Molecular imaging of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC), a deadly cancer characterized by multiple molecular alterations, remains the most lethal cancer, with a 5‐year survival rate of about 3–15%. Early diagnosis and treatment monitoring are essential to improve patient survival. Traditional imaging methods can only provide structural information, but not biological processes. Nuclear medicine imaging combines the high sensitivity of radionuclides with high‐resolution structural imaging to visualize specific targets of PDAC for more accurate and reliable diagnosis and monitoring of therapeutic responses. In this review, we summarize the available literature regarding molecular nuclear medicine imaging in PDAC. We classify the probe targets into two categories, targeting tumor cell membranous and the tumor microenvironment, respectively. We summarize the latest evidence in this field and outline how these emerging strategies could potentially optimize clinical practice.

of the disease. 2 The disease usually causes few symptoms until it reaches an advanced stage, and effective treatment options are limited.For patients with early diagnosis of PDAC, surgical resection has the highest 5-year survival rate, while only 10-15% of patients in the early stages are suitable for surgical resection due to the presence of distant metastasis or local invasion.Although there is a global recognition of the need for prompt diagnosis, 3 with several health-care organizations recommending a shift to early diagnosis, there are several challenges.Existing endoscopic ultrasound (EUS), computed tomography (CT), or magnetic resonance imaging (MRI) are commonly used imaging techniques for pancreatic cancer diagnosis, but these methods still have several limitations. 4In some cases, PDAC can only be diagnosed after surgery (when sufficient tumor material is available for histologic diagnosis), which poses a substantial risk of mortality.
Molecular nuclear medicine imaging uses radionuclides to map and quantify tumor biological processes at the cellular and molecular levels.It plays an important role in diagnosis and treatment of patients with PDAC and has complementary value to conventional imaging techniques. 5Currently, fluorine-18 ( 18 F, T 1/2 = 110 min)fluorodeoxyglucose ( 18 F-FDG) positron emission tomography (PET)/CT is the only molecular imaging technique clinically used for PDAC, which enables whole-body imaging to determine disease staging.However, the sensitivity and specificity of 18 F-FDG PET/CT are low for the detection of small resectable and curable lesions (∼2-3 mm) in pancreatic cancer.Besides that, glucose physiologic uptake by normal and inflammatory tissue can lead to falsepositive findings, which might result in a similar appearance for pancreatitis and PDAC. 6Over the last decade, significant advances have been made in the application of molecular imaging to PDAC.For example, aptamers and nanobodies have been used to elucidate the biological properties of PDAC. 7Furthermore, immuno-positron emission tomography (immunoPET) has encouraged the development of new treatments for use in complex clinical settings. 8These approaches provide an opportunity to gain deep insights into the pathogenesis of PDAC and novel therapeutic targets for PDAC.Indeed, the current approach of targeting specific markers on either the tumor or surrounding stroma with radiotracer molecular imaging is most promising for early detection in pancreatic cancer. 9he significant group of targeted imaging agents currently being developed is based on targeting specific receptors that are upregulated during the development of PDAC. 5 Examples are carbohydrate antigen 19-9 (CA19-9), which is approved by the US Food and Drug Administration (FDA) as a secreted biomarker for detecting PDAC, 10 or tumor stromal targets such as fibroblast activating protein (FAP) 11 and programmed cell death ligand 1 (PD-L1). 12arly detection of tumor and early resection or targeted therapy is the greatest hope for patient survival.New strategies for PDAC diagnoses at an early stage as well as therapeutic monitoring are urgently needed to make a significant clinical impact. 13In the current review, we will present the latest evidence for molecular nuclear medicine imaging of PDAC based on tumor cell membra-nous and stromal targets (Figure 1).We also highlight some of potentially clinically translatable probes in this review.

Targeting tumor cell membranous biomarkers
Most PDAC occurs in precursor lesions that are transformed into invasive carcinoma through a multistep process, progressing in a stepwise process through the acquisition of genetic alterations, and eventually to overt PDAC. 13he altered processes in PDAC lead to the expression of specific biomarkers that could serve as targets for tumorspecific imaging. 14Potential biomarkers for tumor-specific targeting must have certain properties, including localization to cell membranes, diffuse upregulation through tumor tissue, and significant upregulation compared to normal and surrounding tissue expression, 5 which can usually distinguish tumors from normal tissues.

Carcinoembryonic antigen
Carcinoembryonic antigen (CEA) is a cell-surface binding glycoprotein that belongs to the CEA family. 22While CEA is highly expressed in most pancreatic cancers, its expression level is low in normal pancreatic tissues.The target selection criteria assessed it as a promising and suitable molecular target for pancreatic cancer-specific imaging. 23Girgis and colleagues developed 124 I-labeled CEA-targeted chimeric single-chain variable fragment (scFv)-Fc (H310A) antibody fragment imaging agent.The diagnostic efficiency of the probe was explored in CEApositive and -negative cellular-derived xenografts (CDX) using microPET/CT imaging.The imaging results showed an average tumor/blood ratio of 4. The ratio of positive to negative tumor was greater than 17, supporting the imaging potential of the targeted CEA probe in pancreatic cancer (Figure 3A). 24

