Extracellular vesicles (EVs) are critical mediators of intercellular communication in the tumor microenvironment and play an essential role in tumor growth and metastasis. MUC1 has been identified in tumor cell-derived EVs, but its function in that context has not been well investigated. We show that MUC1 is highly enriched in EVs derived from pancreatic cancer cell lines and patient tumors but not normal pancreas. Pretreatment of mice with tumor derived MUC1 positive EVs promoted tumor cell growth and metastasis in vivo. MUC1 positive EVs enhanced tumor cell growth, motility and invasion in vitro. Proteomic profiling revealed that MUC1-positive EVs contain protein cargo distinct from MUC1-negative EVs, which implicates MUC1 in selective EV cargo regulation. We demonstrate that the cytoplasmic tail of MUC1 expressed in pancreatic cancer cells interacts with programmed cell death 6-interacting protein (ALIX) and influences EV biogenesis and loading of specific oncogenic cargo (PhosphoTyr416-Src) that are known to associate with MUC1. EVs from SRC inhibitor Bosutinib treated cells showed reduced effects on cell viability, migration, and invasion of MUC1 knockout cells as compared to EVs from untreated cells. Thus, MUC1-positive EVs containing activated Src promoted tumor cell proliferation and in vivo tumor growth, migration, and metastasis of orthotopic pancreatic cancer cells. Collectively, our findings identify a previously unrecognized role for MUC1 in influencing EV composition and function: influencing loading of oncogenic protein cargoes into EVs. Our data highlight a novel mechanism controlling tumor-specific EV cargo loading and demonstrate that oncogenic cargo in MUC1-associated EVs contributes to pancreatic cancer progression.

Local and systemic immunosuppression are prominent features of pancreatic cancer, rendering anti-tumor effector cells inactive and immunotherapeutic approaches ineffective. The spleen, an understudied point of antigen-presentation and T cell priming in humans, holds particular importance in pancreatic cancer due to its proximity to the developing tumor. As main effectors of antigen presentation, dendritic cells display antigens to lymphocytes, thereby bridging the innate and adaptive immune response. While tumor-infiltrating anti-inflammatory dendritic cells have been described, splenic dendritic cells have historically just been considered to stimulate the anti-tumor immune response. Here, we describe, for the first time, the presence of an immunosuppressive, tolerogenic IDO1+ dendritic cell subset in the spleens of pancreatic cancer patients that likely contributes to systemic immunosuppression that is associated with pancreatic ductal adenocarcinoma. Network analysis of scRNA seq data reveals extensive communication networks between the identified tolerogenic DC cluster and numerous immune cell populations in the spleen. Interactions with innate and adaptive immune cells suggest a broad influence on leukocyte trafficking and immune regulation within the spleen microenvironment. The identification of signaling pathways involving AHR and IDO-1, CCL19, NECTIN2, CLEC2D, and others elucidates potential mechanisms underlying the immunosuppressive functions of this cell type.

