Tumor-Initiating Cells (TICs), or Cancer Stem Cells (CSCs), are considered a subpopulation of cells within a tumor that are particularly aggressive because when isolated, these cells can form secondary tumors. Along these lines, experiments in which fragments of tumors, derived from primary patient samples, are re-implanted into nude mice, the majority of cells within the fragment die. However, day 3 after re-implantation, a small subpopulation emerges that is able to seed a new tumor. We hypothesized that these cells are unique and rare within the original tumor and consequently present an opportunity to identify novel markers that can identify these aggressive tumor cells. After sorting live human cells from tumor fragments three days after passaging, we used single cell PCR to determine the extent of heterogeneity within the fragment population. We, in fact, found that there is a subpopulation that highly expresses markers of proliferation and stemness. This is in contrast to the major population within the fragment that is enriched for genes associated with hypoxia and stress response. Interestingly, we found that the hypoxia responsive gene, CA9, does not correlate with other markers of hypoxia, such as VEGF, when analyzing cells derived from the fragment. Instead, CA9 was highly expressed among cells that were also enriched for markers such as Ki67, survivin, and Lgr5. To confirm if CA9 does identify a more proliferative, stem-like population, we have sorted CA9+ and CA9- cells from three day PDX fragments and analyzed using DNA microarrays and real-time PCR. We have consistently found, among several different PDX lines, that passaging of tumors followed by sorting CA9+ cells enriches for cells with increased levels of Lgr5, ASCL2, CD133, and Ki67. This is also validated through IHC analysis in which the majority of the cells that are positive for Ki67 are also positive for CA9. We are continuing to investigate the tumorigenicity of these cells by reinjecting CA9+ and CA9- cells from fragments back into immune-compromised mice. Furthermore, we are building a gene list derived from the microarrays to inspect by single cell PCR if any of these genes associate with Lgr5 in a grown tumor. Thus, we believe this is a functionally relevant and novel method for identify novel TIC markers.

Citation Format: Julia Friedman, Wenyan Zhong, Christine Loreth, Veronica Diesl, Xin Han, Justin Lucas, Andrea Hooper, Vlad Buklan, Edward Rosfjord, Danielle Leahy, Judy Lucas, Maximillian Follettie, Kim Arndt. CA9 expression highly correlates with cancer stem cell markers during passaging of PDX lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3487. doi:10.1158/1538-7445.AM2014-3487