Carcinoembryonic antigen-related cell adhesion molecule 6
CEA-related cell adhesion molecule 6 (CEACAM-6), a member of the CEA family, is a glycosylphosphatidylinositol anchored cell membrane surface protein that is expressed at 20-25 times higher levels in pancreatic cancer tissues than in normal pancreatic tissues. 25High Tumor uptake was significantly lower in 18 F-FDG than in 89  expression of CEACAM-6 is involved in the proliferation, angiogenesis, invasion, migration, and chemoresistance of pancreatic cancer cells. 26Niu et al. developed three probes of radionuclide 64 Cu-labeled with three different sizes of antibodies (single-domain antibody 2A3 of 16 kDa, heavy chain antibody 2A3-mFc of 80 kDa, and full length antibody 9A6 of 150 kDa), all three probes were highly uptake by BxPC-3 subcutaneous xenograft tumor (Figure 3B, C).Rapid BxPC-3 tumor uptake and quick whole-body clearance were seen with 64 Cu-labeled 2A3.After 24 h of probes injection, region of interest (ROI) data showed tumor uptake rates of 64 Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-2A3-mFc and 64 Cu-DOTA-9A6 were 98.2 ± 6.12%ID/g and 57.8 ± 3.73%ID/g.The heavy chain antibody 2A3-mFc has a higher tumor uptake rate and more favorable pharmacokinetics (lower liver uptake and shorter cir-culating half-life) than the full length antibody 9A6 (Figure 3D-G).The results indicated that the heavy chain antibody 2A3-mFc has great potential to be developed for imaging and treatment of pancreatic cancer targeting CEACAM-6. 271.3  Receptor tyrosine kinases

Epidermal growth factor receptor
Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein belonging to the ErbB family of receptor tyrosine kinases. 28EGFR is overexpressed in most malignancies, including PDAC, and its expression in tumor tissue differs markedly from that in normal pancreatic tissue. 29EGFR is activated by binding to a variety of growth factors, such as epidermal growth factor, epiglotins, and transforming growth factorα (TGF-α).After ligand binding, EGFR will be involved in the activation of pathways that promote tumor proliferation, malignant transformation, metastasis, and drug resistance. 30The development of molecular probe imaging agent based on EGFR is of great significance in improving precise diagnosis and guiding targeted therapy.Boyle et al. reported that the 64 Cu-labeled F(ab') 2 fragment of humanized anti-EGFR antibody panitumumab localized specifically in patient-derived pancreatic cancer xenografts in mice, allowing tumor visualization by small-animal PET/CT at 24 or 48 h post injection. 31ET imaging with another 64 Cu-labeled anti-EGFR antibody ( 64 Cu-cetuximab ipPET) could identify ≥ 3 mm tumor lesions in pancreas and contributed to image-guided surgery resection of small residual tumors.32 Further, Hihara and colleagues reported that the intraperitoneal radioimmunotherapy using the 64 Cu-labeled anti-EGFR antibody (cetuximab) improved survival time in peritoneal dissemination mouse models with AsPC-1 cancer cell lines.33

Insulin-like growth factor-1 receptor
Insulin-like growth factor-1 receptor (IGF-1R) is a transmembrane receptor tyrosine kinase and is involved in tumor cell growth, apoptosis, angiogenesis, and chemotherapy resistance through activation of MAPK and PI3K-AKT pathways. 34Although IGF-1R is expressed at a low level in normal tissues, it is upregulated in most cancers including prostate, lung, breast, and pancreatic cancers. 35It has been reported that high expression levels of IGF-1R were associated with a higher tumor grade and poorer survival in patients with pancreatic cancer. 36At the moment, several therapeutic strategies, including tyrosine kinase inhibitors (TKIs) and receptor targeting antibodies, have been evaluated but with results largely disappointing. 37One of the main reasons is the lack of predictive biomarkers to accurately select patient. 38Noninvasive imaging of IGF-1R allows for more accurate stratification of patients and more effective monitoring of treatment response in patients with PDAC.England et al. developed a tracer 89 Zr-Df-1A2G11 based on IGF-1R monoclonal antibody (1A2G11) to perform PET imaging in subcutaneous pancreatic cancer cell tumor models (Figure 4A). 39It was found that tumor accumulation of 89 Zr-Df-1A2G11 depended on the expression level of IGF-1R.Conversely, 89 Zr-labeled nonspecific human IgG showed minimal uptake in IGF-1R positive tumor xenografts, suggesting that the accumulation of 89 Zr-Df-1A2G11 was highly specific.

Human epidermal receptor 3
Human epidermal receptor 3 (HER3) belongs to the human epidermal receptor family of receptor tyrosine kinases, which activates downstream signaling pathways associated with it by binding to ligands. 40Several HER3-targeted therapeutic antibodies under preclinical and clinical evaluation are being explored for PET imaging.Patritumab is a first-in-class fully human anti-HER3 monoclonal antibody that binds to the HER3 extracellular domain and promotes receptor internalization.Lockhart et al. developed a patritumab based 64 Cu-labeled imaging probe and evaluated the dosimetry, apparent receptor occupancy, and safety of patients with advanced solid tumors, including 2 PDAC patients (NCT01479023). 41his study demonstrated the feasibility of 64 Cu-DOTApatritumab imaging for HER3 expression.However, the clinical application was unsatisfactory due to the low uptake of HER3-expressing lesions. 41In recent years, several other HER3 targeting vectors, including HER3specific antibody fragments and affibody, have been developed and used as PET imaging probes.Seribantumab is another fully human anti-HER3 monoclonal antibody, and in a preclinical study, [ 89 Zr]Zr-DFO-seribantumab and [ 89 Zr]Zr-DFO-seribantumab-F(ab') 2 had the highest tumor-to-background ratio at 96 and 48 h after injection, respectively. 42Despite lower tumor uptake, the best imaging contrast with gallium-68 ( 68 Ga, T 1/2 = 1.1 h)labeled affibody molecule [ 68 Ga]Ga-Z HER3 was observed because of the fastest clearance of blood and normal organs.However, it is noteworthy that HER3 is relatively mild overexpression in tumors and is highly expressed in healthy organs, making HER3 still a challenging molecular imaging target.

