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The organization of immune cells in human tumors is not well understood. Immunogenic tumors harbor spatially-localized multicellular ‘immunity hubs’ defined by expression of the T cell-attracting chemokinesCXCL10/CXCL11and abundant T cells. Here, we examined immunity hubs in human pre-immunotherapy lung cancer specimens, and found that they were associated with beneficial responses to PD-1-blockade. Immunity hubs were enriched for many interferon-stimulated genes, T cells in multiple differentiation states, andCXCL9/10/11+ macrophages that preferentially interact with CD8 T cells. Critically, we discovered the stem-immunity hub, a subtype of immunity hub strongly associated with favorable PD-1-blockade outcomes, distinct from mature tertiary lymphoid structures, and enriched for stem-like TCF7+PD-1+ CD8 T cells and activatedCCR7+LAMP3+ dendritic cells, as well as chemokines that organize these cells. These results elucidate the spatial organization of the human intratumoral immune response and its relevance to patient immunotherapy outcomes.

Cell fate transitions observed in embryonic development involve changes in three-dimensional genomic organization that provide proper lineage specification. Whether similar events occur within tumor cells and contribute to cancer evolution remains largely unexplored. We modeled this process in the pediatric cancer Ewing sarcoma and investigated high-resolution looping and large-scale nuclear conformation changes associated with the oncogenic fusion protein EWS-FLI1. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, EWS-FLI1 depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns and large-scale compartment configuration matching those observed in mesenchymal stem cells, a candidate Ewing sarcoma progenitor. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs.

The current high mortality of human lung cancer stems largely from the lack of feasible, early disease detection tools. An effective test with serum metabolomics predictive models able to suggest patients harboring disease could expedite triage patient to specialized imaging assessment. Here, using a training-validation-testing-cohort design, we establish our high-resolution magic angle spinning (HRMAS) magnetic resonance spectroscopy (MRS)-based metabolomics predictive models to indicate lung cancer presence and patient survival using serum samples collected prior to their disease diagnoses. Studied serum samples were collected from 79 patients before (within 5.0 y) and at lung cancer diagnosis. Disease predictive models were established by comparing serum metabolomic patterns between our training cohorts: patients with lung cancer at time of diagnosis, and matched healthy controls. These predictive models were then applied to evaluate serum samples of our validation and testing cohorts, all collected from patients before their lung cancer diagnosis. Our study found that the predictive model yielded values for prior-to-detection serum samples to be intermediate between values for patients at time of diagnosis and for healthy controls; these intermediate values significantly differed from both groups, with an F1 score = 0.628 for cancer prediction. Furthermore, values from metabolomics predictive model measured from prior-to-diagnosis sera could significantly predict 5-y survival for patients with localized disease.

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.

Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. Here we define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma, an aggressive cancer driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 displays a distinct DNA binding pattern that requires the EWSR1 domain and promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures observed in primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of regulatory circuits that promote neural crest-related developmental programs. Taken together, our study elucidates the epigenetic mechanisms utilized by EWSR1-ATF1 to establish regulatory networks in CCS, and points to precursor cells in the neural crest lineage as candidate cells of origin for these tumors.

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.