Abstract 5177: Heterogeneity of pancreatic ductal adenocarcinoma visualized
Authors: Veenstra Veronique , Damhofer Helene , van Leusden Tom , Kessler Jan , Medema Jan Paul , van Laarhoven Hanneke , Vermeulen Louis , Bijlsma Maarten 
Published: August 2015

The outcome of pancreatic ductal adenocarcinoma (PDAC) remains dismal. Factors that contribute to the lethality of this disease include its intrinsic resistance to therapeutics, an aggressive growth pattern, and a large degree of both inter- as well as intratumor heterogeneity. All these features have been attributed (at least in part) to the presence and function of a population of pluripotent cells called cancer stem cells. Despite the alleged importance of these cells for clinical outcome, the methods that have been used to study these cells in PDAC are troubled by some serious caveats. Defining important quantitative parameters for these cells in an unbiased, marker-agnostic, way is called for and we aim to achieve this by unbiased tracing methods in patient derived xenografts.

A panel of PDX-derived lines generated in our institute was labeled using the LeGO (Lentiviral Gene Ontology; Weber et al. 2012 Nat. Prot) system to enable tracking of single cell clones by color. The system utilizes three vectors coding for red, green, and blue fluorophores much like a television uses these colors to generate an almost infinite range of colors in the visible range. Nuances in color and intensity are generated by the many possible combinations of fluorophores, the different integration numbers of the genes coding for them, and the variations in expression levels determined by for instance the integration sites. The end result is a population of cells in which each cell is endowed with a unique color. This will allow detection of the offspring of such a cell by identifying clones within a structural unit in for instance a tumor, or in a culture dish, of similar color. The ability to give rise to such a structure is then, depending on the experimental context and outcome, considered a proxy for stem cell function.

Several primary lines have proven amenable to LeGO-marking. These lines showed stable color patterns during culturing in vitro as analyzed by continuous FACS measurements, and tumors derived from marked cells did not show a difference to umarked tumors as assessed by gross histology and growth rate. We are currently testing the ability of these cells to faithfully occupy and mark structural units in PDAC, much like intestinal stem cells would in a lineage tracing experiment in the gut, and derive numbers for stem cell number as well as replacement rate. Experiments are currently being performed to assess the system's potential to measure parameters associated with stemness, and possibly also differentiation status of the original tumor, as the cells grow in vitro. (Radio)chemotherapy regimens are included to formally address the purported resistance of cancer stem cells to these treatments without having to rely on an a priori chosen marker. Furthermore, more detailed comparison should reveal use of LeGO-derived data to measure the intra- and intertumor heterogeneity in PDAC tumor biology in our panel of PDXs.

Citation Format: Veronique Veenstra, Helene Damhofer, Tom van Leusden, Jan Kessler, Jan Paul Medema, Hanneke van Laarhoven, Louis Vermeulen, Maarten Bijlsma. Heterogeneity of pancreatic ductal adenocarcinoma visualized. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5177. doi:10.1158/1538-7445.AM2015-5177