Hepatocyte growth factor receptor
Hepatocyte growth factor receptor (HGFR), also known as Met, is a transmembrane tyrosine kinase receptor that acts through stimulation of hepatocyte growth factor. 43everal inhibitors targeting Met or its downstream signaling pathway components have been tested over the past few years with unsatisfactory results for the poor patient selection and compensatory changes in signaling networks. 44Crizotinib and cabozantinib, TKIs with known activity against Met and other kinases, decreased PDAC cell line viability in vitro.However, capmatinib, the TKI with the lowest IC 50 for Met, had no activity in PDAC lines. 45Met-specific onartuzumab was labeled with 89 Zr and then evaluated in orthotopic PDAC xenograft models (Figure 4B). 45The results showed that [ 89 Zr]Zr-DFOonartuzumab was selectively uptaken by mice xenograft models transplanted with high-expression Met PDAC lines (BxPC-3) but not in mice transplanted with lowexpression Met PDAC lines (MIA PaCa-2).Although significant tumor growth delay and overall survival benefits were observed in both BxPC-3 and MIA PaCa-2 transplanted animals when treated with lutetium-177 ( 177 Lu, T 1/2 = 6.7 d)-labeled onartuzumab, the benefit was more pronounced and sustained in Met high-expression PDAC line (BxPC-3) transplanted animals.While overexpression of Met is not predictive of Met-directed TKI response, immunoPET can detect Met overexpression in vivo and predict treatment response to Met-selective radioligand therapy.

Cluster of differentiation
As potential targets for tumor diagnosis and therapy, differentiated antigen clusters (CD) have been studied extensively and intensively for a long time.Numerous CD molecules (e.g., CD20 and CD38) have shown potential as molecular imaging targets for hematological malignancies. 46In this review, we will describe in depth some of the markers investigated in PDAC.

CD54
CD54, also named intercellular cell adhesion molecule-1 (ICAM-1), is a member of the immunoglobulin superfamily. 15As a cell surface glycoprotein, CD54 is mainly expressed on endothelial cells and immune system cells and is involved in tumor cell extravasation and recruitment of immune cell populations. 47The overexpression of CD54 in pancreatic cancers makes it a particularly attractive target for PET imaging of PDAC. 48i et al. dual-labeled an ICAM-1 mAb to develop a molecular probe denoted as [ 89 Zr]Zr-DFO-ICAM-1-IR800. 49 PET/near-infrared fluorescence (NIRF)/ Cerenkov luminescence imaging (CLI) were conducted to validate the performance and biodistribution of the probe in subcutaneous and orthotopic PDAC models (Figure 5A-F

CD71
CD71 (also named transferrin receptor, TfR) is a cell membrane-associated glycoprotein, that is involved in iron uptake through its interaction with transferring and is involved in regulating cell growth. 51A total of 93% of pancreatic tumor cells showed positive CD71 expression compared with normal pancreatic tissue, 52 making CD71 an attractive molecule for targeted therapy of pancreatic cancer.For effective CD71 targeting therapy using anti-CD71 antibodies, noninvasive imaging methods to evaluating CD71 expression and antibody accumulation in cancer will provide useful information for selecting suitable patients who will benefit from treatment.Sugyo engineered radioiodine-125 ( 125 I, T 1/2 = 59.7 d)-labeled and 89 Zr-labeled anti-TfR antibodies (TSP-A01, TSP-A02, TSP-A03, and TSP-A04) first. 53Of the four antibodies, 125 I-labeled TSP-A01 showed the highest binding to MIA PaCa-2 cells with high-expression CD71, but not to murine A4 cells with low-expression CD71.Accordingly, 89 Zrlabeled TSP-A01 clearly delineated MIA PaCa-2 xenografts in vivo, but not A4 xenografts.Subsequently, they reported an almost complete response to yttrium-90 ( 90 Y, T 1/2 = 64.1 h)-labeled TSP-A01 radioimmunotherapy in MIA PaCa-2 tumors. 54KRAS mutations are present in >90% of PDAC and are linked through their oncogenic cascade to many signaling pathways including extracellular regulated kinase (ERK) and MYC. 55TfR is a key downstream event of MYC.Based on this, Henry et al. developed 89 Zrlabeled Tf and demonstrated that uptake of [ 89 Zr]Zr-Tf was higher in mice bearing KRAS mutant tumors than in mice that withdrew from carcinogenic KRAS. 56BRD4 inhibitors have been used in clinical trials to treat tumors with KRAS mutations.Results of Henry and colleagues showed that BRD4 inhibitors significantly reduced the uptake of [ 89 Zr]Zr-Tf in drug-treated mice compared with vehicle-treated mice.This preclinical research implied that [ 89 Zr]Zr-Tf may be a valuable tool in PDAC to evaluate oncogene status and target engagement of MYC-and ERK-targeted therapy.

