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Pancreatic ductal adenocarcinoma lethality can be attributed to a combination of rapid metastatic dissemination and intrinsic resistance to conventional therapies. Our prior studies using single cell RNA-seq in pancreatic circulating tumor cells (CTCs) revealed that these “seeds of metastasis” had a biphenotypic state of both epithelial and mesenchymal features. This suggested that CTCs are highly plastic cells that exist in an intermediate state along epithelial (E) to mesenchymal (M) transition (EMT). Analysis of CTC markers in primary PDAC tumors revealed that this subpopulation of cancer cells was concentrated at the interface of tumor cells and stromal cancer associated fibroblasts (CAFs). To evaluate the contribution of CAFs in PDAC EMT plasticity, we utilized patient derived PDAC-CAF co-culture preclinical models demonstrating that PDAC EMT heterogeneity was modulated by the density of CAFs and partially driven by TGF-b secretion. Moreover, analysis of patient derived PDAC tumor spheres treated with FOLFIRINOX (FFX) demonstrated selection or induction of EMT changes that were also found in patient tumors that were resected after neoadjuvant FFX. Altogether, our collective work along with others demonstrates the importance of EMT plasticity in PDAC cell metastatic propensity and the ability to resist cytotoxic chemotherapy. A multi-institutional randomized Phase II trial supported by SU2C-Lustgarten Foundation evaluating the role of the TGF-b modulating activities of losartan on locally advanced PDAC response to chemotherapy and suppression of metastatic dissemination has neared completion. This 3 arm (n=40 per arm) trial of neoadjuvant FFX, FFX + losartan, or FFX+ losartan + nivolumab (anti-PD1). Here, we have performed EMT RNA in situ hybridization in all post-treatment resection specimens to determine if there is a difference in EMT proportions between the different arms of the study. As an orthogonal unbiased approach, we have utilized the NanoString GeoMx Digital Spatial Profiler whole transcriptome assay (18,000+ protein coding genes) on each of the resection specimens with analysis of the tumor cells, CAFs, and immune cell separately for multiple regions of interest in these specimens. This provides deeper molecular insight of the PDAC tumor cells that resisted neoadjuvant therapy, the changes in the surrounding CAFs, and the modulation of immune infiltrates that might differ between each arm of the study. We anticipate the results of the trial will be completed before the conference and initial correlative analysis of spatial transcriptomics and RNA-ISH will be presented. Citation Format: Amaya Pankaj, Michael J. Raabe, Bidish Patel, Evan R. Lang, Joshua Kocher, Katherine H. Xu, Linda T. Nieman, Alec C. Kimmelman, David P. Ryan, Theodore S. Hong, William L. Hwang, Martin Aryee, David T. Ting. Characterizing the effects of neoadjuvant therapy in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR004.

Aberrant transcription of the repeat RNAs is a common feature in epithelial cancers including PDAC, but the function of these non-coding RNAs in cancer development is relatively unexplored. We have found that these repeat RNAs are sensed and replicate like retroviruses, and now have identified the ability of these viral-like elements to be transmitted from cancer cells through extracellular vesicles (EVs). PDAC-derived EVs applied to cancer-associated fibroblasts (CAFs) activates interferon-stimulated genes (ISGs) and is able to drive CAFs towards an inflammatory CAF (iCAF) phenotype with concordant loss of myofibroblast CAF (myCAF) marker genes. Using in-vitro transcription, we demonstrate that individual repeat RNAs (HSATII, HERVK (env), LINE-1 5’UTR and LINE-1 3’UTR) are sufficient to induce ISG response in CAFs with HSATII and HERVK (env) having the most potent ISG response. In contrast, PDAC cells were found to induce epithelial-mesenchymal transition (EMT) with loss of epithelial gene expression. To determine the potential sensor of HSATII repeat RNAs, we utilized CRISPR/Cas9 knockout of the viral RNA sensors RIG-I, MDA5, and MAVS in PDAC and CAF cells. Notably, these sensors were important for PDAC repeat RNA sensing and response, but not in CAF cells. Evaluating the innate immune pathway further downstream, we used genetic knockout of IRF3 with CRISPR/Cas9 knockout and find significant downregulation of key EMT genes that are shared with myCAF markers (ACTA2, FN1, SERPINE1). Interestingly, HSATII RNA activated IRF3 dependent