Pancreatic adenocarcinoma is generally diagnosed at later stages as disseminated disease. There are no effective treatments for the majority of cases and, thus PanAdo is associated with a high mortality rate [1
]. The development of targeted imaging agents that can non-invasively detect, aid in diagnosis, and determine therapy response could improve therapeutic outcomes. As few effective treatment options are available for metastatic pancreatic cancer, the development of targeted therapeutic agents could reduce systemic toxicity, while delivering a higher dosage locally to the tumor, potentially decreasing patient mortality.
Our approach has been to identify putative cell-surface markers through gene expression profiling of DNA microarray data generated from patient tumor and unaffected tissue samples. Once identified as being expressed at the level of mRNA, it is essential that expression be validated at the level of protein. For this study, these validations were performed using IHC of tumor and normal tissue microarrays. Cell-surface markers are ideal for targeting since the agent is not required to cross the plasma membrane prior to binding to the target. Once bound to the cell-surface, the agent may be rapidly internalized by receptor mediated endocytosis or more passively by pinocytosis [19
]. Hence, allowing for the intracellular accumulation of imaging contrast or therapeutic payload. It is worth noting that our targeting approach does not require intervening with the biological function of our targets, rather, the cell-surface marker is used more as a landing-pad for attachment and internalization of the targeted agent.
Cell-surface targets (170) were identified as being highly and broadly expressed in PanAdo but not expressed in normal pancreas. Two of these targets, ABCC3 and TLR2, were validated for protein expression. ATP-binding cassette, subfamily C, member 3 (ABCC3) is highly and broadly expressed in PanAdo, and has generally low expression in normal tissues, except that it is expressed in adrenal tissue (). Expression in adrenal tissue is a concern. However, ABCC3 may be useful for diagnostic imaging of PanAdo using non-toxic imaging agents, or for methods of localized delivery of therapy. ABCC3 is a membrane transport protein that is a member of the MRP multidrug resistance subfamily and is known to mediate taxane resistance in breast cancer [20
]. ABCC3 has no known ligands, thus, ligand development will require sophisticated approaches such as the screening of one-bead one-compound random peptide sequence ligand libraries [21
], or phage display [22
]. Toll-like receptor 2 (TLR2) is expressed in immune cells for the recognition of microbial infections with subsequent immune system activation, and is also expressed in cancer [23
]. Palmitoylated synthetic ligands for binding TLR2 are known [24
]. In this study we report low to moderate expression in a few additional non-vital normal tissues. However, the moderate TLR2 expression in the kidney cortex and adrenal gland may be of concern for its use in agents with associated toxicity.
A limitation to the sole use of DNA microarray for expression profiling of tissue samples is that the tissue contains a mixture of tumor and host cell types found within a given sample. Or conversely, a determination of non-expression in normal tissue may be misleading if the marker is expressed in a small subset of cell types. An example of this is found in the IHC determination that TLR2 is expressed in a small subset of normal pancreas cells (), despite the fact that the DNA array data were scored as non-expressing. Some of these cells have stem cell morphologies (). TLR2 has recently been reported as a marker for human renal and mouse embryonic stem cells [25
]. The percentage of cells expressing TLR2 in normal pancreas is low, with only 2% scoring moderate or strong positive by IHC. Thus, TLR2 is a potential target for diagnostic imaging of pancreatic tumors and for methods of delivering tumor localized treatment.
A number of strategies are being developed for delivering tumor localized treatment using targets that are also expressed in normal tissues. Toxicity can be decreased by locally activating therapy at the site of the tumor. These approaches may be used without targeting, but targeting to the tumor can increase the local concentration of the agent prior to activation through binding and cellular uptake enhancing the therapeutic effect. An example of this approach includes agents designed for imaging by photoacoustic tomography (PAT) and local delivery of treatment by photothermal ablation [27
]. Other examples include agents that are size restricted to remain within the normal vasculature, but are not restricted from leaving the tumor vasculature due to enhanced vascular permeability and retention (EPR) [28
]; photodynamic therapy, where light can be directed to the tumor site locally activating therapy [29
]; and agents that combine ligands for heterologous markers into heterobivalent constructs allowing for specific targeting of to tissues that express all markers relative to tissues expressing only a subset of the markers [31
This study exemplifies the need for thorough characterization and validation of tumor markers prior to expending the considerable time, resources and effort required for development of targeted imaging and therapeutic agents. Quantifying mRNA expression is a useful initial screen, but validation of protein expression is essential. These results also illustrate the difficulty in finding the “ideal” marker that is highly and broadly expressed amongst all cancer of a given type, but is not expressed in vital tissues or organs of drug clearance. It appears that a number of markers, a cocktail, may be required to cover all types of PanAdo, and that development of these markers into imaging and therapeutic agents will require creative approaches to minimize toxicities in normal tissues.