CD142
CD142, also known as tissue factor (TF), is a transmembrane glycoprotein known to be overexpressed during tumor growth and metastasis. 57Abnormal expression of TF in PDAC is correlated with tumor stage and overall survival, making it a promising imaging target.ALT-836 is a chimeric mAb targeting human TF.Studies have reported the feasibility of 64 Cu-NOTA-ALT-836 and 89 Zr-Df-ALT-836 immunoPET in revealing the expression of TF in PDAC mouse models. 58 developed 64 Cu-NOTA-FVIIai (active site inhibited FVII) and demonstrated that immunoPET imaging using this tracer identified TF in both subcutaneous and orthotopic BxPC-3 tumors. 60Besides that, another anti-TF monoclonal antibody 1849 was labeled with 111 In by Sugyo et al. and tested by immuno-single photon emission computed tomography (SPECT). 61Results showed that 111 In-1849 was exceptionally uptake by TF-positive BxPC-3 models but not by TF knock-out negative models.TF-targeted immunoPET imaging has the potential to improve diagnosis and select therapeutic strategies for personalized therapy of patients with pancreatic cancer.

CD147
CD147 is a conserved membrane protein that is highly expressed in many malignancies. 62The expression level of CD147 mRNA in PDAC tissues was 2-3.5 times higher than that in normal pancreatic tissues, pancreatic neoplasms, and chronic pancreatitis. 63CD147 induces the expression of matrix metalloproteinases (MMP-1, MMP-2, MMP-9) and vascular endothelial growth factors, and involved in tumor proliferation, angiogenesis, invasion, and metastasis. 64Based on the high expression and significant role of CD147 in PDAC, it could be served as a potential target for diagnosing and treatment of pancreatic cancer.
ImmunoPET imaging by the 89 Zr-labeled anti-CD147 monoclonal antibody 059-053 clearly visualized subcutaneous and orthotopic CD147 high-expression xenograft tumors. 64heir subsequent research demonstrated that combined treatment using gemcitabine and radioimmunotherapy with 90 Y-labeled 059-053 significantly inhibited tumor growth and prolonged survival with tolerable toxicity, 65 suggesting a promising therapeutic option for pancreatic cancer.

CD318
CD318, also known as CUB-domain-containing protein 1 (CDCP1), is a single-pass, glycosylated transmembrane protein transmitting cancer-promoting signals through other receptors, metabolic mediators, and key intracellular signal transducers. 66Martinko et al. showed that the receptor density of CD318 was commensurate with reported values for other highly abundant therapeutic targets such as HER2, prostate-specific membrane antigen (PSMA) and somatostatin receptor 2 (SSTR2), 67 which makes it easily accessible to exogenous ligands for imaging or treatment applications. 68Kryza et al. radiolabeled anti-CD318 antibody 10D7 with 89 Zr and compared its diagnostic efficacy with IgG-89 Zr (Figure 5G, H). 66 The authors reported that 10D7- 89 Zr localized relatively small (∼100 mm 3 ) subcutaneous and orthotopic tumors more clearly than IgG- 89 Zr.Moroz et al. radiolabeled CD318specific recombinant human antibody 4A06 with 89 Zr, 225 Ac, and 177 Lu. 68 Similarly, authors found that 89 Zrlabeled 4A06 was effective in detecting tumor autonomous antigen expression in PDAC animal models including two PDX models.Subsequently, they showed that single or fractional doses of 177 Lu-labeled 4A06 significantly reduced pancreatic cancer tumor volume compared to controls.A single dose of 225 Ac-labeled 4A06 also suppressed tumor growth, but the inhibitory effect was not as good as 177 Lulabeled 4A06.Although CD318 has not been clinically tested and is relatively unexplored as a drug target, preclinical data suggested that CD318 has potential as a target for delivery of PET imaging agents and/or treatment of PDAC.

Integrin α v β 6
Integrins are involved in cell proliferation, adhesion, invasion, migration, and the dysregulation of their expression and signaling pathways may promote cancer progression. 69Integrin α v β 6 is an epithelial-specific integrin with α-and β-subunits, that is a receptor for the extracellular matrix (ECM) proteins fibronectin, vitronectin, as well as the latency-associated peptide of transforming growth factor-β (TGF-β). 70Integrin α v β 6 is overexpressed in 95% PDAC and has been linked with a poor prognosis in PDAC. 71 68 Ga-cycratide and 18 F-FDG at 7 months postoperatively in a patient with pancreatic cancer, with red arrows suggesting a retroperitoneal soft tissue mass and a higher SUV mean value for 18 F-FDG than 68 Ga-cycratide.However, CECT reviewed 3 months later suggested an inflammatory lesion rather than a recurrence.Reproduced with permission from Ref. [73], Copyright 2020, SNMMI.
significantly uptaken by both primary and metastatic sites.The results suggested that even in this type of cancer with dense stroma, imaging agents could penetrate the tumor.Recent work by Feng et al. showed that α v β 6 targeted 68 Ga-labeled cyclic peptide was comparable to 18 F-FDG in diagnostic imaging and postoperative tumor recurrence monitoring (Figure 6). 731.6Trophoblast cell-surface antigen 2 Trophoblast cell-surface antigen 2 (Trop-2), a transmembrane glycoprotein that is highly expressed in many human epithelial malignancies and associated with disease progression and the development of metastases, 74 making it a vital target for cancer diagnosis, treatment, and therapeutic monitoring.Sacituzumab govitecan is a Trop-2 directed antibody drug conjugate (ADC) has been approved for the treatment of metastatic triple-negative breast cancer and uroepithelial carcinoma with some therapeutic effect. 75Indeed, accurate patient screening plays an important role in tumor targeted therapy.Anti-Trop-2 antibody (AF650) was labeled with 89 Zr for immunoPET imaging of PDAC with overexpression of Trop-2. 76PET imaging and biodistribution studies demonstrated that 89 Zr-labeled AF650 could noninvasively monitor Trop-2positive subcutaneous xenograft of PDAC.Further, 89 Zr-DFO-AF650 effectively delineated primary tumors in an in situ BxPC-3 tumor model (Figure 7A), with a high correlation between PET imaging and biological distribution.Although a recent phase I/II multicenter trial demonstrated antitumor efficacy and acceptable tolerability of Trop-2-directed ADC in patients with advanced epithelial cancers (NCT01631552). 7716 PDAC patients enrolled in the trial did not show a satisfactory treatment response.Therefore, effective treatments still need to be explored.
1.1.7Claudin 18.2 Claudin 18.2 (CLDN18.2), a member of the Claudin family, has four transmembrane domains and is abnormally expressed in a variety of cancers including gastric cancer, pancreatic cancer, and a fraction of nonsmall cell lung cancer. 78Recently, a phase II clinical trial (NCT01630083) demonstrated that anti-CLDN18.2monoclonal antibody zolbetuximab combined with firstline chemotherapy showed good therapeutic effects for CLDN18.2-positiveadvanced or recurrent gastric cancers and gastroesophageal junction cancers. 79Anti-human CLDN18.2ADCs and CD3-bispecific modalities exhibited in vitro cytotoxicity in CLDN18.2-positiveBxPC-3 cells and inhibited tumor growth of PDAC models. 78Hu7v3-Fc was CLDN18.2-specifichumanized variable domain of heavy chain antibody and generated by Zhong et al. for imaging and treatment. 80Compared with 89 Zr-Zolbetuximab (the benchmark anti-CLDN18.2monoclonal antibody), 89 Zrlabeled hu7v3-Fc exhibited better tumor penetration and faster tumor uptake in mouse subcutaneous xenograft model.