EMT genes in PDAC cells, but induced IRF3 degradation in CAF cells that results in loss of myCAF gene expression. Furthermore, we found that conditioned media from HSATII transfected CAF activates EMT-related gene expression (ACTA2, FN1, SERPINE1) in PDAC cell lines, which indicates an cell extrinsic mechanism to augment EMT induction in PDAC cells. We utilized next generation spatial transcriptomic platforms NanoString GeoMx and CosMx to understand the spatial distribution of repeat RNAs in human PDAC tumors. We find that repeat RNAs can be found as a gradient from PDAC cells to the surround tumor microenvironment consistent with delivery of these RNA species. Analysis of over 300,000 individual cells in 3 PDAC tumor specimens, we find that high repeat PDAC cells have lost epithelial gene expression and high repeat CAFs have lost myCAF gene expression. Altogether, these findings support the “infection” of repeat RNAs disrupts cellular identity in both tumor cells and the CAF microenvironment as a mechanism for tumor progression. Citation Format: Eunae You, Luli Zou, Patrick Danaher, Ildiko E. Phillips, Michael J. Raabe, Bidish Patel, Amaya Pankaj, Khrystyna North, Sean Kim, Youngmi Kim, Martin Aryee, David T. Ting. Repeat RNA dysregulation of cellular states in the pancreatic cancer microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C038.

BACKGROUND: The hepatocellular carcinoma (HCC) tumor microenvironment (TME) is composed of a complex ecosystem dominated by cancer cells and the endothelial cells that line tumor blood vessels. Although many genomic drivers have been identified and at least three transcriptional subsets have been proposed, these efforts have not yet led to novel therapies or otherwise significantly impacted management options. Single cell transcriptional profiling has generated deep insights into the multiple heterogeneous cell types within tissues, but the spatial context of these data is lost during single cell processing. Spatial transcriptomic approaches aim to bridge the gap between dissociative single cell technologies and in situ histopathological characterization.METHODS: To gain insight into potential in situ cancer-endothelial crosstalk interactions, we utilized the Nanostring GeoMx spatial transcriptomics platform with the Cancer Transcriptome Atlas ~1800 gene oligonucleotide probe panel to generate tumor (Arginase+) and blood vessel (CD31+) areas of interest (AOI) gene expression profiles from formalin-fixed, paraffin-embedded archival tissue specimens obtained from HCC resection specimens. Oligonucleotides released from each microscopic AOI were then captured, processed by DNA sequencing, and analyzed using custom computational pipelines.RESULTS: Using the 119 ROI containing data from both tumor and vessels that passed quality control filters, we performed unbiased hierarchical clustering of both the tumor and vessel areas of interest (AOI) within each ROI using the most highly variable genes for each AOI set and identified at least 3 clusters within each AOI type (tumor and vessel). Based on gene ontology analysis of the tumor AOIs, the two subsets were distinguished by unique immune and inflammatory-related genes. Analogous ontology-based characterization of the vessel AOIs demonstrated two groups: 1) an interferon-activated, inflamed progenitor, and immune checkpoint-associated cluster; and 2) a TGF-beta and oxidative stress-associated cluster. Notably, both vessel clusters also contained significant numbers of leukocyte genes, concordant with the intimate relationship of the vasculature and immune system. Canonical correlation analysis (CCA) utilizing both the most variable genes within each AOI set showed significant correlated gene sets within tumor AOIs and vessel AOIs, implying biologically significant interactions in multiple signaling pathways.CONCLUSIONS: Spatial transcriptomic profiling enables an understanding of cell-cell interactions in situ that can uncover biologically distinct tumor and blood vessel niches within the HCC microenvironment. Subsequent efforts will be focused on functionally assessing the spatially linked cancer and endothelial cell phenotypes with the goals of developing improved prognostic and predictive biomarkers and generating novel drug targets. Citation Format: Joseph W Franses, Michael J Raabe, Amaya Pankaj, Bidish Patel, Avril Coley, Irun Bhan, Martin Aryee, David T Ting. Spatial transcriptomic profiling to characterize the tumor-vascular interactome of hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO016.