The goal of this study was to develop a bioluminescent mouse model of PDAC metastasis that would facilitate rapid and relatively inexpensive in vivo
analyses of putative PDAC metastasis genes. There is significant need for such a model given that PDAC is a highly metastatic, deadly malignancy for which there are currently no cures or effective treatments (1
). Although a number of elegant, genetically engineered mouse models of PDAC exist that effectively mimic the human disease (33
), they are expensive and time consuming to generate. They are also unsuitable for efficiently evaluating large numbers of genes with suspected roles in PDAC tumor formation and metastasis. Here, we describe the development and characterization of a novel mouse xenograft model of PDAC that employs BLI to longitudinally track metastatic colonization and tumor growth in vivo
Three frequently studied human PDAC cell lines (Panc-1, MiaPaCa-2 and BxPC-3) expressing luciferase were generated and tested in this model. This allowed for non-invasive, serial imaging of tumor growth in vivo following intracardiac injection and hematogenous distribution of the cells in scid mice. BxPC-3 cells displayed the fastest rates of tumor onset and lethality, and the highest efficiency of tumor formation. Their rapid rate of tumor growth would be amenable for testing genetic alterations predicted to slow tumor metastasis, but not as useful for those that would accelerate the process. However, the use of BxPC-3 cells in the model was complicated by their tendency to form non-systemic tumors restricted to the thoracic region. Our data suggested this was due to aggressive tumor growth rather than failed injections into the heart, nonetheless it diminishes their utility.
Of the three cell types, MiaPaCa-2 cells behaved optimally in the metastasis model. They displayed efficient metastatic colonization (~90% of mice injected formed tumors), a moderate rate of tumor onset experimentally suitable for in vivo studies, and a wide distribution of systemic tumors. Tumors developed at organ sites commonly colonized by human PDAC metastases, including the liver, adrenal glands and celiac plexus. MiaPaCa-2 tumors also formed in the mammary fat pads and craniofacial region, including lower jaw bone and lymph nodes, and one was even found invading the pancreas and spleen. While these overall patterns of tumor distribution do not exactly mimic the clinical spectrum of human PDAC metastasis, more than one-third of tumors developed at metastatic sites normally seen in PDAC patients.
A key element of our model is the use of intracardiac injections into the left ventricle to introduce the bioluminescent pancreatic cancer cells directly into the arterial circulation. This approach circumvents early steps of metastasis, namely invasion and intravasation, but it does evaluate the extravasation, metastatic colonization and growth of tumor cells at clinically relevant sites of metastasis. Indeed, it has been used to successfully model metastasis of retinoblastoma, breast and prostate cancers, among others (13
). Importantly, our experiments are the first to show that the ARF tumor suppressor effectively inhibits the process of pancreatic tumor cell colonization in vivo
The fact that ARF had no suppressive effect on in vitro
proliferation of MiaPaCa-2 cells is interesting and indicates it is interfering with other aspects of tumor colonization. In that regard, in vitro
assays showed that ARF significantly impairs the migration and invasion of MiaPaCa-2 cells. The decreased invasiveness of ARF-expressing cells through endothelial cell monolayers in vitro
would be expected to greatly reduce the ability of those cells to extravasate in vivo
, thereby reducing their ability to form tumors. Such results are consistent with findings that a high percentage of human pancreatic ductal adenocarcinomas, which are highly motile and metastatic, have inactivated ARF
The anti-migratory and anti-tumor effects of ARF observed in this study were independent of the p53 tumor suppressor, a primary effector of ARF (29
), since MiaPaCa-2 cells express a mutated form of the protein. This supports in vitro
evidence in other cancer cell types that ARF functions independent of p53 to inhibit tumor cell migration. Specifically, ARF suppresses the in vitro
migration and invasion of lung, colon and hepatocellular carcinoma (HCC) cell lines that lack functional p53 (21
). In the HCC study, ARF selectively impaired tumor cell migration and invasion without affecting their proliferation (28
), as seen here in MiaPaCa-2 cells. Notably, these p53-independent effects of ARF required its ability to bind and inhibit CtBP2. Our studies show that ARF employs the same mechanism here to limit pancreatic cancer cell migration in vitro
, suggesting that the interplay between CtBP2 and ARF may play a critical and significant role in controlling PDAC metastasis in vivo
The robust selective pressure against ARF expression in vivo
, which was not seen in adherent cultures in vitro
, was remarkable. Specifically, ARF expression was dramatically reduced in all tumors derived from the ARF group of mice. In two of the four tumors analyzed, a nearly complete lack of both ARF and GFP was observed suggesting that non-infected (~12%) or poorly infected cells in the population had a discernable tumorigenic advantage. By comparison, two other tumors from the ARF group of mice retained GFP yet lost ARF expression. Given that ARF and GFP were ectopically expressed from a bicistronic retroviral construct, this implied a specific mechanism for ARF destabilization is activated in vivo
. Indeed, we found ARF is down-regulated by the proteasome in these cells and there is an inverse correlation between levels of ARF and ULF, its recently discovered ubiquitin ligase (32
). Since ULF levels appear unchanged in the tumor-derived versus non-injected adherent cells, ULF activity (not levels) and/or other ubiquitin ligases may be turned on by paracrine factors in vivo
, thereby facilitating ARF destruction. Simple loss of cell adherence may also contribute to ARF down-regulation since soft agar plating likewise led to loss of exogenous ARF expression (supplemental Fig S1
). Interestingly, no correlation between ULF and ARF expression was seen in soft agar colonies, unlike in the tumor-derived cells, suggesting different factors may be involved.
In summary, although much progress has been made with genetic modeling in defining key alterations that promote the pancreatic ductal adenocarcinoma, this cancer remains one of the deadliest and most difficult to treat in humans. This necessitates a more comprehensive understanding of molecular mechanisms required for PDAC development and metastasis. To address that need, we created and validated a novel mouse model of PDAC metastasis that will facilitate economically feasible and rapid in vivo evaluation of candidate genes that control PDAC metastasis. This system should also provide a powerful platform for pre-clinical testing of unique chemotherapies that can effectively treat PDAC.