Neurotensin receptor 1
Neurotensin receptor (NTR), a G protein-coupled receptor, is a well-validated marker of PDAC. 81NTR1 is the most expressed NTR, present in 75 to 90% of PDAC, but not in normal pancreatic tissue or chronic pancreatitis. 82eurotensin (NT) is the endogenous ligand of NTR1 and NT-20.3 is an NT(6−13) analogue, showed an excellent affinity for NTR1. 83After demonstrating binding affinity to cell lines, 84 preclinical researches showed that NT-20.3-basedprobes were excellent candidates for imaging and therapy of NTR-1-positive PDAC models. 85Building on previous success, a recent small trial performed by Hodolic et al. on three patients with PDAC showed that 68 Ga-NT-20.3was safe and well tolerated. 86Moreover, immunoPET imaging showed the uptake of 68 Ga-NT-20.3 in PDAC of all the three patients and in the liver metastases of two patients.Recently, dual-modality imaging tools, both 64 Cu-AmBaSar-NT/IRDye800-NT and [ 68 Ga] Ga-1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA)-Lys(Cy5**)-AEEAc-[Me-Arg 8 , Tle 12 ]-NT(7-13), enabled immunoPET and fluorescence imaging of NTR-1-positive PDAC models and facilitated direct imageguided surgical resection of tumors. 82,87In addition, Wang et al. reported a direct chelation method for the simple preparation of NTR-specific PET agent [ 18 F]AlF-NOTA-NT. 88ImmunoPET imaging showed that the radiotracer was exceptionally accumulated in NTR1-positive models but not in animals after the administration of a blocking agent (Figure 7B).Although approximately 30% of patients still lack NTR overexpression, the tracers may probably serve as agents "confirming the presence of NTR," which could assist identify the patient population who most likely to benefit from radioimmunotherapy.
A preliminary clinical study used a 177 Lu-labeled NTR1 antagonist (3BP-227) to evaluate NTR1 expression and the feasibility of treatment of patients with PDAC. 89 177 u-3BP-227 was well tolerated by all of the six patients and tumor uptake was reported in five of the six patients included.Of three patients who received a therapeutic dose (5.1-7.5 GBq), one achieved a partial response with significant improvement in symptoms and quality of life.Imaging agents based on NTR1 have improved the diagnostic accuracy of PDAC and hold promise for patient screening and treatment monitoring for NTR1-targeted therapy. 89

Targeting the tumor microenvironment
Unlike other solid tumors, PDAC is characterized by a distinct tumor microenvironment (TME) that dynamically evolves during tumor progression, and the TME around PDAC may make up to 90% of the tumor bulk.Numerous preclinical and clinical studies have provided substantial evidence that acellular and cellular components of TME contribute to the development of pancreatic tumors and provide a homeostatic response to injury. 90The PDAC TME consists of cancer-associated fibroblasts (CAFs), vasculature, ECM proteins, and infiltrating immune cells. 91 I G U R E 8 ImmunoPET imaging of FAP in patients with PDAC.(A) MIP images of 18 F-FDG and 68 Ga-FAPI.(B) Axial images of the CECT scan (upper image), 18 F-FDG PET (middle image), and 68 Ga-FAPI PET (lower image) of the same patient.Suspicious FAPI accumulation in projection on perihepatic lymph node was shown by 68 Ga-FAPI PET.(C) Biopsy of pulmonary lesion confirmed PDAC metastasis, which was revealed by FAPI PET/CT.Reproduced with permission from Ref. [11], Copyright 2021 SNMMI.