Abstract Introduction: Adult stem cell dysregulation can initiate cancer. Pancreatic ductal glands (PDGs) act as a stem cell niche for the pancreatic main duct epithelium (MD). Previous studies have shown that Tff2+ cells within the PDGs give rise to progeny that migrate to the MD epithelium during inflammation. In human IPMN, the PDGs have been shown to comprise the basal proliferative compartment of these neoplasms. Here we investigate the effect of oncogenic activation and tumor suppressor deletion within Tff2+ PDG cells. Methods: We utilized Tff2CreERT2;Rosa26mT/mG;KrasLSL-G12D;P53fl/fl mice (TTKP) to activate oncogenes in Tff2+ cells and GFP tag this lineage in vivo and ex vivo. Cre activity was induced by tamoxifen injection in TTKP experimental or Tff2CreERT2;Rosa26mT/mG (TT) control mice. Pancreata were collected at pre-determined time points post-tamoxifen with GFP tagged, Tff2 lineage cells quantified and compared between groups at each timepoint. For ex vivo experiments, ductal cells were isolated from tamoxifen-naïve TTKP pancreata via magnetic-assisted cell sorting, grown in 3D culture and induced via 4OHT. GFP+ cells were sorted, cultured, and orthotopically implanted in athymic nude mice. Tumors were collected before reaching 20mm in diameter. Spatial RNA sequencing (spRNAseq) was performed on frozen tissue sections and analyzed with R package Seurat. Cross-species analysis utilized human PDAC scRNAseq reference datasets. Results: TTKP mice developed lineage-traced PDAC eight weeks after tamoxifen induction. In vivo lineage tracing showed a significant increase of GFP+ Tff2-lineage cells in PDGs and MD between TTKP and TT mice at all analyzed time points (4-, 6- and 8-weeks post tamoxifen, ≥ 3 mice each). GFP-positive PDGs exhibited progressive PanIN-like changes before PDAC formation. Tff2-PDG cells in TT mice function as transient amplifying cells, expanding within PDGs and to the MD during inflammation and contracting upon resolution. Oncogenic activation (sans inflammation) in Tff2-PDG cells led to sustained GFP+ cell presence in PDGs and MD, indicating increased stem-like behavior preceding neoplasia. Orthotopic organoid experiments resulted in GFP+ tumor formation. SpRNAseq analysis indicated enrichment of "Ductal cell type 2" in TTKP tumors, particularly overlying the main duct epithelium. Conclusion: Our study demonstrates that dysregulation of the Tff2-lineage in PDGs can initiate PDAC. Oncogenic activation in Tff2+ cells increases their stemness as indicated by lineage expansion in PDGs and MD preceding PanIN and PDAC formation. Orthotopic organoid implantation experiments further validate the tumor-initiating capacity of Tff2+ ductal cells. Spatial transcriptomics analysis confirms the presence of similar cell types identified in human scRNAseq studies within TTKP tumors. These findings strongly support the role of PDGs as a potential niche of origin for human PDAC and provide additional insights into an under-studied disease initiating process. Citation Format: Kyle L. McAndrews, Pinaki Mondal, Dulce Maroni, Dongdong Wang, Michael A. Hollingsworth, Sarah P. Thayer. Activation of oncogenes within Tff2 expressing cells of the pancreatic ductal glands results in increased lineage stemness preceding tumorigenesis: Insights from an inducible mouse model and derived organoids [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C055.

Pancreatic ductal adenocarcinoma (PDAC) is highly lethal. MUC4 (mucin4) is a heavily glycosylated protein aberrantly expressed in PDAC and promotes tumorigenesis via an unknown mechanism. To assess this, we genetically knocked out (KO) MUC4 in PDAC cells that did not express and did express truncated O-glycans (Tn/STn) using CRISPR/Cas9 technology. We found that MUC4 knockout cells possess less tumorigenicity in vitro and in vivo, which was further reduced in PDAC cells that express aberrant overexpression of truncated O-glycans. Also, MUC4KO cells showed a further reduction of epidermal growth factor receptors (ErbB) and their downstream signaling pathways in truncated O-glycan expressing PDAC cells. Tn-MUC4 specific 3B11 antibody inhibited MUC4-induced ErbB receptor and its downstream signaling cascades. MUC4 knockout differentially regulates apoptosis and cell cycle arrest in branched and truncated O-glycan expressing PDAC cells. Additionally, MUC4KO cells were found to be more sensitive to gemcitabine treatment. They possessed the upregulated expression of hENT1 and hCNT3 compared to parental cells, which were further affected in cells with aberrant O-glycosylation. Taken together, our results indicate that MUC4 enhances the malignant properties and gemcitabine resistance in PDAC tumors that aberrantly overexpress truncated O-glycans via altering ErbB/AKT signaling cascades and expression of nucleoside transporters, respectively.

