Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with only a 9% five-year survival rate in the United States. A hallmark of PDAC is the dramatic infiltration of fibroblasts and immune cells to the cancer site, which form a complex tumor microenvironment. This extensive stromal remodeling composes the majority of the tumor volume and plays a critical role in both cancer initiation and maintenance. Therefore, there is a profound need to better understand the unique relationships between tumor cells, fibroblasts, and immune cells in order to better predict how this balance might be favorably shifted for clinical applications. Arguably, the most defining molecular characteristic of PDAC is oncogenic KRAS, a mutation that has been observed in >90% of assayed tumors. Because KRAS operates at the apex of many molecular pathways that impact extracellular signaling, our group is interested in exploring the impact that modulating PDAC’s oncogenic Kras activity has on stromal cells. To this end, we have used single-cell RNA sequencing (scRNAseq) to assess transcriptional changes in murine tumor-associated macrophages (TAMs) directly cocultured with murine PDAC cells harboring a doxycycline-inducible oncogenic KrasG12D cassette. The benefit of using scRNAseq is that we can detect changes in cellular activity not only more immediately than would be possible at the protein level, but also with single-cell resolution, giving us the sensitivity to parse out subpopulations of TAMs with varied phenotypes. We conducted these experiments in parallel with identical cultures that also included murine cancer-associated fibroblasts (CAFs) to examine whether or not the presence of CAFs affected TAM transcription synergistically with or independent of epithelial oncogenic Kras. By comparing our cells to a manually curated list of common myeloid markers, we observed three distinct TAM phenotypes: regardless of Kras activity status, one TAM phenotype was present in all cultures that included CAFs and two other TAM phenotypes were found in all groups where CAFs were absent. Differential gene expression analysis of TAM populations cultured with or without KrasG12D yielded 241 significant genes when CAFs were also present in the system, but only one differentially expressed gene, Actb, when CAFs were excluded. When differential gene expression analysis was then done between TAMs grown with or without CAFs but across the same KrasG12D activity context, 19 genes were significant in the context of KrasG12D depletion and 278 genes were significant when KrasG12D was present. In summary, our data have begun to dissect the intricacies of the PDAC tumor microenvironment by stressing the impact that both oncogenic Kras and fibroblast invasion have on TAM activity. We hope that continued research into this intercellular network will allow for more precise therapeutic opportunities that consider the pancreatic tumor in its entirety.
Citation Format: Katelyn Donahue, Yaqing Zhang, Veerin Sirihorachai, Stephanie The, Arvind Rao, Marina Pasca di Magliano. Using single-cell RNA sequencing to assess the impact of pancreatic oncogenic Kras on macrophage gene expression in vitro [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A10.