1.2.1
Targeting CAFs-FAP In the tumor mesenchyme, CAFs are a key component in stimulating cancer cell growth and invasion. 92In PDAC, CAF accounts for more than 90% of the tumor volume.A distinctive feature of CAFs is the overexpression of FAP, a type II membrane-bound glycoprotein with dipeptidyl peptidase and endopeptidase activities. 93Over the past few years, a series of radioactive tracers based on FAP inhibitors (FAPI) have been developed.Since the application of 68 Ga-labeled FAPI in 2018, 94 68 Ga-FAPI PET/CT has significantly improved imaging sensitivity in many malignancies, including pancreatic cancer, when compared with 18 F-FDG PET/CT.Two retrospective studies demonstrated that 68 Ga-FAPI might be better than 18 F-FDG and CECT in diagnosing of PDAC (Figure 8). 11,95Recently, a headto-head clinical study showed that 68 Ga-labeled FAPI-04 PET/MR might be superior to 18 F-FDG in detecting suspected lymph node metastases. 96Diagnosis value was also demonstrated in some distant metastatic lesions (e.g., liver, bone, muscle). 97In addition, Ferdinandus et al. observed signs of tumor response in PDAC patients by radioligand therapy with 90 Y-labeled FAPI-46. 98However, rapid clearance and insufficient tumor retention hinder 177 Lu-FAPI-46 further clinical application. 99Subsequently, two albumin binder-conjugated FAPI radiotracers (named as TEFAPI-06 and TEFAPI-07) derived from FAPI-04 were developed by Xu et al. to extend blood circulation in vivo. 100The binding affinities of 68 Ga-TEFAPI-06 and 68 Ga-TEFAPI-07 to FAP were similar to that of 68 Ga-FAPI-04.Compared with 177 Lu-FAPI-04, SPECT/PET imaging and biodistribution studies showed significantly enhanced tumor accumulation and retention of -177 Lu-labeled TEFAPI-06 and TEFAPI-07.Moreover, in pancreatic cancer PDX animal models, 177 Lu-labeled TEFAPI-06/TEFAPI-07 showed significant tumor growth inhibition compared with 177 Lu-FAPI-04 treatment group.Highly selective tumor uptake may open up new applications for noninvasive tumor characterization, staging, and radioligand therapy. 101

Targeting the angiogenesis pathway
Although PDAC is considered to be a hypovascular cancer type, angiogenesis is a necessary process to provide adequate nutrients and oxygen. 102In the past few years, though a variety of antiangiogenic agents have failed in late-stage clinical trials of PDAC, 103 other vascular targeting approaches with the purpose of vascular normalization and tumor immunosensitization have shown promise in preclinical models. 104ImmunoPET imaging targeting angiogenesis can specifically monitor the early effects of antiangiogenesis therapy.

CD105
CD105, a receptor of TGF-β, is highly expressed in most tumor neovasculature. 105,106The expression abundance of CD105 is distinctly higher than most other angiogenesisrelated targets, 107 making it an appropriate and potential target for tumor angiogenesis imaging.A bispecific heterodimer targeting both TF and CD105 was synthesized by using a bio-orthogonal "click" reaction between TCO and Tz and then labeled with 64 Cu. 108 ImmunoPET imaging with this tracer displayed significantly enhanced tumor uptake compared with each of their monospecific Fab tracers.Furthermore, dual-targeted imaging can sensitively detect orthotopic pancreatic tumor lesions.Subsequently, a bispecific immunoconjugate based on TF and CD105 Fab′ antibody fragments was developed and investigated after being dual-labeled with 64 Cu and fluorescent dye. 109ET/fluorescence imaging and the results of biodistribution revealed that dual-labeled heterodimeric imaging agents increased tumor accumulation compared with single-targeted homodimers.These studies indicated the feasibility of PET or PET/NIRF technology for noninvasive imaging of CD105 expression.

Integrin α v β 3
Integrin α v β 3 is highly expressed in tumor angiogenic endothelial cells and is closely associated with angiogenesis and metastasis in malignant tumors. 110Arginineglycine-aspartic acid (Arg-Gly-Asp, RGD) peptides are common sequence in natural α v β 3 receptor ligands.Over the past decade, RGD-based PET tracers have been used in clinical practice and to visualize angiogenesis in many cancers. 111As a potential target for PDAC visualization, research has also been conducted.[c(Arg-Gly-Asp-D-Tyr-Lys)] is an RGD analogue, and 125 I-labeled this cyclic peptide could bound to and internalized in rat pancreatic CA20948 tumor cells in vitro. 112Furthermore, the authors reported receptor-specific accumulation of 111 Inlabeled analogue in CA20948 tumor.Multimodal molecular imaging, including integriSense fluorescence molecular tomography and 68 Ga-NODAGA-RGD PET, enabled noninvasive visualization of PDAC in pancreas-specific oncogenic Kras G12D -activated mouse models. 113

Prostate-specific membrane antigen
Targeting PSMA has shown promising results in the diagnosis and treatment of prostate cancer. 114In addition to its confirmed expression in more than 90% of prostate cancers, 115 PSMA has also been found in tumor-associated neovascular endothelial cells in most nonprostate solid cancers including PDAC.Ren and colleagues reported that both PSMA protein and mRNA levels were upregulated in human PDAC samples. 116Moreover, the expression of PSMA was associated with the prognosis of patients with PDAC.In a case report, a patient was evaluated for prostate cancer recurrence by applying PSMA PET/CT imaging with high radiotracer uptake of a soft tissue mass at the pancreatic site.PDAC was later confirmed by histopathology. 117Subsequently, a clinical prospective comparative study showed that 68 Ga-PSMA PET/CT had high specificity (90.5% versus 65.4%) and accuracy (92.5% versus 72.5%) in noninvasive diagnosis of primary pancreatic malignancies compared to 18 F-FDG PET/CT (Figure 9). 118PSMA may be a potential imaging and therapeutic target for patients with pancreatic cancer.