Abstract The efficacy of current oncotherapeutics is largely limited by an inability to access avascular tissues, which is in part responsible for forty years of stagnant pancreatic cancer statistics where the median survival remains a mere six months. Oncolytic bacteria such as Clostridium novyi-NT overcome this challenge with its ultrasensitive, innate affinity for hypoxic/necrotic areas found at the center of solid tumors and their metastases. While preclinical and clinical data from intratumoral injections of C. novyi-NT are promising, many tumors are inaccessible to such injections. Preclinical trials of analogous IV injections have uncovered other obstacles such as rapid clearance of C. novyi-NT by the immune system independent of septic complications. To mitigate rapid clearance, CRISPR/Cas9n was used to genetically modify a non-toxic form of C. novyi-NT to express a tumor targeting RGD peptide on the spore surface. Through this novel, first of its kind, methodology, spores with stronger affinity to a surface coated with the targeted binding partner of RGD, aVb3 integrin, have been generated. Importantly, there was no statistically significant difference in the genetically modified spore’s capacity for sporulation or germination when compared to unmodified C. novyi-NT spores, nor was a difference in lytic capacity observed, suggesting no relevant off-target effects from genomic modification. Biodistribution and efficacy of non-toxic RGD-modified spores was evaluated in an immunocompetent, syngeneic, pancreatic cancer murine model. Ongoing efforts to characterize the biodistribution and efficacy of the intravenously injected RGD-modified C. novyi-NT include the application of multiplex immunofluorescence, laser microdissection, and live, whole animal imaging. Supported as a pilot project by funds from NIH COBRE grant 1P20GM109024, Doctoral Dissertation Funds to KMD from NDSU, and by discretionary funds from investigators at UNMC. Citation Format: Kaitlin M. Dailey, Krysten Vance, Kyle McAndrews, Reed I. Jacobson, Jandro Delgado, Paige R. Johnson, Taylor M. Woolery, Megan Orr, Jiha Kim, Sanku Mallik, Kenneth W. Bayles, Michael A. Hollingsworth, Amanda E. Brooks. Development of an RGD CRISPR-modified Clostridium novyi NT spores as an intravenous oncotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-037.

Lrig1, an epithelial stem cell marker and tumor suppressor, is uniquely expressed within PDGs in pancreas. PDGs are an epithelial progenitor compartment and likely compartment-of-origin for PDAC. Here, in vivo GFP-lineage tagging of Lrig1 expressing PDG cells identify GFP tagged cells in mesenchyme but not in the main duct epithelium, identifying a cell within a PDG epithelial compartment capable of EMT to regenerate its own micro-environment. Mechanisms regulating this EMT switch results from a loss of Lrig1. Characterization of the GFP-positive mesenchyme and PDG cells confirms EMT. ScRNASeq of epithelial spheroids revealed enhanced mesenchymal features in Lrig1 deficient cells, while Lrig1 overexpression abrogates mesenchymal differentiation. In vivo, in vitro and ex vivo studies reveal the Lrig1 loss within this stem cell compartment results in enhanced EGF and TGF-β signaling, expansion of stem cell, proliferative and mesenchymal markers in addition to the up-regulation of a master regulator of stemness and EMT, PSPC1. In vivo murine models of PDAC (KPC) which tag and lose Lrig1 in the PDG compartment results in the expansion of the PDG stem cell compartment which now resembles low-grade PanINs in addition to the formation of an extensive GFP-positive desmoplasia. In vitro, Lrig1 regulates proliferation and EMT in cancer cell lines. These studies identify Lrig1 as a regulator of stemness and EMT in regeneration and cancer.

Caecincola cookorum n. sp. is described from the intestine of white crappie (Pomoxis annularis) collected from B. A. Steinhagen Reservoir in Tyler County, Texas, U. S. A. Worms of the new species possess ceca that extend past the posterior testis, whereas members of the other species in the genus (Caecincola autumnae, Caecincola latostoma, Caecincola longiscens, Caecincola parvulus, and Caecincola wakullata) have ceca that terminate before the end of the posterior testis. This is the second species of Caecincola described from white crappie from rivers draining into the Gulf of Mexico (C. longiscens from Mississippi being the other), and it is possible that a large fraction of the species-level diversity in this genus remains to be discovered.