of diseased conditions, ACE2 PET showed the feasibility in capturing the subtle changes.Presently, the COVID-19 pandemic has spurred even more extensive research on ACE2, particularly its involvement in host cell entry and subsequently an array of dysfunction and clinical manifestations.ACE2 is widely expressed in human tissues, as visually shown in normalized ACE2 PET images; in this way, the high ACE2 expression led to susceptibility of tissue, and further facilitated increased SARS-CoV-2 cellular entry and viral replication. 27In both acute phase and post-COVID, ACE2 fluctuation displayed organ-specific patterns, for example of the most susceptible liver, which was manifested of a significant decline in acute phase but an excessive rebound for recovery, meanwhile, the similar pattern in mice was reported in our previous study. 28 68Galabeled cyclic DX600 peptide-based PET for noninvasive detection of ACE2 changes has been reported by Parker in evaluating SARS-CoV-2 infection to various organs, and understanding how ACE2 modulation correlates with susceptibility and organ injury. 24SARS-CoV-2-induced ACE2 dysregulation counteracted many ACE-mediated physiological and pathophysiological effects, ranging from blood pressure regulation, cardiovascular function, cell death, and proliferation to host responses to a variety of insults, presumably the critical mechanism for subsequent systemic disorders. 5,29Additionally, patients with pre-existing comorbidities such as hypertension, diabetes, cardiovascular diseases, and chronic obstructive pulmonary disease are more likely to display a severe course and to have higher mortality rates, might result from the fact that ACE2 was key modulator in several pathologic conditions. 30herefore, relying on ACE2-specific molecular imaging to dynamically and comprehensively monitor ACE2 fluctuation potentially provides idea for ACE2-target therapy. 5n addition to circulating RAAS, there are also local RAS in many organs and tissues throughout the body, such as brain, ovary, uterus, testis, adrenal gland, submandibular gland, and bone marrow, which affect critical steps of physiological and pathological production via autocrine,

F I G U R E 6
A 55-year-old patient with liver metastases from rectal cancer underwent 18 F-FDG PET/CT and 68 Ga-cyc-DX600 positron emission tomography/magnetic resonance (PET/MR) successively.The liver metastatic lesions were of circular increased uptake in 18 F-FDG PET/CT A, but decreased uptake in angiotensin converting enzyme 2 (ACE2) PET/MR B.

F I G U R E 7
A 78-year-old man diagnosed as suspected lung cancer with liver metastases from rectal cancer underwent 18 F-FDG PET/CT and angiotensin converting enzyme 2 (ACE2) positron emission tomography/magnetic resonance (PET/MR) successively for preoperative evaluation.The lung primary lesions and metastases were of high uptake in 18 F-FDG PET/CT A, and comparable uptake with normal lung tissue in ACE2 PET/MR B. paracrine and intracrine pathways. 31Hemopoietic system was often over-activated in kinds of diseases, and the hypothesis of a local RAS in the bone marrow hematopoietic tissue was proposed in 1996. 32Consequently, anemiainduced ACE2 up-regulation was sensitively recorded in ACE2 PET.
Renin angiotensin system can heavily affect tumor growth and development. 33,34ACE2 expression in tumor tissues such as gallbladder, liver, and lung is lower than that in adjacent tissues, suggesting that ACE2 served as a tumor suppressor gene in these tumors, 35 ACE2 PET was therefore of the feasibility in diagnosing aggressiveness and already proved in Figures 6 and 7. Concomitantly to aggressiveness, the expression level of ACE2 is correlated with progression and prognosis of many tumors, such as breast cancer, non-small cell lung cancer, prostate cancer, nasopharyngeal carcinoma, and so on, for which detection of ACE2 activity via ACE2 PET may be a useful diagnostic tool and provide a prognostic indicator for cancer patients, meanwhile, the practical ACE2 expression in different tumors still needs to be confirmed. 36For example of liver, ACE2 can play an antifibrotic role by inhibiting the activation of liver shape cells and has a negative regulatory effect on the progression axis of hepatitis B, cirrhosis and liver cancer.Whether ACE2 plays a positive role in inhibiting the occurrence of liver cancer and as a biological therapeutic target of liver cancer is still worth further investigation.Similarly, as a tumor suppressor gene, ACE2 may affect tumor progression by regulating angiogenesis in gastric adenocarcinoma.Therefore, ACE2 may become a new target of anti-tumorigenesis, and ACE2 PET was helpful in tumor screening and corresponding drug design.
Besides these proved possibilities, there were also some limitations in this research.Although normalized ACE2-specifific molecular imaging protocol was preliminarily established in this research, the number and type of diseases used for validation were limited.Further studies dedicate to evaluating the diagnostic performance in the respective and relevant settings are highly warranted.

CONCLUSION
A standardized 68 Ga-cyc-DX600-based ACE2 PET model was set up in this study, providing visualization of systemic ACE2 expression.Additionally, the feasibility of diagnosis and evaluation of diseased conditions were primarily proved in COVID-19, anemia and malignancies.

A C K N O W L E D G M E N T S
This research was funded by the National Natural Science Foundation of China (grant numbers: 81871390 and 82272040), the First Affiliated Hospital of Naval Medical University (Shanghai Changhai Hospital) "Guhai Plan in the 14th Five-Year Plan period" (grant number: GH145-22).

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

F I G U R E 4
Angiotensin converting enzyme 2 (ACE2) positron emission tomography/magnetic resonance (PET/MR) of two long COVID patients.The diffuse high ACE2 expression in liver and spleen were displayed in a 71-year-old COVID-19 survivor A, and in testis of another long COVID patient B. The comparative analysis between abnormal values (red marks) and reference were presented by interval plots.

F I G U R E 5
Angiotensin converting enzyme 2 (ACE2) positron emission tomography/magnetic resonance (PET/MR) of an anemic patient.Corresponding cross-sectional images (T2WI, PET, and fusion images from top to bottom) at the level of the sternum and ribsparatracheal A, spine and iliac bone B and thigh bone C showed significantly high ACE2 expression.
The detailed characteristics of patients.
TA B L E 1Abbreviation: ACE2, angiotensin converting enzyme 2; INR, international normalized ratio; PET, positron emission tomography.a Diffuse higher uptake.b High.c Low. d Focal lower uptake.