Targeting the ECM protein
ECM is a major component of the TME, 119 which has long been associated with the regulation of cancer progression (e.g., migration and invasion).Fibronectin, including extradomain B fibronectin (EDB-FN), is highly expressed in the ECM and neovasculature, promoting cancer invasion and drug resistance. 120EDB-FN was found to be overexpressed in PDAC, but not in pancreatitis or normal pancreatic tissues, 121 making it an attractive target for PDAC imaging.A linear peptide ZD2 (Thr-Val-Arg-Thr-Ser-Ala-Asp) was identified by Gao et al. with specific binding to EDB-FN. 122 68Ga-NOTA-ZD2 immunoPET imaging delineated pancreatic cancer tumors of 10 mm or smaller in size with minimal background noise in normal tissues.NJB2, a high-affinity nanobody, was validated by Jailkhani et al. specific for the alternatively spliced EIIIB (EDB) domain of fibronectin.7a Subsequently, NJB2 was labeled by 64 Cu and immunoPET imaging showed that it can detect not only PDAC tumors but also early pancreatic lesions (pancreatic intraepithelial neoplasias), outperforming 18 F-FDG (Figure 10).

Targeting infiltrating immune cells
Although immunotherapies, such as immune checkpoint inhibitors (ICIs) or engineered T cells, have not been shown to be effective, there is increasing evidence that combinations of these and other strategies can turn on immunotherapy for PDAC. 123Tumor-infiltrating CD8 T cells expressing programmed cell death protein 1 (PD-1) are often impaired in terms of antitumor functions. 124In tumor TME, the PD-1/PD-L1 signaling pathway prevents effector T cells from recognizing and killing tumor cells, leading to immune cell escape. 125PD-1/PD-L1 pathway targeted immunoPET imaging can not only help identify the appropriate patient population for immunotherapy, but also provide a powerful biomarker for predicting and monitoring tumor response to anti-PD-1/anti-PD-L1 antibody combination therapy.Anti-PD-1 and anti-PD-L1 antibodies were labeled with 64 Cu and PET imaging showed that the tumor-to-blood ratio of [ 64 Cu]Cu-NOTA-anti-PD-L1 was over 40 times higher than that of 64 Cu-labeled anti-PD-1 or IgG controls in orthotopic pancreatic tumor models. 12In addition, by coinjection of excessive unlabeled anti-PD-L1, the background signal of the spleen of 64 Cu-labeled anti-PD-L1 was significantly weakened, thus, clearly delineating an in situ pancreatic tumor.The clinical-stage molecular imaging tracers targeting tumor cell membranous and the TME biomarkers are summarized in Supporting Information Table S1, while the corresponding tracers in preclinical stages are summarized in Supporting Information Table S2.

CONCLUSIONS AND FUTURE PERSPECTIVES
Early detection and accurate assessment of treatment response are needed to enable rapid clinical decision making. 126The disadvantage of anatomic imaging using ultrasound, CT, and MRI is that it is challenging to differentiate malignant lesions from nonmalignant cysts, especially for smaller lesions. 127Changes in the expression of functional tumor biomarkers may occur earlier than changes in lesion size as assessed by conventional morphological imaging. 126In recent decades, molecular nuclear medicine imaging, especially immunoPET imaging, has become an increasingly popular approach to help clinicians locate biomarkers and potential therapeutic targets. 128Specific and highly expressed biomarkers are expected to be the potential targets of immunePET imaging.One of the histological hallmark features of PDAC is the pronounced TME that dynamically evolves in the process of tumor progression. 90TME around PDAC may make up to 90% of the tumor bulk.In recent years, novel stroma-targeted approaches are emerging that may help to improve the devastating prognosis of PDAC patients. 90Therefore, biomarkers on either tumor or surrounding stroma containing extracellular domains are excellent targets for early imaging, patient screening, and therapeutic monitoring for patients with PDAC.Over the past decade, the construction and application of excellent immunoPET probes based on these biomarkers have promoted the development of molecular imaging in pancreatic cancer.The selection of tumor-targeting vectors is an important part of probe construction.
Several important properties, including high clinical relevance and high specificity, make antibodies suitable candidates for tumor-targeting vectors. 129Radiolabeled mAb-based imaging of patients with pancreatic tumors has been reported for as long as 20 years. 130However, they are captured by reticuloendothelial system with high uptake by the liver and spleen, which complicates the distinction of liver metastasis and primary PDAC from physiological uptake. 126Long blood circulation times as well as slow tumor accumulation of full antibodies further limit the potential clinical application. 131For this reason, it has become a common practice to construct imaging probes using smaller antibody fragments such as Fab, F(ab') 2 , scFv, and single-domain antibody or named nanobody.In general, small antibody fragments show shorter blood circulation times and higher tumor accumulation. 132Furthermore, fragmented antibodies may penetrate solid tumors more homogeneously and efficiently, it is vital for PDAC with the dense fibrotic TME around. 133It is worth mentioning that, small size, high affinity, high antigen specificity, and low immunogenicity, make nanobody "magic bullets" for molecular cancer imaging. 134Recently, a bivalent nanobody caplacizumab used in the treatment of patients with thrombotic thrombocytopenic purpura has been approved by FDA and the European Medicines Agency, promoting the application of structural domain antibodies in clinical and research. 135Despite the favorable pharmacokinetics, nanobody-based radiotracers have very high kidney accumulation due to the renal clearance of the excess substances, which is an important reason affecting the clinical translation of the probes.Several factors, including the sequence of the nanobody, conjugation method, and specific receptors in the glomeruli, may lead to the high kidney retention of the devel-oped radiotracers.Site-specific conjugation, PEGylation, removal of the polyhistidine tag, as well as coinfusion of gelofusine and Lys could be used to circumvent this phenomenon. 8s a companion diagnostic tool for PDAC, molecular nuclear medicine imaging, especially immunoPET imaging has obvious advantages over traditional anatomical imaging.From obtaining tumor phenotypic information to continuously and longitudinally monitoring the dynamic evolution of relevant targets, immunoPET imaging has the potential to noninvasively capture complete tumor heterogeneity, like a "virtual biopsy." 8,136With the application of immunotherapy in the past decade, immunoPET with ICIs can also be used as a reliable biomarker for predicting and monitoring responses in patients with PDAC. 12In addition to being a quantitative, specific, and sensitive imaging method, another potential role for immunoPET is guiding antibody drug development. 137It can not only assist in the discovery of antibody drugs, but also provide clinical evaluation of the antibody drugs. 8With the discovery of appropriate biomarkers and the transformation and application of immune PET imaging strategies, immune PET has great prospects in optimizing the clinical management of PDAC.

