Using a phage display screen and exploiting the experimental merits of a refined genetically engineered mouse model of PDAC, we successfully generated a multimodal nanoparticle-based targeted imaging agent, PTP-NP, that allows imaging of PDAC in the background of normal, mucinous, and ductal metaplasia of the pancreas. The imaging agent has potential use for both MRI and endoscopy in high-risk patients. Important issues in the development of improved diagnostics for PDAC include the need to distinguish pancreatic neoplasia from regions of pancreatic damage and the need for methods that identify earlier stages of tumor progression [31
]. The imaging probes identified in this study home to the neoplasm while showing no appreciable colocalization with adjacent areas or acinar-ductal metaplasia. This specificity could be used to possibly reduce “false-positives” in diagnostic tests. Further, these new imaging probes bind to PanINs as well as to advanced cancers. The capacity to detect such premalignant lesions could enable the development of new approaches in the management of this disease. Although liver and kidney uptake is high, the tomographic imaging techniques that would be used with this probe (i.e., MRI, single photon emission computed tomography [SPECT]/CT, or optical) would allow the resolution of the pancreas in the context of both organs.
In addition to the development of novel molecularly targeted imaging agents, phage display screening and modified immunoprecipitation permitted the identification of membrane-localized plectin-1 as a potential new biomarker for PDAC. Differential protein processing and/or trafficking, identified using proteomic approaches, represents a potential class of biomarkers that would be missed if looking at cDNA expression data only or using whole-cell proteomics methods. For example, the binding partner of clone 15 identified from this screen represents an additional biomarker that may shed light on aberrant molecular pathways contributing to PDAC pathogenesis.
Plectin-1 is a high molecular weight protein (500 kDa) that links intermediate filaments to microtubules and microfilaments and also anchors the cytoskeleton, the plasma, and nuclear membranes (reviewed in [29
]). We have shown that plectin-1 levels are low in normal pancreatic ductal cells, but its expression is upregulated in PanINs and remains elevated in PDAC. Perhaps more importantly, plectin-1 exhibits distinct cytoplasmic and nuclear localization in normal fibroblasts, whereas an aberrant expression on the cell membrane is observed in PDAC. Studying the mechanisms of protein upregulation, differential trafficking, and whether plectin-1 contributes to disease progression are important future experiments. Notable in this regard, recent publications illustrate that plectin-1 can be recruited to the membrane during epithelial cell transformation [32
]. Altered subcellular localization of plectin-1 is also observed in the autoimmune condition, paraneoplastic pemphigus, and in the associated lymphoproliferative neoplasm, Castleman disease [33
]. Plectin-1 has a number of important roles in signal transduction, influencing Rho activity [34
], and serving as a scaffold for proteins involved in protein kinase C (PKC) [35
] and AMP-activated protein kinase signaling pathways [36
]. Thus, plectin-1 in PDAC may have an impact on signaling pathways that regulate cell migration, polarity, and energy metabolism.
Noninvasive imaging has particular applications in high-risk groups—hereditary PDAC kindreds and new-onset diabetes patients—who are currently targets of screening for pancreatic cancer. Despite the increased risk in these individuals, the incidence of pancreatic cancer will only be ~0.4%–0.6% [37
], hence surgery, which carries substantial morbidity and mortality, is not typically carried out prophylactically. Traditional imaging such as CT scan or MRI often do not detect PDAC lesions until they have reached a size at which many tumors have already metastasized—rendering surgery ineffective. There is, consequently, a considerable need for a new imaging modality that would accurately identify the presence of PDAC at an earlier point in its evolution—when surgery is effective. Other settings for noninvasive imaging of incipient cancers include patients with cystic neoplasms: intraductal papillary mucinous neoplasms (IPMN) and mucinous cystic neoplasms (MCN). These tumors are often benign, however a subset progress to PDAC. Furthermore, there may be utility for these approaches in postsurgical screening for recurrence and screening prior to surgery to determine exact tumor extension more accurately. Finally, we envision that the approach could be clinically valuable in differential diagnosis, i.e., patients presenting with pancreatitis, jaundice, or upper abdominal pain. In screening of high-risk groups, it will be important to be able to distinguish low-grade PanINs, which are present in many healthy individuals, from high-grade PanINs and carcinoma in situ. The ideal probes would recognize lesions of PanIN-3 and higher since these are thought to have very high potential for progressing to invasive PDAC. Further work will be required to define whether the plectin-1 targeted nanoparticles discriminate between low-grade PanINs and high-grade PanINs. Translational studies can be conducted in patients undergoing resection; the rapid homing of the agent to tumors and subsequent clearance from the body makes this technically feasible.
Genetically engineered mouse models of human cancers effectively recapitulate many of the molecular, biological, and clinical features of the human disease [11
]. Recent genomics studies of mouse and human cancers have established that cross-species analysis can serve as an effective filter in identifying recurrent alterations [38
]. Our studies here show that the utility of such mouse models extends to the development of molecularly targeted imaging agents. We identified conserved markers of early disease in screens that took advantage of mouse cell lines derived from the early stages of cancer development and primary pancreatic ductal cells. Furthermore, the known kinetics of tumor progression of our mouse model facilitated testing of the imaging probes at defined stages of tumorigenesis. The approaches described here may be broadly applicable to the discovery of cancer biomarkers predictive of disease stage, prognosis, or presence of specific genetic alterations.