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Aberrant expression of repeat RNAs in pancreatic ductal adenocarcinoma (PDAC) mimics viral-like responses with implications on tumor cell state and the response of the surrounding microenvironment. To better understand the relationship of repeat RNAs in human PDAC, we performed spatial molecular imaging at single-cell resolution in 46 primary tumors, revealing correlations of high repeat RNA expression with alterations in epithelial state in PDAC cells and myofibroblast phenotype in cancer-associated fibroblasts (CAFs). This loss of cellular identity is observed with dosing of extracellular vesicles (EVs) and individual repeat RNAs of PDAC and CAF cell culture models pointing to cell-cell intercommunication of these viral-like elements. Differences in PDAC and CAF responses are driven by distinct innate immune signaling through interferon regulatory factor 3 (IRF3). The cell-context-specific viral-like responses to repeat RNAs provide a mechanism for modulation of cellular plasticity in diverse cell types in the PDAC microenvironment., Competing Interests: Declaration of interests D.T.T. and B.D.G. are co-founders with equity and consulting fees from ROME Therapeutics, a company developing drugs targeting repetitive elements, but this work was not supported by ROME Therapeutics. D.T.T. received in kind research support and a speaking honorarium from NanoString Technologies, and in kind research support from ACD-Biotechne, which was used in this work. M.J.A. is a co-founder of, owns equity in, and receives consulting fees from SeQure Dx, unrelated to this work. M.J.A. receives consulting fees from Chroma Medicine, unrelated to this work. P.D., N.S., B.M.H., K.N., S.E.C., T.K.K., Y.C., and Y.K. are or were employees of NanoString Technologies. D.T.T. has received consulting fees from PanTher Therapeutics, AstraZeneca, Sonata Therapeutics, Moderna, abrdn, Astellas, and Leica Biosystems Imaging; is a founder and has equity in PanTher Therapeutics and TellBio, Inc.; and is on the scientific advisory board with equity for ImproveBio, Inc., which are not related to this work. D.T.T. receives research support from Sanofi, AVA LifeScience GmbH, and Incyte, which was not used in this work. B.D.G. is a consultant or received honoraria for Darwin Health, Merck, PMV Pharma, Merck, Bristol-Meyers Squibb, and Chugai Pharmaceuticals and has research funding from Bristol-Meyers Squibb., (Copyright © 2024 Elsevier Inc. All rights reserved.)

To thrive, cancer cells must navigate acute inflammatory signaling accompanying oncogenic transformation, such as via overexpression of repeat elements. We examined the relationship between immunostimulatory repeat expression, tumor evolution, and the tumor-immune microenvironment. Integration of multimodal data from a cohort of pancreatic ductal adenocarcinoma (PDAC) patients revealed expression of specific Alu repeats predicted to form double-stranded RNAs (dsRNAs) and trigger retinoic-acid-inducible gene I (RIG-I)-like-receptor (RLR)-associated type-I interferon (IFN) signaling. Such Alu-derived dsRNAs also anti-correlated with pro-tumorigenic macrophage infiltration in late stage tumors. We defined two complementary pathways whereby PDAC may adapt to such anti-tumorigenic signaling. In mutant TP53 tumors, ORF1p from long interspersed nuclear element (LINE)-1 preferentially binds Alus and decreases their expression, whereas adenosine deaminases acting on RNA 1 (ADAR1) editing primarily reduces dsRNA formation in wild-type TP53 tumors. Depletion of either LINE-1 ORF1p or ADAR1 reduced tumor growth in vitro. The fact that tumors utilize multiple pathways to mitigate immunostimulatory repeats implies the stress from their expression is a fundamental phenomenon to which PDAC, and likely other tumors, adapt., Competing Interests: Declaration of interests B.D.G. received honoraria from Merck, Bristol Meyers Squibb (BMS), and Chugai Pharmaceuticals; research funding from BMS and Merck; and has been a compensated consultant for Darwin Health, Merck, PMV Pharma, Shennon Biotechnologies, Synteny AI, and ROME Therapeutics, of which he is a co-founder. A.S. has consulted for PMV Pharma and ROME Therapeutics, and he holds stock options of ROME Therapeutics. D.T.T. has received consulting fees from ROME Therapeutics, Sonata Therapeutics, Leica Biosystems Imaging, PanTher Therapeutics, and abrdn. D.T.T is a founder and has equity in ROME Therapeutics, PanTher Therapeutics, and TellBio, Inc., which is not related to this work. D.T.T is on the advisory board with equity for ImproveBio, Inc. D.T.T. has recieved honorariums from Astellas, AstraZeneca, Moderna, and Ikena Oncology that are not related to this work. D.T.T. recieves research support from ACD-Biotechne, AVA LifeScience GmbH, Incyte Pharmaceuticals, Sanofi, and Astellas, which was not used in this work. D.T.T.’s interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict-of-interest policies. S.W.L. is a consultant and holds equity in Blueprint Medicines, ORIC Pharmaceuticals, Mirimus, Inc., PMV Pharmaceuticals, Faeth Therapeutics, and Fate Therapeutics. J.L. received research funding from ROME Therapeutics, Ribon Therapeutics, and Refeyn; he received compensation from Transposon, ROME, and Oncolinea. C.A.I.-D. has received research support from Bristol Meyers Squibb., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells)., Experimental Design: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids., Results: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors., Conclusions: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer., (©2024 American Association for Cancer Research.)

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC., (Copyright © 2019 Elsevier Inc. All rights reserved.)