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 1
Molecular nuclear medicine imaging targets for PDAC on either tumor cells or surrounding stroma.

F I G U R E 3
ImmunoPET probes targeting carcinoembryonic antigen family.(A) ImmunoPET imaging of 124 I-labeled CEA-targeted chimeric single-chain variable fragment (scFv)-Fc antibody fragment in three CEA-high expression pancreas cancer models.Reproduced with permission from Ref. [24], Copyright 2011 Springer Berlin Heidelberg.(B) Structure of single domain antibody 2A3 (left), heavy chain antibody 2A3-mFc (middle), and full-length IgG 9A6 (right).(C) Multitemporal imaging results of the above three antibodies labeled by 64 Cu as well as a negative IgG control in BxPC3 tumor-bearing mice.Tumors were shown by white arrows.(D-G) Region of interest (ROI) analysis of the PET imaging at different time points after tracer injection.64Cu-DOTA-2A3-mFc showed significantly high accumulation in tumor at 24 h after injection.Reproduced with permission from Ref.[27], Copyright 2012, Elsevier.

F I G U R E 6
ImmunoPET imaging of integrin α v β 6 expression using a68 Ga-labeled integrin α v β 6 targeted cyclic radiotracer.(A) PET images obtained at 0.5 h after injection of68 Ga-cycratide in orthotopic pancreatic cancer models without (left) or with (right) blocking doses of cold cycratide.(B) Hematoxylin and eosin staining of tumor tissues harvested from orthotopic tumor model.(C) Anti-integrin α v β 6 immunofluorescence staining of tumor tissues harvested from orthotopic tumor model.(D) Contrast-enhanced CT (CECT) and PET/CT results of

F I G U R E 7
ImmunoPET probes targeting Trop-2 and NTR1.(A) Representative MIP images of 89 Zr-labeled AF650 in orthotopic pancreatic cancer models at various time points, and representative MIP/CT and PET/CT images at 120 h; tumor sites are indicated by white dashed circles.Reproduced with permission from Ref. [76], Copyright 2021, Springer Berlin Heidelberg.(B) ImmunoPET imaging of [ 18 F]AlF-NOTA-NT demonstrated prominent uptake of the tracer in AsPC-1 and Panc-1 tumors (top row) but not in the NT blocking group (bottom row).Reproduced with permission from Ref. [88], Copyright 2018, American Chemical Society.

F I G U R E 9 F I G U R E 1 0
The performance of68 Ga-PSMA PET/CT and 18 F -FDG PET/CT in benign pancreatic lesion.(A) MIP image of18 F-FDG PET/CT shows a focal area of tracer activity in the region of pancreas (white arrow) while the MIP image of68 Ga-PSMA PET/CT (F) shows no tracer activity in the region of the pancreas.CECT images in axial (B) and coronal planes (G) show a heterogeneously enhancing lesion in the region of the head and uncinate process of the pancreas (white arrow) with fat stranding and foci of calcifications.Fused 18 F-FDG PET/CT images in axial (C) and coronal planes (H) show the presence of focal intense 18 F-FDG uptake, while fused68 Ga-PSMA PET/CT images in axial (D) and coronal planes (I) show faint PSMA uptake in the pancreatic mass.(E and J) Cytology images showed sheets of benign ductal cells with no evidence of malignancy.Reproduced with permission from Ref.[118], Copyright 2020, European Society of Radiology.ImmunoPET probe targeting fibronectin.64Cu-labeled NJB2 PET/CT could clearly delineate tumors (P and S) in KPC mouse model of PDAC (A iv), even for PanIN lesions (A ii and A iii, arrowheads).Although18 F-FDG clearly detected a large pancreatic tumor (B viii) in a mouse with PDAC, 18 F-FDG did not clearly show PanIN lesions (B vi and B vii).B, bladder; K, kidney; S, fibrosarcoma; P, PDAC tumor.Reproduced with permission from Ref. [7a], Copyright 2019 National Academy of Sciences.
This work was supported in part by the National Key Research and Development Program of China (Grant number 2021YFA0910000 and 2020YFA0909000), the National Natural Science Foundation of China (Grant number 82171972 and 82001878), and the Shanghai Rising-Star Program (Grant number 20QA1406100).